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Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. Here, we present preliminary results of a Surface Acoustic Wave (SAW) biosensor with potential to detect Ebola virus in multiple, unprocessed sample types including blood, serum, saliva, and feces within 5-10 min.
Ebola virus is a class A select agent filovirus that was first identified in Zaire in 1976 and named after the River Ebola in Zaire [7]. Ebola outbreaks are sustained through person-to-person transmission from direct contact with infected people, bodily fluids, or contaminated clothes and linens [7]. Early detection of this highly contagious virus would allow for improved infection control measures and early treatment at specialized facilities. The current mortality rate from epidemic Ebola ranges from 40% to 90% and could be greatly reduced with early, point-of-care diagnostics [8,9]. Mathematical models suggest that the total direct costs of the present outbreak range from $82 million to $356 million in the three most effected countries; early diagnosis and treatment will also reduce aggregate healthcare costs [10].
Current approaches to Ebola identification include antigen-capture ELISA testing, IgM ELISA, RT-PCR, virus isolation, electron microscopy, and serologic testing for IgM or IgG antibodies. Quantitative measurement of the viral load also has prognostic importance, with a much higher case fatality rate in patients with viral loads over 10 million copies per milliliter [11]. Current tests for Ebola, especially PCR-based techniques, are generally limited by the need for added reagents, refrigeration, and the need for specially trained laboratory personnel. Lateral flow assays can provide rapid and fairly inexpensive qualitative results, but are unable to quantify viral load.
Our group has developed a point-of-care biosensor that employs surface acoustic waves for label-free pathogen detection without the need for any sample preparation [12][13][14]. This biosensor platform detects target antigens in the presence of confounding analytes and in various types of media, thus eliminating complicated sample preparation protocols. Such acoustic wave sensors can achieve limits of detection of <50 pg/cm 2 , which is an order of magnitude lower than detection limits for other marker-free systems such as optical surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) devices [15].
With this sensor platform, viral detection is rapid (with acoustic wave signal detection within 2 min), and the entire detection protocol (from collecting the test sample to obtaining a positive or negative test result) requiring about 5-10 min. Such technological benefits would be ideal for rapid, point-of-care identification and diagnosis of the Ebola virus, especially in environments with minimal or overloaded infrastructure, such as in public health or emergency response situations. Here, we discuss initial modifications of the biosensor to demonstrate detection of Ebola and its potential utility to combat this recurrent epidemic.
Fabrication and Functionalization of the SAW Biosensor
The sensor chips were prepared using lithium tantalate (36°, y-cut, x-propagating LiTaO3) wafers by lithographic deposition and patterning of inter-digital transducers (IDT) and waveguide layers as previously described [12]. Briefly, cleaned LiTaO3 wafers were patterned with four IDT patterns with sets of transducers and delay lines using negative tone photoresist AZ2020 (AZ Electronic Materials, Branchburg, NJ, USA). Then, a metallization step was performed with 5000 Å aluminum using an electron-beam evaporator (Temescal, Wilmington, MA, USA). The wafer was placed in an acetone bath to lift off the photoresist and any excess aluminum, followed by an acetone spray as needed and by the following rinses in methanol, isopropyl alcohol, and deionized water. This process was repeated for ground metallization, bus lines, and contact pads. Next, a waveguide layer of 5000 Å silicon dioxide (SiO2) was deposited as a film on the wafer using lift-off plasma enhanced chemical vapor deposition (Oerlikon Versaline, Pfaeffikon, Switzerland). The oxide was coated with hexamethyldisilazane in a vacuum oven, and positive tone photoresist AZ4330 (AZ Electronic Materials) was used to form a photoresist mask on the wafer having exposed portions. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science<|endoftext|>Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. Here, the surface of the piezoelectric substrate was sensitized with a monoclonal antibody specific for Ebola virus. A binding event causes a change in surface mass and results in a phase shift of the signal wave propagating across the sensor surface. Biochemical interactions occurring on the sensor surface can be quantified by measuring this change in phase shift (Δφ).
The overall schematic concept of the biosensor device for detecting the Ebola virus is shown in Figure 1. The biosensor includes a test lane including antibodies selective for the target bioagents (i.e., the Ebola virus in this study), as well as a reference lane including a control IgG1 antibody (Figure 1a). Measurements of Δφ were performed for both the test lane and the reference lane by use of an output interface device (connected to the SAW biosensor) and a laptop computer (Figure 1b). (b) The detection system for research use with fluidic housing surrounding the biosensor, an output interface device connected to the biosensor, and a laptop computer. The sensor is powered by disposable AA, and further miniaturization of the output interface is possible for field use. In addition, the fluidic housing and biosensor can be provided as a disposable module that can be detached from the output interface and then decontaminated prior to disposal.
Imaging Analysis of the Ebola Zaire Antigen Sample
Fully intact Ebolavirus is highly pathogenic and designated BSL-4 material. For this particular study, we employed inactivated and disrupted Zaire (Mayinga strain) Ebola virus (BSL-1 material), which is available for research purposes under non-BSL-4 conditions. Thus, transmission electron microscopy studies were conducted to identify the size distribution of the fragmented viral particles. Representative images are provided in Figure 2. Shown is a TEM image of a 1:20 diluted sample (with PBS), which includes a majority of particles that are less than 10 nm and the presence of larger aggregates between about 50 nm to 300 nm (Figure 2a). Rare filamentous structures, with diameter of about 30 nm (Figure 2b) were also noted. Intact Ebola virus has a filamentous structure with diameter of 80 nm and length greater than 950 nm [18]. TEM of the inactivated virus sample shows highly disrupted, fragmented particles that form aggregates (Figure 2a), rather than the elongated filaments observed in intact filovirus. When filamentous structures were observed (Figure 2b), the cross-section diameter was less than half of the diameter expected for intact filovirus [18]. While the predominant species in the inactivated virus sample are disrupted particles, the biosensor phase shift response is likely to be much greater for an infectious Ebola sample containing intact virus. Based on our previous study of HIV detection [16] and the relative masses of intact HIV and Ebola [18,19], we would estimate a 5-10× greater sensitivity for intact Ebola virus compared to intact HIV. As a first approximation, we assume a log-linear correlation between mass and phase shift. As particles of greater mass will provide a greater response, the sensor is likely to detect more massive, intact viral particles at lower concentration.
SAW Biosensor Detection of the Ebola Zaire Sample
In analogy to our previous report on the detection of HIV-1 and HIV-2 [16], we functionalized the SAW biosensor with monoclonal antibodies specific for Zaire (Mayinga) strain of Ebola Virus. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science
|
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|
Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. Such acoustic wave sensors can achieve limits of detection of <50 pg/cm 2 , which is an order of magnitude lower than detection limits for other marker-free systems such as optical surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) devices [15].
With this sensor platform, viral detection is rapid (with acoustic wave signal detection within 2 min), and the entire detection protocol (from collecting the test sample to obtaining a positive or negative test result) requiring about 5-10 min. Such technological benefits would be ideal for rapid, point-of-care identification and diagnosis of the Ebola virus, especially in environments with minimal or overloaded infrastructure, such as in public health or emergency response situations. Here, we discuss initial modifications of the biosensor to demonstrate detection of Ebola and its potential utility to combat this recurrent epidemic.
Fabrication and Functionalization of the SAW Biosensor
The sensor chips were prepared using lithium tantalate (36°, y-cut, x-propagating LiTaO3) wafers by lithographic deposition and patterning of inter-digital transducers (IDT) and waveguide layers as previously described [12]. Briefly, cleaned LiTaO3 wafers were patterned with four IDT patterns with sets of transducers and delay lines using negative tone photoresist AZ2020 (AZ Electronic Materials, Branchburg, NJ, USA). Then, a metallization step was performed with 5000 Å aluminum using an electron-beam evaporator (Temescal, Wilmington, MA, USA). The wafer was placed in an acetone bath to lift off the photoresist and any excess aluminum, followed by an acetone spray as needed and by the following rinses in methanol, isopropyl alcohol, and deionized water. This process was repeated for ground metallization, bus lines, and contact pads. Next, a waveguide layer of 5000 Å silicon dioxide (SiO2) was deposited as a film on the wafer using lift-off plasma enhanced chemical vapor deposition (Oerlikon Versaline, Pfaeffikon, Switzerland). The oxide was coated with hexamethyldisilazane in a vacuum oven, and positive tone photoresist AZ4330 (AZ Electronic Materials) was used to form a photoresist mask on the wafer having exposed portions. The exposed portions of SiO2 was etched with reactive ion etching to access electrical contact pads. Finally, the resultant wafer was diced to form individual chips, in which photoresist from dies was removed by rinsing in acetone, methanol, and isopropanol.
Antibodies were individually patterned on sensing lanes as previously described [16]. Briefly, sensor chips were coated with toluene and 3-glycidyloxypropyl trimethoxysilane (90%/10%) in an oven at 60 °C for 1.5 h and then rebaked at 100 °C for 1 h. Each lane was coated with antibodies at a concentration of 10 µg/mL in phosphate-buffered saline (PBS). The following antibodies were used: mouse monoclonal antibodies (IgG2a isotype) specific for Ebola (AB-EB-MAB1, anti-Ebola virus monoclonal antibody 1; BEI Resources, Manassas, VA, USA); and mouse IgG1 antibody isotype control (F(ab')2 fragment) (ab37426, monoclonal isotype control; Abcam, Cambridge, MA, USA). Typically, two lanes were functionalized with antibodies specific for the target analyte, and two lanes were functionalized with isotype control antibodies; the latter are referred to as reference lanes.
Provenance and Handling of Ebola Virus Strain Zaire (Mayinga) Inactivated
The Ebola virus antigen sample (NR-31807; BEI Resources, Manassas, VA, USA) consists of inactivated and highly disrupted viral particles and was used as provided. Fully intact Ebolavirus is highly pathogenic and designated BSL-4 material. For research purposes, the supplier provides inactivated and disrupted Zaire (Mayinga) Ebola virus (BSL-1 material), which was prepared as follows. As stated by the supplier, Zaire (Mayinga) Ebola virus from infected Vero E6 pellets was suspended in 50 mM sodium borate, gamma irradiated (5 × 10 6 rads total dose) on dry ice, and sonicated. Culture cell debris were removed by centrifugation. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science<|endoftext|>Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. The exposed portions of SiO2 was etched with reactive ion etching to access electrical contact pads. Finally, the resultant wafer was diced to form individual chips, in which photoresist from dies was removed by rinsing in acetone, methanol, and isopropanol.
Antibodies were individually patterned on sensing lanes as previously described [16]. Briefly, sensor chips were coated with toluene and 3-glycidyloxypropyl trimethoxysilane (90%/10%) in an oven at 60 °C for 1.5 h and then rebaked at 100 °C for 1 h. Each lane was coated with antibodies at a concentration of 10 µg/mL in phosphate-buffered saline (PBS). The following antibodies were used: mouse monoclonal antibodies (IgG2a isotype) specific for Ebola (AB-EB-MAB1, anti-Ebola virus monoclonal antibody 1; BEI Resources, Manassas, VA, USA); and mouse IgG1 antibody isotype control (F(ab')2 fragment) (ab37426, monoclonal isotype control; Abcam, Cambridge, MA, USA). Typically, two lanes were functionalized with antibodies specific for the target analyte, and two lanes were functionalized with isotype control antibodies; the latter are referred to as reference lanes.
Provenance and Handling of Ebola Virus Strain Zaire (Mayinga) Inactivated
The Ebola virus antigen sample (NR-31807; BEI Resources, Manassas, VA, USA) consists of inactivated and highly disrupted viral particles and was used as provided. Fully intact Ebolavirus is highly pathogenic and designated BSL-4 material. For research purposes, the supplier provides inactivated and disrupted Zaire (Mayinga) Ebola virus (BSL-1 material), which was prepared as follows. As stated by the supplier, Zaire (Mayinga) Ebola virus from infected Vero E6 pellets was suspended in 50 mM sodium borate, gamma irradiated (5 × 10 6 rads total dose) on dry ice, and sonicated. Culture cell debris were removed by centrifugation. The virus was confirmed non-viable (killed or inactivated) by inoculation of cell culture (10 days on Vero cells) followed by a second passage (10 days on Vero cells). The concentration of the Ebola virus antigen sample was provided in plaque-forming units (PFU/mL) by the supplier, indicating the concentration of viable viral particles prior to inactivation, and allowing for comparison across different animal models and experimental conditions [17].
The Ebola antigen samples were analyzed by scanning electron microscopy at the University of New Mexico Health Sciences Center Electron Microscopy Facility (Albuquerque, NM, USA) to roughly determine the size distribution of the degraded viral particles. Ebola samples were stored in aliquots of 50 µL in screw cap safety microtubes at −80 °C until ready for use.
When ready to be tested, the samples were thawed on ice and supplemented with phosphate buffered saline (PBS; 137.0 mM NaCl, 2.7 mM KCl, 10.0 mM Na2HPO4·2H2O, 2.0 mM KH2PO4, pH 7.4 with HCl). All Ebola antigen samples were prepared fresh for each experiment.
Virus Detection Using the SAW Biosensor
Detection of Ebola Zaire antigen was conducted in BSL-2 certified biosafety cabinets. Data expressed as phase shift (Δφ, expressed in degrees) were recorded with a custom acquisition program developed using Visual Studio (Microsoft) as previously described [12]. In brief, data from each IDT delay line were collected for each lane and acquired simultaneously as a occasion of time of acquisition. The biosensor supports a surface shear wave, and the IDT detects the wave by transducing the mechanical wave into an electrical signal. This electrical signal (expressed as voltage information) was converted to phase (φ, expressed in degrees) using the custom acquisition program. Data from reference lanes were subtracted from the data from Ebola test lanes at each time point to determine the specific Ebola signal. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science
|
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|
Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. This preliminary study demonstrates SAW biosensor detection of the Ebola virus in a concentration-dependent manner. The detection limit with this methodology is below the average level of viremia detected on the first day of symptoms by PCR. We observe a log-linear sensor response for highly fragmented Ebola viral particles, with a detection limit corresponding to 1.9 × 104 PFU/mL prior to virus inactivation. We predict greatly improved sensitivity for intact, infectious Ebola virus. This point-of-care methodology has the potential to detect Ebola viremia prior to symptom onset, greatly enabling infection control and rapid treatment. This biosensor platform is powered by disposable AA batteries and can be rapidly adapted to detect other emerging diseases in austere conditions.
Introduction
The 2014 Ebola virus outbreak is the largest in history, with widespread transmission in multiple West African countries and sporadic cases in Europe and North America [1][2][3][4]. The massive public health response has been limited, in part, by the inability to rapidly detect the presence of Ebola virus in potential patients living in remote areas [5]. The early symptoms of Ebola overlap with symptoms of endemic malaria and other febrile illnesses [6].
Rapid, point-of-care detection of the Ebola virus could enable early quarantine and halt future epidemics and pandemics [5,6]. While point-of-care nucleotide amplification tests exist [5], these are limited by the need for multiple reagents, refrigeration, and specialized personnel. Here, we present preliminary results of a Surface Acoustic Wave (SAW) biosensor with potential to detect Ebola virus in multiple, unprocessed sample types including blood, serum, saliva, and feces within 5-10 min.
Ebola virus is a class A select agent filovirus that was first identified in Zaire in 1976 and named after the River Ebola in Zaire [7]. Ebola outbreaks are sustained through person-to-person transmission from direct contact with infected people, bodily fluids, or contaminated clothes and linens [7]. Early detection of this highly contagious virus would allow for improved infection control measures and early treatment at specialized facilities. The current mortality rate from epidemic Ebola ranges from 40% to 90% and could be greatly reduced with early, point-of-care diagnostics [8,9]. Mathematical models suggest that the total direct costs of the present outbreak range from $82 million to $356 million in the three most effected countries; early diagnosis and treatment will also reduce aggregate healthcare costs [10].
Current approaches to Ebola identification include antigen-capture ELISA testing, IgM ELISA, RT-PCR, virus isolation, electron microscopy, and serologic testing for IgM or IgG antibodies. Quantitative measurement of the viral load also has prognostic importance, with a much higher case fatality rate in patients with viral loads over 10 million copies per milliliter [11]. Current tests for Ebola, especially PCR-based techniques, are generally limited by the need for added reagents, refrigeration, and the need for specially trained laboratory personnel. Lateral flow assays can provide rapid and fairly inexpensive qualitative results, but are unable to quantify viral load.
Our group has developed a point-of-care biosensor that employs surface acoustic waves for label-free pathogen detection without the need for any sample preparation [12][13][14]. This biosensor platform detects target antigens in the presence of confounding analytes and in various types of media, thus eliminating complicated sample preparation protocols. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science<|endoftext|>Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. Ebola virus antigens were detected in PBS solutions over a 2.5 log reference range at concentrations corresponding to 1.0 × 10 4 PFU/mL to 3.0 × 10 6 PFU/mL prior to virus inactivation. The lowest concentration is below the average viremia level of 3 × 10 4 RNA copies per mL observed in Ebola patients on the first day of disease symptoms [20].
Representative phase shift data are provided in Figure 3. The IgG control lane accounts for matrix effects and non-specific binding of potentially interfering species.
Detection of Ebola virus resulted in a concentration-dependent increase with Δφ values ranging from 0.20 ± 0.04 to 4.46 ± 0.86, corresponding approximately to 1.6 × 10 4 PFU/mL to 6.5 × 10 6 PFU/mL ( Figure 4). There was a log-linear relationship between viral load and Δφ for this concentration range of viral particles with a correlation coefficient R 2 of 0.92. The linear range and correlation coefficients compare favorably to those recently reported for qRT-PCR [21]. A limit of detection (LOD) of 1.9 × 10 4 PFU/mL was calculated for Ebola virus by linear regression and using the average background noise Δφ value of 0.31. These results indicate that the prototype SAW biosensor rapidly detects the Zaire strain of Ebola antigens in a defined buffer with detection limits below the average viremia level at onset of clinical symptoms.
Conclusions/Outlook
We have demonstrated an adaptable, label-free sensing system for the rapid detection of bioagents and show its use to detect the Ebola Zaire virus. We have previously shown the effectiveness of this system in detecting the Bacillus anthracis bacteria simulant, Bacillus thuringiensis [13]; the Coxsackie virus [12]; the Sin Nombre hantavirus [12]; the Francisella tularensis bacteria [22] and the Human Immunodeficiency Virus types 1 and 2 [16]. This study further extends the capability of this SAW biosensor platform to accommodate the rapid, label-free, and specific detection of the Ebola Zaire virus.
As fully intact Ebola virus is highly contagious, we used an inactivated virus in order to conduct this study under non-BSL-4 conditions. However, in a first responder or real world scenario, the sample would not necessarily have to be inactivated and could be tested directly, providing the sensor was contained in a controlled environment.
Sample preparation is not required for this label-free sensing methodology, simplifying use in field conditions without centralized laboratories or refrigeration. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science
|
[{"added": "2016-05-12T22:15:10.714Z", "created": "2015-04-01T00:00:00.000", "id": "789840", "metadata": {"extfieldsofstudy": ["Biology", "Computer Science", "Medicine"], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1424-8220/15/4/8605/pdf", "pdf_hash": "60f9b30b0f80eb5d32264ddddb95158fa9066296", "pdf_src": "PubMedCentral", "provenance": "peS2o-0028.json.gz:556", "s2fieldsofstudy": ["Medicine"], "sha1": "60f9b30b0f80eb5d32264ddddb95158fa9066296", "year": 2015}, "source": "pes2o/s2orc", "version": "v3-fos-license"}, {"added": "2016-05-12T22:15:10.714Z", "created": "2015-04-01T00:00:00.000", "id": "789840", "metadata": {"extfieldsofstudy": ["Biology", "Computer Science", "Medicine"], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.mdpi.com/1424-8220/15/4/8605/pdf", "pdf_hash": "60f9b30b0f80eb5d32264ddddb95158fa9066296", "pdf_src": "PubMedCentral", "provenance": "peS2o-0028.json.gz:556", "s2fieldsofstudy": ["Medicine"], "sha1": "60f9b30b0f80eb5d32264ddddb95158fa9066296", "year": 2015}, "source": "pes2o/s2orc", "version": "v3-fos-license"}]
|
Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. Representative images are provided in Figure 2. Shown is a TEM image of a 1:20 diluted sample (with PBS), which includes a majority of particles that are less than 10 nm and the presence of larger aggregates between about 50 nm to 300 nm (Figure 2a). Rare filamentous structures, with diameter of about 30 nm (Figure 2b) were also noted. Intact Ebola virus has a filamentous structure with diameter of 80 nm and length greater than 950 nm [18]. TEM of the inactivated virus sample shows highly disrupted, fragmented particles that form aggregates (Figure 2a), rather than the elongated filaments observed in intact filovirus. When filamentous structures were observed (Figure 2b), the cross-section diameter was less than half of the diameter expected for intact filovirus [18]. While the predominant species in the inactivated virus sample are disrupted particles, the biosensor phase shift response is likely to be much greater for an infectious Ebola sample containing intact virus. Based on our previous study of HIV detection [16] and the relative masses of intact HIV and Ebola [18,19], we would estimate a 5-10× greater sensitivity for intact Ebola virus compared to intact HIV. As a first approximation, we assume a log-linear correlation between mass and phase shift. As particles of greater mass will provide a greater response, the sensor is likely to detect more massive, intact viral particles at lower concentration.
SAW Biosensor Detection of the Ebola Zaire Sample
In analogy to our previous report on the detection of HIV-1 and HIV-2 [16], we functionalized the SAW biosensor with monoclonal antibodies specific for Zaire (Mayinga) strain of Ebola Virus. Ebola virus antigens were detected in PBS solutions over a 2.5 log reference range at concentrations corresponding to 1.0 × 10 4 PFU/mL to 3.0 × 10 6 PFU/mL prior to virus inactivation. The lowest concentration is below the average viremia level of 3 × 10 4 RNA copies per mL observed in Ebola patients on the first day of disease symptoms [20].
Representative phase shift data are provided in Figure 3. The IgG control lane accounts for matrix effects and non-specific binding of potentially interfering species.
Detection of Ebola virus resulted in a concentration-dependent increase with Δφ values ranging from 0.20 ± 0.04 to 4.46 ± 0.86, corresponding approximately to 1.6 × 10 4 PFU/mL to 6.5 × 10 6 PFU/mL ( Figure 4). There was a log-linear relationship between viral load and Δφ for this concentration range of viral particles with a correlation coefficient R 2 of 0.92. The linear range and correlation coefficients compare favorably to those recently reported for qRT-PCR [21]. A limit of detection (LOD) of 1.9 × 10 4 PFU/mL was calculated for Ebola virus by linear regression and using the average background noise Δφ value of 0.31. These results indicate that the prototype SAW biosensor rapidly detects the Zaire strain of Ebola antigens in a defined buffer with detection limits below the average viremia level at onset of clinical symptoms.
Conclusions/Outlook
We have demonstrated an adaptable, label-free sensing system for the rapid detection of bioagents and show its use to detect the Ebola Zaire virus. We have previously shown the effectiveness of this system in detecting the Bacillus anthracis bacteria simulant, Bacillus thuringiensis [13]; the Coxsackie virus [12]; the Sin Nombre hantavirus [12]; the Francisella tularensis bacteria [22] and the Human Immunodeficiency Virus types 1 and 2 [16]. This study further extends the capability of this SAW biosensor platform to accommodate the rapid, label-free, and specific detection of the Ebola Zaire virus.
As fully intact Ebola virus is highly contagious, we used an inactivated virus in order to conduct this study under non-BSL-4 conditions. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science<|endoftext|>Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. However, in a first responder or real world scenario, the sample would not necessarily have to be inactivated and could be tested directly, providing the sensor was contained in a controlled environment.
Sample preparation is not required for this label-free sensing methodology, simplifying use in field conditions without centralized laboratories or refrigeration. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
==
Domain: Biology Medicine Computer Science<|endoftext|>Rapid Detection of Ebola Virus with a Reagent-Free, Point-of-Care Biosensor
Surface acoustic wave (SAW) sensors can rapidly detect Ebola antigens at the point-of-care without the need for added reagents, sample processing, or specialized personnel. The data for quantitative measurement of specific Ebola particle detection refer to the phase shift (Δφ), which corresponds to the difference between the reference and Ebola signals that is measured continuously after addition of a viral antigen sample Ebola antigen samples of various concentrations were prepared in 100 µL of PBS and applied to the biosensor. Binding of viral antigens to the sensor surface resulted in a net phase shift of the signal Δφ (expressed in degrees on the y-axis) along the time plot (on the x-axis). While phase shift differences associated with specific Ebola signal are apparent and stable within seconds of sample addition, we continue measuring up to 5 min after sample addition to assure signal stability and reproducibility. All measurements represent multiplicates of at least three, and data points are represented as means ± standard error of the mean (SEM).
Results and Discussion
We present here the initial development and characterization of a biosensor for point-of-care, quantitative detection of the Ebola virus. This device enables log-linear, concentration-dependent detection of disrupted viral particles in the clinically relevant range. Overall sensitivity and limit of detection are dependent on the mass of the species that is bound to the sensor surface; this biosensor is likely to be orders of magnitude more sensitive for intact, infectious Ebola viral samples [15].
Design Elements of Surface Acoustic Wave Biosensor
The biosensor was adapted to allow for sensitive detection of the Ebola virus. The biosensor has a planar, piezoelectric substrate containing inter-digital transducers (IDTs) [12,13]. The piezoelectric substrate propagates horizontally polarized surface shear waves, and such waves are induced by applying an alternating voltage to the IDTs at a high frequency (generally between 80 and 400 MHz). The surface shear waves are characterized by a particular resonant frequency that is sensitive to changes on the sensor surface.
Specificity for the target bioagents is obtained by functionalizing the surface of the substrate (Figure 1a). Here, the surface of the piezoelectric substrate was sensitized with a monoclonal antibody specific for Ebola virus. A binding event causes a change in surface mass and results in a phase shift of the signal wave propagating across the sensor surface. Biochemical interactions occurring on the sensor surface can be quantified by measuring this change in phase shift (Δφ).
The overall schematic concept of the biosensor device for detecting the Ebola virus is shown in Figure 1. The biosensor includes a test lane including antibodies selective for the target bioagents (i.e., the Ebola virus in this study), as well as a reference lane including a control IgG1 antibody (Figure 1a). Measurements of Δφ were performed for both the test lane and the reference lane by use of an output interface device (connected to the SAW biosensor) and a laptop computer (Figure 1b). (b) The detection system for research use with fluidic housing surrounding the biosensor, an output interface device connected to the biosensor, and a laptop computer. The sensor is powered by disposable AA, and further miniaturization of the output interface is possible for field use. In addition, the fluidic housing and biosensor can be provided as a disposable module that can be detached from the output interface and then decontaminated prior to disposal.
Imaging Analysis of the Ebola Zaire Antigen Sample
Fully intact Ebolavirus is highly pathogenic and designated BSL-4 material. For this particular study, we employed inactivated and disrupted Zaire (Mayinga strain) Ebola virus (BSL-1 material), which is available for research purposes under non-BSL-4 conditions. Thus, transmission electron microscopy studies were conducted to identify the size distribution of the fragmented viral particles. In summary, the SAW biosensor is a versatile platform that shows promise to revolutionize rapid pathogen detection and enable early treatment in public health and emergency responses.
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Domain: Biology Medicine Computer Science
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. Response of auxin-related transcripts to IAA, decapitation, and NPA treatments.
Supplemental Table S1. List of protein sequences used in phylogeny reconstruction.
==
Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In contrast to SL response mutants, grafts between the wild type and SL-deficient mutants in pea show a minor effect of the scion on gene expression in the rootstock, possibly due to the restoration of SL content by the wildtype graft partner. To investigate the scenario for d27, we analyzed gene expression in the hypocotyl and rootstock of plants reciprocally grafted with the wild type (Fig. 6D). In d27 self-grafts, MAX3 and MAX4 transcripts were up-regulated, consistent with feedback. (D27 transcripts were also highly overexpressed, but this may be primarily a result of misregulation of a nonfunctional transcript.) As with wild-type rootstocks of pea grafted to mutants in SL biosynthesis (Dun et al., 2009b), expression in the root was not significantly affected by the genotype of the shoot in either reciprocal graft in Arabidopsis. As in pea, this may be because wild-type rootstocks can restore branching, and presumably the long-distance feedback signal, to normal levels in d27 shoots. Notably, in grafts involving d27, the three auxin-responsive transcripts did not exhibit a statistically significant difference from wild-type self-grafts, suggesting that auxin transport and/or signaling is not substantially affected in d27 mutants. This outcome may be due to the relatively moderate phenotype of d27.
DISCUSSION
The identification of AtD27 as the ortholog of OsD27 is supported by phenotypic, physiological, and genetic data. From a genetic point of view, we identified the sequence with greatest similarity to OsD27 ( Fig. 1; Supplemental Fig. S1), obtained homozygous T-DNA insertion Atd27 mutant lines (Fig. 2), and complemented this mutant phenotype to the wild type with overexpression of AtD27 ( Fig. 3; Supplemental Fig. S2). Like the d27 mutant in rice, Atd27 affects the number of axillary shoots and plant height (Fig. 3). Interestingly, the Atd27 mutant is also similar to Osd27 in the sense that both mutants are intermediate in phenotype compared with their respective wild types and other characterized SL mutants (Fig. 3). As expected, Atd27 responds to GR24 supplied through the roots ( Fig. 3; Supplemental Fig. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. We checked D27 gene expression in hypocotyls after various treatments known to modulate auxin content: auxin (indole-3-acetic acid [IAA]) addition; decapitation; decapitation followed by IAA addition; addition of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA); and combined addition of IAA and NPA. D27 expression showed similar trends to that of MAX3 and MAX4 across these treatments, although, as above, the magnitude of the response was less (Fig. 6C). Classical auxin-responsive genes, IAA1, IAA5, and PIN1, also responded in a similar manner in these samples, but with varied magnitudes (Supplemental Fig. S5). The only difference between trends among the treatments for the classical auxin response genes compared with the SL genes was that exogenous IAA added to NPAtreated plants fully restored and indeed overcompensated for the NPA, whereas the SL genes were not fully restored back to control levels in this IAA+NPA treatment. This raises the possibility of auxin and nonauxin effects of NPA regulation of MAX3, MAX4, and D27 gene expression.
In pea, there is evidence that feedback regulation of MAX3 and MAX4 orthologs can occur over long distances in a graft-transmissible and localized manner (Foo et al., 2005;Johnson et al., 2006;Dun et al., 2009b). In Arabidopsis, a wild-type shoot can suppress the elevated MAX3 and MAX4 expression in a max2 rootstock ), indicative of a downwardly mobile feedback signal. Nevertheless, in Arabidopsis, the local effect of max2 in the rootstock is greater than that of the long-distance signaling from shoots. In similar experiments in pea, the effect of the scion appears greater than the local effect of the rootstock (Foo et al., 2005;Johnson et al., 2006). In contrast to SL response mutants, grafts between the wild type and SL-deficient mutants in pea show a minor effect of the scion on gene expression in the rootstock, possibly due to the restoration of SL content by the wildtype graft partner. To investigate the scenario for d27, we analyzed gene expression in the hypocotyl and rootstock of plants reciprocally grafted with the wild type (Fig. 6D). In d27 self-grafts, MAX3 and MAX4 transcripts were up-regulated, consistent with feedback. (D27 transcripts were also highly overexpressed, but this may be primarily a result of misregulation of a nonfunctional transcript.) As with wild-type rootstocks of pea grafted to mutants in SL biosynthesis (Dun et al., 2009b), expression in the root was not significantly affected by the genotype of the shoot in either reciprocal graft in Arabidopsis. As in pea, this may be because wild-type rootstocks can restore branching, and presumably the long-distance feedback signal, to normal levels in d27 shoots. Notably, in grafts involving d27, the three auxin-responsive transcripts did not exhibit a statistically significant difference from wild-type self-grafts, suggesting that auxin transport and/or signaling is not substantially affected in d27 mutants. This outcome may be due to the relatively moderate phenotype of d27.
DISCUSSION
The identification of AtD27 as the ortholog of OsD27 is supported by phenotypic, physiological, and genetic data. From a genetic point of view, we identified the sequence with greatest similarity to OsD27 ( Fig. 1; Supplemental Fig. List of protein sequences used in phylogeny reconstruction.
==
Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. The alignment was conservatively trimmed using PFAAT ( [URL]) to remove regions of poor homology, including putative N-terminal plastid transit peptides that show poor conservation. MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003) was used to infer Bayesian trees (random start tree, four chains of temperature 0.2 in each of four independent runs, WAG substitution matrices (Whelan and Goldman, 2001), and four discrete categories of g-distribution substitution rate). Computational analysis was performed using CIPRES Science Gateway version 3.1 ( [URL]/), and phylograms were generated using Dendroscope version 2.7.4 ( [URL]. informatik.uni-tuebingen.de/software/dendroscope).
For comparisons between multiple treatments and a control, one-way twosided ANOVAs (Dunnett's t test) were performed as described in the figure legends. The overdispersed data shown in Figure 3C were analyzed using the more appropriate nonparametric Mann-Whitney U tests. When transcript expression data spanned several orders of magnitude (Fig. 6A), data were log transformed prior to analysis to control for large differences in variance between groups. Statistical analysis was performed with SAS Enterprise Guide 4.3.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number NM_202019.1 (AtD27). Protein accession numbers are listed in Supplemental Table S2.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Alignment of selected D27 orthologs from angiosperms.
Supplemental Figure S5. Response of auxin-related transcripts to IAA, decapitation, and NPA treatments.
Supplemental Table S1. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. Similarly, the relationship between MAX3 and MAX4 could not be determined via grafting (Booker et al., 2005). Instead, biochemical studies have shown that MAX3/CCD7 and MAX4/CCD8 likely act sequentially in carotenoid cleavage (Schwartz et al., 2004). The precise functions of MAX3 and MAX4 were only recently elucidated following the identification of D27 as a b-carotene isomerase (Alder et al., 2012). In vitro, the combination of purified D27, CCD7, and CCD8 is sufficient to convert b-carotene into carlactone, while exogenous carlactone is sufficient to rescue the high-tillering phenotype of rice d27, ccd7, and ccd8 mutants (Alder et al., 2012). Thus, there are at least three enzymatic steps in the plastid before the generation of a mobile SL precursor (Fig. 7). These findings are consistent with our grafting studies. At present, it is unclear whether carlactone itself can exit the plastid and mobilize and whether further modifications by subsequent enzymes are required for the transport of the branching signal from root to shoot.
Like the d27 mutant in rice, which is substantially SL deficient, the Arabidopsis d27-1 shows a weaker phenotype than other branching mutants in the MAX/ D/RMS pathway. Our grafting studies support the notion of residual bioactive branching inhibition capacity in d27, because d27 rootstocks can partly inhibit branching in max1 and max4 scions down to the level of d27 control plants (Fig. 4). Based on our phylogenetic analysis, it is possible that another D27-like protein can perform the isomerization of all-transb-carotene in the absence of functional D27. Although the function of At1g64680 is unknown, the predicted protein has been localized to the chloroplast envelope and/or thylakoids by liquid chromatographytandem mass spectrometry analysis (Ferro et al., 2010), while its closest homolog in rice, encoded by Os08g0114100, is weakly predicted to be plastidic by TargetP software (Emanuelsson et al., 2007). Alternatively, nonenzymic isomerization may provide a small amount of 9-cis-b-carotene substrate for MAX3/ CCD7, allowing only limited levels of SL to be synthesized in the d27 mutant (despite enhanced MAX3 and MAX4 expression). However, the rice d27 mutant does not contain any detectable SL, either by liquid chromatography-mass spectrometry or a bioassay based on Orobanche seed germination (Lin et al., 2009), which may suggest that any residual SLs are below the limit of detection in d27. On the other hand, max1 and max4 mutants, which exhibit a strong branching phenotype, still contain detectable levels of orobanchol . This contradiction means that the basis for the relatively mild branching phenotype of d27 in both rice and Arabidopsis is currently unclear. Therefore, detailed biochemical analyses of the d27 Figure 7. Position of D27 within the SL biosynthesis and signaling pathway. Enzymatic steps are denoted with solid arrows, transport steps with dashed arrows, and signaling steps with thick arrows. AtD14 may be a SL receptor, a hydrolytic enzyme that processes SL into a further active compound, or a combination of both; for a discussion, see Scaffidi et al. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. The N terminus of Arabidopsis D27 is sufficient for plastid targeting. A and E, Epifluorescence micrographs of onion epidermis transiently transformed with plasmids encoding a full-length D27-GFP protein fusion (A) or the first 50 amino acids of D27 fused to the N terminus of GFP (E). B and F, Plastids are positively identified by coexpression of a protein fusion between the full-length Rubisco small subunit from pea (SSU) and RFP. C and G, The merged images confirm colocalization of the two fluorescent signals. Insets in A to C provide magnification and show clustering of plastids around the cell nucleus. D and H, Brightfield micrographs of the same field of view to provide the cell outline. Bars = 50 mm (main images) and 20 mm (insets).
were 5-fold down-regulated in axr1 mutants, D27 transcripts were only moderately (albeit significantly) less abundant. Consequently, although the magnitude of expression changes for D27 are less than for MAX3, the response is similar and therefore may involve common regulatory components.
Levels of MAX3 and MAX4 transcripts in the hypocotyl decrease in response to removal of the shoot tip (decapitation) by decreasing expression levels ). This presumably reduces SL levels in order to allow buds below the decapitation site to grow and compensate for the missing shoot tip. A proportion of the decrease in gene expression is due to auxin depletion in the hypocotyl after removal of the shoot tip (the shoot tip being the main source of auxin for the plant). We checked D27 gene expression in hypocotyls after various treatments known to modulate auxin content: auxin (indole-3-acetic acid [IAA]) addition; decapitation; decapitation followed by IAA addition; addition of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA); and combined addition of IAA and NPA. D27 expression showed similar trends to that of MAX3 and MAX4 across these treatments, although, as above, the magnitude of the response was less (Fig. 6C). Classical auxin-responsive genes, IAA1, IAA5, and PIN1, also responded in a similar manner in these samples, but with varied magnitudes (Supplemental Fig. S5). The only difference between trends among the treatments for the classical auxin response genes compared with the SL genes was that exogenous IAA added to NPAtreated plants fully restored and indeed overcompensated for the NPA, whereas the SL genes were not fully restored back to control levels in this IAA+NPA treatment. This raises the possibility of auxin and nonauxin effects of NPA regulation of MAX3, MAX4, and D27 gene expression.
In pea, there is evidence that feedback regulation of MAX3 and MAX4 orthologs can occur over long distances in a graft-transmissible and localized manner (Foo et al., 2005;Johnson et al., 2006;Dun et al., 2009b). In Arabidopsis, a wild-type shoot can suppress the elevated MAX3 and MAX4 expression in a max2 rootstock ), indicative of a downwardly mobile feedback signal. Nevertheless, in Arabidopsis, the local effect of max2 in the rootstock is greater than that of the long-distance signaling from shoots. In similar experiments in pea, the effect of the scion appears greater than the local effect of the rootstock (Foo et al., 2005;Johnson et al., 2006). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. Contaminating DNA was removed with Turbo DNA-free (Ambion) or DNase (Macherey-Nagel or Qiagen), and RNA was quantified with a NanoDrop 1000 spectrophotometer.
In the case of Figure 2B, Figure 6A, and Supplemental Figure S2C, cDNA was generated from 0.5 mg of total RNA in a 10-mL reaction using the iScript cDNA Synthesis kit (Bio-Rad). Quantitative RT-PCR was performed on a Roche LC480 using LightCycler 480 SYBR Green Master Mix (Roche) in 5-mL reactions. Cycle conditions were as follows: 95°C for 10 min; then 45 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s; followed by melt-curve analysis.
Crossing point values were calculated under high confidence. For each biological replicate, two technical replicates of each real-time PCR were examined, and the mean crossing point value was used to calculate expression relative to an internal reference gene using the formula (E gene ) 2Cp_gene /(E ref ) 2Cp_ref , where E is the primer efficiency and Cp represents the crossing point. Primer efficiencies were determined in separate runs using serial dilutions of pooled cDNA.
Phylogenetic Analysis
Protein sequences with significant homology to D27 were identified with BLASTP searches of GenBank protein databases using the rice (Oryza sativa ssp. japonica) D27 amino acid sequence as a query ( [URL]). Over 90 sequences were identified with a highly significant E-value score (E 210 or smaller). To reduce the complexity of the analysis, 62 sequences were selected on the basis of wide taxonomic coverage while also providing sufficient support for each clade. Sequences were further screened for duplication and truncation. Full-length sequences were then aligned using MAFFT ( [URL]:// mafft.cbrc.jp/alignment/software) using the default settings. The alignment was conservatively trimmed using PFAAT ( [URL]) to remove regions of poor homology, including putative N-terminal plastid transit peptides that show poor conservation. MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003) was used to infer Bayesian trees (random start tree, four chains of temperature 0.2 in each of four independent runs, WAG substitution matrices (Whelan and Goldman, 2001), and four discrete categories of g-distribution substitution rate). Computational analysis was performed using CIPRES Science Gateway version 3.1 ( [URL]/), and phylograms were generated using Dendroscope version 2.7.4 ( [URL]. informatik.uni-tuebingen.de/software/dendroscope).
For comparisons between multiple treatments and a control, one-way twosided ANOVAs (Dunnett's t test) were performed as described in the figure legends. The overdispersed data shown in Figure 3C were analyzed using the more appropriate nonparametric Mann-Whitney U tests. When transcript expression data spanned several orders of magnitude (Fig. 6A), data were log transformed prior to analysis to control for large differences in variance between groups. Statistical analysis was performed with SAS Enterprise Guide 4.3.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number NM_202019.1 (AtD27). Protein accession numbers are listed in Supplemental Table S2.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Alignment of selected D27 orthologs from angiosperms.
Supplemental Figure S5. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. The AtD27 protein is predicted to be 264 amino acids long and shares 48% pairwise identity with OsD27 (excluding the predicted plastid transit peptides; Supplemental Fig. S1). At present, the D27 protein family does not possess any annotated InterPro domains and belongs to a "domain of unknown function" family (Pfam: DUF4033) of 91 eukaryotic and eubacterial sequences, which are widely represented in Figure 1A. However, the rice D27 protein binds a nonheme iron cofactor, which presumably is required for redox-based activity as a b-carotene isomerase (Lin et al., 2009;Alder et al., 2012).
AtD27 Is Required for the Regulation of Axillary Branching
A search of available germplasm collections for mutations in AtD27 revealed three possible T-DNA insertion alleles among the GABI-Kat collection (Kleinboelting et al., 2012). Of these, two were confirmed to carry a T-DNA insertion within the At1g03055 locus. A T-DNA copy resides within the fifth exon in GK-134E08 and in the seventh exon in GK-774D06 ( Fig. 2A). Reverse transcription (RT)-PCR analysis showed that both alleles result in the accumulation of incomplete AtD27 transcripts, consistent with the predicted T-DNA location (Fig. 2B). However, only one of these two alleles resulted in a clear mutant phenotype: in plants homozygous for the GK-134E08 insertion, adult plants exhibited a substantial increase in axillary rosette branches relative to wild-type controls, while GK- Figure 1. At1g03055 encodes the Arabidopsis ortholog of rice D27. A, Bayesian inference phylogeny produced from an alignment of D27-like protein sequences from land plants, green algae, and cyanobacteria. Topology support for nodes is given by posterior probability. Terminals are labeled with the genus name followed by a sequence identifier, as detailed in Supplemental Table S2. The tree is rooted on the cyanobacterial clade, based on their ancestral relationship to chloroplasts in photosynthetic eukaryotes. B, Exon structure of AtD27, MtD27, and OsD27 mRNAs. A block arrow denotes each exon. Numbers indicate the length of each exon in bp. Protein-coding sequences are indicated by gray fill, and white fill denotes noncoding untranslated regions. The two nucleotides on either side of each exon-exon interface are indicated, with splice junctions denoted by a forward slash (/). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. patens. Loss of PpCCD8 activity leads to severely compromised SL profiles and impaired control of protonemal branching, which in turn limits the ability to sense neighboring colonies (Proust et al., 2011). As the P. patens genome also encodes a D27 ortholog, multiple D14 homologs, and a single MAX2 ortholog, it appears that the SL biosynthesis and signaling system is conserved among land plants. The availability of complete genome sequences for the streptophyte algae, combined with an assessment of their ability to produce SLs, will help in determining the origin of SL signaling.
We have contributed further to the understanding of the D27 protein by demonstrating that the Arabidopsis version of D27 functions upstream of MAX1 in the production of SL-related signal(s) and displays similar feedback control compared with other genes encoding plastid-localized SL biosynthesis proteins. AtD27 controls a graft-transmissible signal because branching can be inhibited in Atd27 mutant shoots by grafting to wild-type rootstocks (Fig. 4). Whereas max1 mutant rootstocks retain this inhibitory function, max4 rootstocks do not. This is clear evidence in support of D27 acting upstream of MAX1. On the other hand, the grafting data do not permit the ordering of D27 with respect to MAX4, presumably because the carotenoid-derived substrates of D27 and MAX4 are unable to leave the plastid (Alder et al., 2012). Similarly, the relationship between MAX3 and MAX4 could not be determined via grafting (Booker et al., 2005). Instead, biochemical studies have shown that MAX3/CCD7 and MAX4/CCD8 likely act sequentially in carotenoid cleavage (Schwartz et al., 2004). The precise functions of MAX3 and MAX4 were only recently elucidated following the identification of D27 as a b-carotene isomerase (Alder et al., 2012). In vitro, the combination of purified D27, CCD7, and CCD8 is sufficient to convert b-carotene into carlactone, while exogenous carlactone is sufficient to rescue the high-tillering phenotype of rice d27, ccd7, and ccd8 mutants (Alder et al., 2012). Thus, there are at least three enzymatic steps in the plastid before the generation of a mobile SL precursor (Fig. 7). These findings are consistent with our grafting studies. At present, it is unclear whether carlactone itself can exit the plastid and mobilize and whether further modifications by subsequent enzymes are required for the transport of the branching signal from root to shoot.
Like the d27 mutant in rice, which is substantially SL deficient, the Arabidopsis d27-1 shows a weaker phenotype than other branching mutants in the MAX/ D/RMS pathway. Our grafting studies support the notion of residual bioactive branching inhibition capacity in d27, because d27 rootstocks can partly inhibit branching in max1 and max4 scions down to the level of d27 control plants (Fig. 4). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. (2012). [See online article for color version of this figure. ] mutant, intact or grafted, compared with other mutant and wild-type lines may be useful in evaluation of the levels and profiles of endogenous SLs and their potential correlations with phenotypic responses.
Interestingly, despite the complete rescue of d27 shoots by grafting to wild-type rootstocks, the expression of D27 is not greatest in the rootstock. Indeed, its expression in roots and hypocotyls of 3-week-old plants is greatly and slightly reduced, respectively, compared with shoots (Fig. 6A). This regulation is a point of difference with MAX3 and MAX4, which are both highly expressed in the hypocotyl. In a further contrast, MAX3 is relatively weakly expressed in roots compared with shoots, while MAX4 is again highly expressed. The expression patterns that we observed are fully consistent with meta-profiles derived from multiple microarray experiments (Supplemental Fig. S4). Recently, it was found that two GRAS-type transcription factors, NSP1 and NSP2, regulate D27 expression and SL levels in Medicago and rice . In Medicago, the nsp1 mutant does not produce any detectable SL while the nsp2 mutant exhibits a specific deficiency in didehydro-orobanchol, and these deficiencies correlate with a decrease in MtD27 transcripts in both mutants. Interestingly, MtCCD7 and MtCCD8 expression was unaffected in nsp1 and nsp2 mutants , suggesting that the carotenoid cleavage steps in SL biosynthesis are under distinct transcriptional control from the initial isomerization step involving D27. This finding is consistent with our data from Arabidopsis. Although it is unclear whether MtD27 transcription is directly regulated by NSP1 and NSP2, the conservation between rice and Medicago of this regulatory system implies that a similar mechanism might operate in Arabidopsis and beyond. The identification of putative NSP1 and NSP2 orthologs in Arabidopsis (Zhu et al., 2006) opens the door for reverse genetics to address this question.
In contrast to the differences in relative expression among different tissues, the SL biosynthesis genes D27, MAX3, and MAX4 show similar trends in response to auxin-related treatments and in different mutant backgrounds. Although D27 transcripts show lower magnitudes of response across all experiments, they show similar trends to other SL biosynthesis transcripts. D27 expression is enhanced in the hypocotyl in SL-deficient or SL response mutant backgrounds, and it appears to be up-regulated by auxin. The auxin response is indicated by reduced expression in axr1 mutants and in response to auxin addition or depletion via decapitation and NPA treatment. As shown from grafted plants, d27 mutants also show enhanced expression of MAX3 and MAX4, which is again consistent with SL deficiency causing the up-regulation of these genes. Restoration of SL content in the mutant shoots by wild-type rootstocks means we cannot easily test whether a component of this feedback is graft transmissible . List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. Like MAX3 and MAX4 SL biosynthesis genes, D27 is expressed in the vasculature. D27 also has strong expression in axillary buds (Lin et al., 2009). Recent in vitro evidence suggests that D27 is a b-carotene isomerase that converts all-trans-b-carotene into 9-cis-b-carotene, which is then further processed by CCD7 and CCD8 into a SL precursor termed carlactone (Alder et al., 2012). If this biochemical activity is also the case in planta, it is likely that D27 acts upstream of MAX1 in the control of the biosynthesis of a mobile shoot-branching signal. The availability of an equivalent d27 mutant in a species amenable to grafting would allow a more direct test of this hypothesis. Moreover, it will allow an extended genetic, biochemical, and physiological analysis of SL biosynthesis and its regulation. In this survey, we identify the Arabidopsis ortholog of OsD27 and characterize it with respect to its SL response, expression profile, and regulation and use grafting to place it within the SL biosynthesis pathway.
At1g03055 Is Orthologous to OsD27
The Arabidopsis genome encodes three proteins with superficial similarity to rice OsD27. To identify the likely ortholog of OsD27, we performed a phylogenetic analysis of D27-like proteins from land plants, green algae, and cyanobacteria. The eukaryotic sequences broadly grouped into three distinct clades, two of which (clades 1 and 2) only contained representatives from land plants, while the third (clade 3) also contained members from chlorophyte algae and diatoms (Fig. 1). With three cyanobacterial proteins serving as an outgroup, the phylogeny suggests that there was a single D27-like gene copy in the common ancestor of the chlorophyte algae and the land plants. Subsequently, the first of two gene duplication events took place in the lineage leading to the land plants, giving rise to clades 1 and 2. The long branches within clade 3, indicative of long periods of sequence divergence, further suggest that this group of proteins arose prior to, and is functionally distinct from, clades 1 and 2. Intriguingly, several gene duplication events have occurred independently within the individual algal lineages, leading to extensive paralogy within clade 3.
The clades specific to land plants, clades 1 and 2, both had strong Bayesian posterior support, and the two groups together formed a superclade that was also strongly supported. Clades 1 and 2 each contained a representative sequence from the moss Physcomitrella patens, indicating that each group derived from a second gene duplication event that occurred during the early evolution of the land plants. Clade 1 contained OsD27, its ortholog in Medicago truncatula , and an Arabidopsis protein encoded by At1g03055. Notably, two other Arabidopsis proteins with similarity to OsD27, encoded by At1g64680 and At4g01995, unambiguously were assigned to clades 2 and 3, respectively. Thus, we concluded that At1g03055 is the likely Arabidopsis ortholog of OsD27. Nevertheless, the presence of other D27-like proteins in land plant genomes is indicative of potential genetic redundancy.
The AtD27 transcript comprises seven exons, and the exon length and structure are highly conserved with the MtD27 and OsD27 mRNAs, further supporting orthology between the three genes (Fig. 1B). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. (2009). Treatment of plants with GR24 in Phytatrays was performed as described (Brewer et al., 2009). Treatment of plants grown in hydroponic medium has been described previously .
IAA Treatments
Prebolting Ws-4 plants were treated with a lanolin ring around the top of the hypocotyl containing 2.5% dimethyl sulfoxide and 2.5% ethanol (control) with 10 mM IAA and/or 10 mM NPA (an auxin transport inhibitor). Decapitation involved removing the apical meristem and some small leaves/flowers from the shoot apex of each plant. Hypocotyl tissue was harvested 4 h post treatment. Three replicates were used for each genotype, with approximately 20 plants used per replicate.
RNA was isolated from 7-d-old whole seedlings using the Qiagen RNeasy procedure. cDNA was generated from 1 mg of total RNA using Invitrogen SuperScript III. The At1g03055.1 coding sequence was first amplified using primers MW350 59-AAAAAAGCAGGCTATGAACACTAAGCTATCAC-39 and MW351 59-CAAGAAAGCTGGGTCTAATGCTTCACACCGTAGC-39 (translation initiation codon and stop codon, respectively, in boldface) using a proofreading DNA polymerase (Phusion; New England Biolabs). Approximately 250 pg of the PCR product was then reamplified using Gateway attB adapter primers 59-GGGGACAAGTTTGTACAAAAAAGCAGGCT-39 and 59-GGGGACCACTTTGTACAAGAAAGCTGGGT-39 (overlapping regions with MW350 and MW351, respectively, are underlined). The resulting PCR product was cloned into pDONR207 via Gateway-based recombination, and positive clones were confirmed by sequencing. The full-length coding sequence was then transferred to pMDC32 (Curtis and Grossniklaus, 2003) via recombination, generating the final 35S:D27 binary vector. The d27-1 mutant was then transformed by the floral dip method. Transgenic seedlings were selected by growth in continuous light for 7 d on one-half-strength Murashige and Skoog medium containing 25 mg mL 21 hygromycin B.
Transient Transformation and Localization Studies
The full-length D27 coding sequence was reamplified from pDONR207-D27 using primers MW350 and MW353 (59-CAAGAAAGCTGGGTTAT-GCTTCACACCGTAGC-39), in which the native stop codon was deleted. The region of D27 encoding the plastid transit peptide was amplified using primers MW350 and MW352 (59-CAAGAAAGCTGGGTTCTCTTTTG-CAGCCTTGAG-39). Both PCR products were then reamplified with the universal Gateway attB primers described above and cloned into pDONR207. Positive clones of each were then recombined with pMDC83 (Curtis and Grossniklaus, 2003) to generate clones expressing N-D27-mGFP6-C protein fusions. For colocalization, the full-length cDNA of the small subunit of Rubisco from pea (Pisum sativum) was fused to the N terminus of red fluorescent protein (RFP;Carrie et al., 2009).
Plasmids (5 mg) were precipitated onto 1-mm gold particles and biolistically transformed into onion (Allium cepa) epidermis as described previously (Thirkettle-Watts et al., 2003). Images were obtained with an Olympus BX61 epifluorescence microscope equipped with GFP (U-MGFPHQ) and RFP (U-MRFPHQ) filters and manipulated with Olympus cell^R software.
RT-PCR and Quantitative RT-PCR Analysis
Total RNA was extracted using the RNeasy (Qiagen) or NucleoSpin RNA Plant (Macherey-Nagel) procedure. For small tissue samples (Fig. 6D), a modified TRIzol (Invitrogen) procedure was used with subsequent RNeasy cleanup. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. At1g03055 encodes the Arabidopsis ortholog of rice D27. A, Bayesian inference phylogeny produced from an alignment of D27-like protein sequences from land plants, green algae, and cyanobacteria. Topology support for nodes is given by posterior probability. Terminals are labeled with the genus name followed by a sequence identifier, as detailed in Supplemental Table S2. The tree is rooted on the cyanobacterial clade, based on their ancestral relationship to chloroplasts in photosynthetic eukaryotes. B, Exon structure of AtD27, MtD27, and OsD27 mRNAs. A block arrow denotes each exon. Numbers indicate the length of each exon in bp. Protein-coding sequences are indicated by gray fill, and white fill denotes noncoding untranslated regions. The two nucleotides on either side of each exon-exon interface are indicated, with splice junctions denoted by a forward slash (/). [See online article for color version of this figure.] 774D06 homozygotes had a normal branching pattern (Fig. 2, C and D). On the basis of its mutant phenotype, we named the GK-134E08 allele d27-1 (hereafter referred to as d27 for simplicity). The T-DNA insertion in d27 disrupts the predicted protein at Leu-189, comparable to the nonfunctional protein produced in Osd27, which is disrupted after Gly-187 (Supplemental Fig. S1; Lin et al., 2009). These data suggest that AtD27 is necessary for the regulation of axillary bud outgrowth and that the extreme 39 end of the transcript is nonessential for the function of the resulting protein.
To confirm that the d27 lesion was responsible for the increased branching phenotype, we complemented the mutant with a cDNA encoding the predicted D27 protein, expressed under the control of the cauliflower mosaic virus 35S promoter. We isolated four independent transgenic lines, in which the transgene segregated in a ratio consistent with a single Mendelian locus and was expressed approximately 40-fold higher than in the wild type at the transcriptional level (Supplemental Fig. S2). In all four lines, the number of axillary branches and overall plant height were not significantly different from wild-type levels, fully complementing the d27 phenotype (Fig. 3, A and B). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. [See online article for color version of this figure.] 774D06 homozygotes had a normal branching pattern (Fig. 2, C and D). On the basis of its mutant phenotype, we named the GK-134E08 allele d27-1 (hereafter referred to as d27 for simplicity). The T-DNA insertion in d27 disrupts the predicted protein at Leu-189, comparable to the nonfunctional protein produced in Osd27, which is disrupted after Gly-187 (Supplemental Fig. S1; Lin et al., 2009). These data suggest that AtD27 is necessary for the regulation of axillary bud outgrowth and that the extreme 39 end of the transcript is nonessential for the function of the resulting protein.
To confirm that the d27 lesion was responsible for the increased branching phenotype, we complemented the mutant with a cDNA encoding the predicted D27 protein, expressed under the control of the cauliflower mosaic virus 35S promoter. We isolated four independent transgenic lines, in which the transgene segregated in a ratio consistent with a single Mendelian locus and was expressed approximately 40-fold higher than in the wild type at the transcriptional level (Supplemental Fig. S2). In all four lines, the number of axillary branches and overall plant height were not significantly different from wild-type levels, fully complementing the d27 phenotype (Fig. 3, A and B). We performed feeding experiments with GR24, a synthetic SL, to evaluate whether the increased branching phenotype of d27 likely results from impaired SL biosynthesis, as is the case in rice (Lin et al., 2009). When grown in culture vessels on medium containing GR24, d27 plants produced significantly fewer branches than those grown on control medium (Fig. 3C). The SL-deficient mutant max3-11 exhibited a similar response, although its branching phenotype was more severe. In contrast, the SL-insensitive mutant Atd14 showed no response at all to GR24, as demonstrated previously . A similar response to GR24 was seen with d27 and max3-9 plants grown hydroponically (Supplemental Fig. S3). Together, these data demonstrate that D27 is essential for the regulation of axillary branching and that branching inhibition in d27 is limited by SL content rather than SL response.
D27 Acts Upstream of MAX1 in the SL Biosynthetic Pathway
Grafting wild-type rootstocks to max1, max3, or max4 scions restores a wild-type branching pattern to the mutant shoots (Booker et al., 2005). This outcome results from the graft transmission of root-synthesized SL to the shoot presumably via the transpiration stream (Foo et al., 2001;Kohlen et al., 2011). Conversely, grafting with these lines shows that SL biosynthesis also occurs in the shoot, as wild-type shoots have wild-type branching phenotypes even where grafted with mutant rootstocks. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. We performed feeding experiments with GR24, a synthetic SL, to evaluate whether the increased branching phenotype of d27 likely results from impaired SL biosynthesis, as is the case in rice (Lin et al., 2009). When grown in culture vessels on medium containing GR24, d27 plants produced significantly fewer branches than those grown on control medium (Fig. 3C). The SL-deficient mutant max3-11 exhibited a similar response, although its branching phenotype was more severe. In contrast, the SL-insensitive mutant Atd14 showed no response at all to GR24, as demonstrated previously . A similar response to GR24 was seen with d27 and max3-9 plants grown hydroponically (Supplemental Fig. S3). Together, these data demonstrate that D27 is essential for the regulation of axillary branching and that branching inhibition in d27 is limited by SL content rather than SL response.
D27 Acts Upstream of MAX1 in the SL Biosynthetic Pathway
Grafting wild-type rootstocks to max1, max3, or max4 scions restores a wild-type branching pattern to the mutant shoots (Booker et al., 2005). This outcome results from the graft transmission of root-synthesized SL to the shoot presumably via the transpiration stream (Foo et al., 2001;Kohlen et al., 2011). Conversely, grafting with these lines shows that SL biosynthesis also occurs in the shoot, as wild-type shoots have wild-type branching phenotypes even where grafted with mutant rootstocks. To verify that d27 shoots are similarly responsive to a graft-transmissible signal, we generated reciprocal grafts between wildtype ecotype Columbia (Col-0) and d27 and found that wild-type rootstocks could suppress the increased branching phenotype of d27 shoots (Fig. 4). A mutant rootstock had no effect on the phenotype of a wildtype shoot, showing that D27 expression in the shoot is sufficient to inhibit branching (Fig. 4).
It has been shown previously that MAX1 operates downstream of MAX3 and MAX4 in SL biosynthesis, because a max1 rootstock can restore a wild-type branching pattern to max3 and max4 shoots, but not vice versa (Booker et al., 2005). These findings have also demonstrated that a compound intermediate between MAX3/MAX4 and MAX1 is subject to longdistance transport from the root to the shoot. Likewise, the substrates of MAX3 and MAX4 are nonmobile (or are unable to access MAX3/MAX4 in the plastid of a remote cell), because a max3 rootstock fails to complement a max4 shoot, and vice versa (Booker et al., 2005). To investigate the relative position of D27 within the MAX pathway, we generated reciprocal grafts between d27 and either max1 or max4. A max1 rootstock was able to fully restore the branching phenotype of a d27 shoot, demonstrating that MAX1 operates downstream of D27 (Fig. 4). Interestingly, as for max4 discussed below, the branching of max1 scions grafted to d27 rootstocks was reduced to that of d27 self-grafts (Fig. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In summary, these grafting data are consistent with D27 functioning upstream of MAX1 in the biosynthesis of SL.
AtD27 Is Localized to the Plastid
Given the apparent nonmobility of the D27 precursor and the biochemical function of D27, it is likely that the Arabidopsis D27 protein is spatially restricted to the plastid. To determine the likely cellular location of D27, we generated a chimeric construct encoding the full-length D27 protein fused to the N terminus of GFP. Following transient expression in onion (Allium cepa) epidermis, the GFP fluorescence fully overlapped with the red fluorescence from the coexpressed plastid-specific marker (Fig. 5, A-D). No GFP fluorescence was detected beyond the plastid, suggesting that the full-length D27 protein was strictly localized to this organelle. This is in accord with plastid localization of OsD27 (Lin et al., 2009). The D27 protein encodes a predicted N-terminal plastid transit peptide of 47 amino acids (Supplemental Fig. S1). To test whether this presequence is sufficient to direct plastid localization, we generated a translational fusion between the first 50 amino acids of D27 and GFP and subjected onion epidermal cells to transient transformation as before. Again, the fluorescence signals from both the GFP and the plastid-specific marker fully overlapped (Fig. 5, E-H), indicating that D27 possesses a bona fide plastid transit peptide. Together, these data suggest that D27 functions in the plastid, consistent with the demonstrated cellular location of OsD27 in rice and its anticipated activity on a nonmobile precursor.
D27 Transcripts Are Subject to Auxin and Feedback Control D27, MAX3, and MAX4 transcript abundance from public microarray data revealed potential differential expression intensities throughout the plant, with a relative deficit of D27 in root tissue (Supplemental Fig. S4). Quantitative RT-PCR analysis of Col-0 tissues confirmed that D27 transcripts were more abundant in shoot tissue than in roots of 3-week-old wild-type plants, while MAX3 and MAX4 transcripts accumulated most strongly in the hypocotyl and roots (Fig. 6A). In contrast, MAX1 transcript levels varied little over the tissues examined. Consistent with their role in SL response, we found that MAX2 and AtD14 transcripts were evenly expressed across the shoot, hypocotyl, and root (Fig. 6A). Differential spatial expression of D27 compared with MAX3 and MAX4 then led us to investigate whether D27 was under similar feedback control as MAX3 and MAX4.
In all max mutants, levels of MAX3 and MAX4 transcripts in the hypocotyl are 6-to 10-fold higher than in the wild type, indicative of feedback up-regulation . This feedback process is dependent on a combination of auxin and nonauxin Figure 5. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In addition to enhanced shoot branching, SL-deficient and SL response mutants also exhibit reduced stature, suppressed development of the cambium ring in the main stem (secondary growth), and enhanced lateral and adventitious root development (Stirnberg et al., 2002;Arite et al., 2007;Agusti et al., 2011;Kapulnik et al., 2011;Rasmussen et al., 2012). It is also likely that SLs form part of the signaling system used for responding to available soil nutrients. In wild-type plants, SL content is greatly enhanced under phosphate deficiency, which promotes lateral root and root hair lengthening and mycorrhizal associations, while suppressing aerial growth via reduced shoot branching (Umehara et al., 2010;Kapulnik et al., 2011;Kohlen et al., 2011;Ruyter-Spira et al., 2011).
SLs are carotenoid-derived terpenoid lactones. Only four enzymes have been attributed to this biosynthetic pathway, and only three of these have been described in Arabidopsis (Arabidopsis thaliana; Xie et al., 2010). These are encoded by the MORE AXILLARY GROWTH genes MAX1, MAX3, and MAX4. MAX3 and MAX4 are carotenoid cleavage dioxygenases (CCD7 and CCD8, respectively) that act in the chloroplast to cleave a carotenoid substrate. MAX1, a cytochrome P450, acts downstream on a mobile intermediate that can traverse a graft union. In Arabidopsis, MAX3 and MAX4 are largely expressed in the vasculature and function in the shoot and root. MAX3 and MAX4 transcripts are regulated both by auxin and by long-and short-distance feedback systems that depend on SL signaling and 1 This work was supported by the Australian Research Council (grant nos. DP1096717 to S. M. S. and DP110100997 to C. A. B.) and by a University of Western Australia-University of Queensland Bilateral Research Collaboration award (to S. M. S. and C. A. B.). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. With three cyanobacterial proteins serving as an outgroup, the phylogeny suggests that there was a single D27-like gene copy in the common ancestor of the chlorophyte algae and the land plants. Subsequently, the first of two gene duplication events took place in the lineage leading to the land plants, giving rise to clades 1 and 2. The long branches within clade 3, indicative of long periods of sequence divergence, further suggest that this group of proteins arose prior to, and is functionally distinct from, clades 1 and 2. Intriguingly, several gene duplication events have occurred independently within the individual algal lineages, leading to extensive paralogy within clade 3.
The clades specific to land plants, clades 1 and 2, both had strong Bayesian posterior support, and the two groups together formed a superclade that was also strongly supported. Clades 1 and 2 each contained a representative sequence from the moss Physcomitrella patens, indicating that each group derived from a second gene duplication event that occurred during the early evolution of the land plants. Clade 1 contained OsD27, its ortholog in Medicago truncatula , and an Arabidopsis protein encoded by At1g03055. Notably, two other Arabidopsis proteins with similarity to OsD27, encoded by At1g64680 and At4g01995, unambiguously were assigned to clades 2 and 3, respectively. Thus, we concluded that At1g03055 is the likely Arabidopsis ortholog of OsD27. Nevertheless, the presence of other D27-like proteins in land plant genomes is indicative of potential genetic redundancy.
The AtD27 transcript comprises seven exons, and the exon length and structure are highly conserved with the MtD27 and OsD27 mRNAs, further supporting orthology between the three genes (Fig. 1B). The AtD27 protein is predicted to be 264 amino acids long and shares 48% pairwise identity with OsD27 (excluding the predicted plastid transit peptides; Supplemental Fig. S1). At present, the D27 protein family does not possess any annotated InterPro domains and belongs to a "domain of unknown function" family (Pfam: DUF4033) of 91 eukaryotic and eubacterial sequences, which are widely represented in Figure 1A. However, the rice D27 protein binds a nonheme iron cofactor, which presumably is required for redox-based activity as a b-carotene isomerase (Lin et al., 2009;Alder et al., 2012).
AtD27 Is Required for the Regulation of Axillary Branching
A search of available germplasm collections for mutations in AtD27 revealed three possible T-DNA insertion alleles among the GABI-Kat collection (Kleinboelting et al., 2012). Of these, two were confirmed to carry a T-DNA insertion within the At1g03055 locus. A T-DNA copy resides within the fifth exon in GK-134E08 and in the seventh exon in GK-774D06 ( Fig. 2A). Reverse transcription (RT)-PCR analysis showed that both alleles result in the accumulation of incomplete AtD27 transcripts, consistent with the predicted T-DNA location (Fig. 2B). However, only one of these two alleles resulted in a clear mutant phenotype: in plants homozygous for the GK-134E08 insertion, adult plants exhibited a substantial increase in axillary rosette branches relative to wild-type controls, while GK- Figure 1. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In Arabidopsis (Arabidopsis thaliana), SL biosynthesis requires the sequential action of two carotenoid cleavage dioxygenases, MORE AXILLARY GROWTH3 (MAX3) and MAX4, followed by a cytochrome P450, MAX1. In rice (Oryza sativa), the plastid-localized protein DWARF27 (OsD27) is also necessary for SL biosynthesis, but the equivalent gene in Arabidopsis has not been identified. Here, we use phylogenetic analysis of D27-like sequences from photosynthetic organisms to identify AtD27, the likely Arabidopsis ortholog of OsD27. Using reverse genetics, we show that AtD27 is required for the inhibition of secondary bud outgrowth and that exogenous application of the synthetic SL GR24 can rescue the increased branching phenotype of an Atd27 mutant. Furthermore, we use grafting to demonstrate that AtD27 operates on a nonmobile precursor upstream of MAX1 in the SL biosynthesis pathway. Consistent with the plastid localization of OsD27, we also show that AtD27 possesses a functional plastid transit peptide. We demonstrate that AtD27 transcripts are subject to both local feedback and auxin-dependent signals, albeit to a lesser extent than MAX3 and MAX4, suggesting that early steps in SL biosynthesis are coregulated at the transcriptional level. By identifying an additional component of the canonical SL biosynthesis pathway in Arabidopsis, we provide a new tool to investigate the regulation of shoot branching and other SL-dependent developmental processes.
Strigolactones (SLs) are a relatively new class of plant hormones that control multiple facets of plant growth and development. SLs were originally identified from root exudates as organic compounds that stimulated the germination of particular parasitic weeds (Cook et al., 1966;Xie et al., 2010) and were more recently found to promote the symbiosis between plants and mycorrhizal fungi (Akiyama et al., 2005;Dor et al., 2011). The most widely characterized role of SL in plant development is in the inhibition of shoot branching (Dun et al., 2009a;Domagalska and Leyser, 2011). In addition to enhanced shoot branching, SL-deficient and SL response mutants also exhibit reduced stature, suppressed development of the cambium ring in the main stem (secondary growth), and enhanced lateral and adventitious root development (Stirnberg et al., 2002;Arite et al., 2007;Agusti et al., 2011;Kapulnik et al., 2011;Rasmussen et al., 2012). It is also likely that SLs form part of the signaling system used for responding to available soil nutrients. In wild-type plants, SL content is greatly enhanced under phosphate deficiency, which promotes lateral root and root hair lengthening and mycorrhizal associations, while suppressing aerial growth via reduced shoot branching (Umehara et al., 2010;Kapulnik et al., 2011;Kohlen et al., 2011;Ruyter-Spira et al., 2011).
SLs are carotenoid-derived terpenoid lactones. Only four enzymes have been attributed to this biosynthetic pathway, and only three of these have been described in Arabidopsis (Arabidopsis thaliana; Xie et al., 2010). These are encoded by the MORE AXILLARY GROWTH genes MAX1, MAX3, and MAX4. MAX3 and MAX4 are carotenoid cleavage dioxygenases (CCD7 and CCD8, respectively) that act in the chloroplast to cleave a carotenoid substrate. MAX1, a cytochrome P450, acts downstream on a mobile intermediate that can traverse a graft union. In Arabidopsis, MAX3 and MAX4 are largely expressed in the vasculature and function in the shoot and root. MAX3 and MAX4 transcripts are regulated both by auxin and by long-and short-distance feedback systems that depend on SL signaling and 1 This work was supported by the Australian Research Council (grant nos. DP1096717 to S. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In Medicago, the nsp1 mutant does not produce any detectable SL while the nsp2 mutant exhibits a specific deficiency in didehydro-orobanchol, and these deficiencies correlate with a decrease in MtD27 transcripts in both mutants. Interestingly, MtCCD7 and MtCCD8 expression was unaffected in nsp1 and nsp2 mutants , suggesting that the carotenoid cleavage steps in SL biosynthesis are under distinct transcriptional control from the initial isomerization step involving D27. This finding is consistent with our data from Arabidopsis. Although it is unclear whether MtD27 transcription is directly regulated by NSP1 and NSP2, the conservation between rice and Medicago of this regulatory system implies that a similar mechanism might operate in Arabidopsis and beyond. The identification of putative NSP1 and NSP2 orthologs in Arabidopsis (Zhu et al., 2006) opens the door for reverse genetics to address this question.
In contrast to the differences in relative expression among different tissues, the SL biosynthesis genes D27, MAX3, and MAX4 show similar trends in response to auxin-related treatments and in different mutant backgrounds. Although D27 transcripts show lower magnitudes of response across all experiments, they show similar trends to other SL biosynthesis transcripts. D27 expression is enhanced in the hypocotyl in SL-deficient or SL response mutant backgrounds, and it appears to be up-regulated by auxin. The auxin response is indicated by reduced expression in axr1 mutants and in response to auxin addition or depletion via decapitation and NPA treatment. As shown from grafted plants, d27 mutants also show enhanced expression of MAX3 and MAX4, which is again consistent with SL deficiency causing the up-regulation of these genes. Restoration of SL content in the mutant shoots by wild-type rootstocks means we cannot easily test whether a component of this feedback is graft transmissible . However, the majority of the feedback induced by d27 is likely to be local, since d27 rootstocks cause high expression of MAX3 and MAX4 similar to mutant self-grafts even where the scion is the wild type.
The regulation by auxin of a third protein in the SL pathway further supports the hypothesis that SLs act downstream of auxin in the control of shoot branching (Brewer et al., 2009). However, again, very small increases in the expression of PIN1 and IAA5 in d27 mutants (Fig. 6D) indicate enhanced auxin signaling or content, which could be indicative of enhanced auxin transport as observed in Osd27 and several other branching mutants (Lin et al., 2009;Crawford et al., 2010;Domagalska and Leyser, 2011). We have recently modeled long-distance auxin transport in the stem, and these data, in addition to data on auxin transport under high auxin content, demonstrate that wild-type plants do not have reduced auxin transport capacity relative to SL mutants (Brewer et al., 2009;Renton et al., 2012). Rather, in those experiments, the enhanced auxin transport seen in max3 and max4 mutants was more likely to be due to enhanced uptake of the exogenously supplied radiolabeled auxin to the top of the long-distance polar auxin transport stream (Renton et al., 2012).
In summary, d27 represents a new genetic tool for the elucidation of shoot branching mechanisms and their regulation in Arabidopsis. Together with MAX3 and MAX4, D27 acts in the plastid on the production of carlactone, an intermediate in the SL pathway, prior to MAX1. These genes encoding plastid-localized proteins, although expressed somewhat differently at the level of different plant parts, appear to carry conserved properties in terms of the regulation of their expression, particularly by the plant hormone auxin.
Unless otherwise stated, Arabidopsis (Arabidopsis thaliana) plants were grown under fluorescent lamps emitting 100 to 120 mmol photons s 21 m 22 (intensity at the rosette level) in a 6:1:1 mixture of peat, vermiculite, and perlite or in University of California mix with added 60% vermiculite. Plants from the Col-0 background were grown in a 16-h-light/8-h-dark photoperiod and a 22°C light/16°C dark temperature cycle, or in constant 22°C. Ws-4 was initially grown for 2 weeks in 14 h of light/10 h of dark and constant 18°C, in order to compensate for the extreme early flowering in Ws-4 and to match their flowering time with Col-0.
Grafting and Branching Assays
Grafting of Arabidopsis seedlings was performed as described in Brosnan et al. (2007) and as described in Brewer et al. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. M. S. and DP110100997 to C. A. B.) and by a University of Western Australia-University of Queensland Bilateral Research Collaboration award (to S. M. S. and C. A. B.). * Corresponding author; e-mailThe author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christine A. Beveridge ([email protected]).
[C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp. 112.196253 response (Bennett et al., 2006;Brewer et al., 2009;Hayward et al., 2009). Based on impaired SL response in corresponding mutants of Arabidopsis and rice (Oryza sativa), two proteins have been identified as necessary for this process: the F-box protein AtMAX2/ OsD3 (Gomez-Roldan et al., 2008;Umehara et al., 2008) and the a/b-fold hydrolase family protein AtD14/OsD14 (Arite et al., 2009;Waters et al., 2012). Relative to wild-type plants, the SL response mutants produce elevated amounts of SL, presumably due to impaired feedback on SL biosynthesis (Umehara et al., 2008;Arite et al., 2009;Kohlen et al., 2011). Accordingly, MAX3 and MAX4 gene expression is greatly enhanced in max2 mutants . In rice, a plastid-targeted protein, DWARF27 (D27), is required for SL biosynthesis (Lin et al., 2009). As with other SL mutants in rice, the d27 mutant has a highly tillered and dwarf habit, although the phenotype of d27 is intermediate between the wild type and other characterized SL-related mutants. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. S1), obtained homozygous T-DNA insertion Atd27 mutant lines (Fig. 2), and complemented this mutant phenotype to the wild type with overexpression of AtD27 ( Fig. 3; Supplemental Fig. S2). Like the d27 mutant in rice, Atd27 affects the number of axillary shoots and plant height (Fig. 3). Interestingly, the Atd27 mutant is also similar to Osd27 in the sense that both mutants are intermediate in phenotype compared with their respective wild types and other characterized SL mutants (Fig. 3). As expected, Atd27 responds to GR24 supplied through the roots ( Fig. 3; Supplemental Fig. S3).
Our phylogenetic analysis demonstrates that AtD27 belongs to a wider family of proteins that originated early in the evolution of photosynthetic eukaryotes. Although D27-like proteins are found throughout cyanobacteria and green algae, two clades are strictly specific to land plants. Notably, CCD7 (MAX3) and CCD8 (MAX4) form monophyletic clades in land plants, and putative orthologs of both CCD7 and CCD8 are present in Chlamydomonas reinhardtii and other photosynthetic eukaryotes (Ledger et al., 2010;Vallabhaneni et al., 2010;Proust et al., 2011). Thus, the enzymatic machinery necessary for the early steps of SL biosynthesis may have been present in the ancestors of the land plants. Interestingly, all land plants so far examined possess homologs of the proteins required for SL response (i.e. MAX2 and D14), but so far, no homologs have been found in other photosynthetic eukaryotes, including the streptophyte algae, the closest living relatives of the land plants (Waters et al., 2011. Although this finding could reflect incomplete genome sequence data for the streptophyte algae, it is possible that SL signaling coincided with the evolution of the land plants and the concurrent development of complex body plans. As carotenoids are essential pigments associated with light harvesting and photoprotection in oxygenic photosynthetic organisms, carotenoid metabolism is a fundamental requirement for photosynthesis and likely the original function of CCD7-, CCD8-, and D27-like proteins. As multicellularity evolved, we propose that gene duplications permitted the acquisition of novel gene function, and a separate pathway for carotenoid breakdown was co-opted for signaling purposes. Recently, SLs were shown to have a dramatic role in gametophytic development of the moss P. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. The full-length coding sequence was then transferred to pMDC32 (Curtis and Grossniklaus, 2003) via recombination, generating the final 35S:D27 binary vector. The d27-1 mutant was then transformed by the floral dip method. Transgenic seedlings were selected by growth in continuous light for 7 d on one-half-strength Murashige and Skoog medium containing 25 mg mL 21 hygromycin B.
Transient Transformation and Localization Studies
The full-length D27 coding sequence was reamplified from pDONR207-D27 using primers MW350 and MW353 (59-CAAGAAAGCTGGGTTAT-GCTTCACACCGTAGC-39), in which the native stop codon was deleted. The region of D27 encoding the plastid transit peptide was amplified using primers MW350 and MW352 (59-CAAGAAAGCTGGGTTCTCTTTTG-CAGCCTTGAG-39). Both PCR products were then reamplified with the universal Gateway attB primers described above and cloned into pDONR207. Positive clones of each were then recombined with pMDC83 (Curtis and Grossniklaus, 2003) to generate clones expressing N-D27-mGFP6-C protein fusions. For colocalization, the full-length cDNA of the small subunit of Rubisco from pea (Pisum sativum) was fused to the N terminus of red fluorescent protein (RFP;Carrie et al., 2009).
Plasmids (5 mg) were precipitated onto 1-mm gold particles and biolistically transformed into onion (Allium cepa) epidermis as described previously (Thirkettle-Watts et al., 2003). Images were obtained with an Olympus BX61 epifluorescence microscope equipped with GFP (U-MGFPHQ) and RFP (U-MRFPHQ) filters and manipulated with Olympus cell^R software.
RT-PCR and Quantitative RT-PCR Analysis
Total RNA was extracted using the RNeasy (Qiagen) or NucleoSpin RNA Plant (Macherey-Nagel) procedure. For small tissue samples (Fig. 6D), a modified TRIzol (Invitrogen) procedure was used with subsequent RNeasy cleanup. Contaminating DNA was removed with Turbo DNA-free (Ambion) or DNase (Macherey-Nagel or Qiagen), and RNA was quantified with a NanoDrop 1000 spectrophotometer.
In the case of Figure 2B, Figure 6A, and Supplemental Figure S2C, cDNA was generated from 0.5 mg of total RNA in a 10-mL reaction using the iScript cDNA Synthesis kit (Bio-Rad). Quantitative RT-PCR was performed on a Roche LC480 using LightCycler 480 SYBR Green Master Mix (Roche) in 5-mL reactions. Cycle conditions were as follows: 95°C for 10 min; then 45 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s; followed by melt-curve analysis.
Crossing point values were calculated under high confidence. For each biological replicate, two technical replicates of each real-time PCR were examined, and the mean crossing point value was used to calculate expression relative to an internal reference gene using the formula (E gene ) 2Cp_gene /(E ref ) 2Cp_ref , where E is the primer efficiency and Cp represents the crossing point. Primer efficiencies were determined in separate runs using serial dilutions of pooled cDNA.
Phylogenetic Analysis
Protein sequences with significant homology to D27 were identified with BLASTP searches of GenBank protein databases using the rice (Oryza sativa ssp. japonica) D27 amino acid sequence as a query ( [URL]). Over 90 sequences were identified with a highly significant E-value score (E 210 or smaller). To reduce the complexity of the analysis, 62 sequences were selected on the basis of wide taxonomic coverage while also providing sufficient support for each clade. Sequences were further screened for duplication and truncation. Full-length sequences were then aligned using MAFFT ( [URL]:// mafft.cbrc.jp/alignment/software) using the default settings. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. 5, E-H), indicating that D27 possesses a bona fide plastid transit peptide. Together, these data suggest that D27 functions in the plastid, consistent with the demonstrated cellular location of OsD27 in rice and its anticipated activity on a nonmobile precursor.
D27 Transcripts Are Subject to Auxin and Feedback Control D27, MAX3, and MAX4 transcript abundance from public microarray data revealed potential differential expression intensities throughout the plant, with a relative deficit of D27 in root tissue (Supplemental Fig. S4). Quantitative RT-PCR analysis of Col-0 tissues confirmed that D27 transcripts were more abundant in shoot tissue than in roots of 3-week-old wild-type plants, while MAX3 and MAX4 transcripts accumulated most strongly in the hypocotyl and roots (Fig. 6A). In contrast, MAX1 transcript levels varied little over the tissues examined. Consistent with their role in SL response, we found that MAX2 and AtD14 transcripts were evenly expressed across the shoot, hypocotyl, and root (Fig. 6A). Differential spatial expression of D27 compared with MAX3 and MAX4 then led us to investigate whether D27 was under similar feedback control as MAX3 and MAX4.
In all max mutants, levels of MAX3 and MAX4 transcripts in the hypocotyl are 6-to 10-fold higher than in the wild type, indicative of feedback up-regulation . This feedback process is dependent on a combination of auxin and nonauxin Figure 5. The N terminus of Arabidopsis D27 is sufficient for plastid targeting. A and E, Epifluorescence micrographs of onion epidermis transiently transformed with plasmids encoding a full-length D27-GFP protein fusion (A) or the first 50 amino acids of D27 fused to the N terminus of GFP (E). B and F, Plastids are positively identified by coexpression of a protein fusion between the full-length Rubisco small subunit from pea (SSU) and RFP. C and G, The merged images confirm colocalization of the two fluorescent signals. Insets in A to C provide magnification and show clustering of plastids around the cell nucleus. D and H, Brightfield micrographs of the same field of view to provide the cell outline. Bars = 50 mm (main images) and 20 mm (insets).
were 5-fold down-regulated in axr1 mutants, D27 transcripts were only moderately (albeit significantly) less abundant. Consequently, although the magnitude of expression changes for D27 are less than for MAX3, the response is similar and therefore may involve common regulatory components.
Levels of MAX3 and MAX4 transcripts in the hypocotyl decrease in response to removal of the shoot tip (decapitation) by decreasing expression levels ). This presumably reduces SL levels in order to allow buds below the decapitation site to grow and compensate for the missing shoot tip. A proportion of the decrease in gene expression is due to auxin depletion in the hypocotyl after removal of the shoot tip (the shoot tip being the main source of auxin for the plant). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. S3).
Our phylogenetic analysis demonstrates that AtD27 belongs to a wider family of proteins that originated early in the evolution of photosynthetic eukaryotes. Although D27-like proteins are found throughout cyanobacteria and green algae, two clades are strictly specific to land plants. Notably, CCD7 (MAX3) and CCD8 (MAX4) form monophyletic clades in land plants, and putative orthologs of both CCD7 and CCD8 are present in Chlamydomonas reinhardtii and other photosynthetic eukaryotes (Ledger et al., 2010;Vallabhaneni et al., 2010;Proust et al., 2011). Thus, the enzymatic machinery necessary for the early steps of SL biosynthesis may have been present in the ancestors of the land plants. Interestingly, all land plants so far examined possess homologs of the proteins required for SL response (i.e. MAX2 and D14), but so far, no homologs have been found in other photosynthetic eukaryotes, including the streptophyte algae, the closest living relatives of the land plants (Waters et al., 2011. Although this finding could reflect incomplete genome sequence data for the streptophyte algae, it is possible that SL signaling coincided with the evolution of the land plants and the concurrent development of complex body plans. As carotenoids are essential pigments associated with light harvesting and photoprotection in oxygenic photosynthetic organisms, carotenoid metabolism is a fundamental requirement for photosynthesis and likely the original function of CCD7-, CCD8-, and D27-like proteins. As multicellularity evolved, we propose that gene duplications permitted the acquisition of novel gene function, and a separate pathway for carotenoid breakdown was co-opted for signaling purposes. Recently, SLs were shown to have a dramatic role in gametophytic development of the moss P. patens. Loss of PpCCD8 activity leads to severely compromised SL profiles and impaired control of protonemal branching, which in turn limits the ability to sense neighboring colonies (Proust et al., 2011). As the P. patens genome also encodes a D27 ortholog, multiple D14 homologs, and a single MAX2 ortholog, it appears that the SL biosynthesis and signaling system is conserved among land plants. The availability of complete genome sequences for the streptophyte algae, combined with an assessment of their ability to produce SLs, will help in determining the origin of SL signaling.
We have contributed further to the understanding of the D27 protein by demonstrating that the Arabidopsis version of D27 functions upstream of MAX1 in the production of SL-related signal(s) and displays similar feedback control compared with other genes encoding plastid-localized SL biosynthesis proteins. AtD27 controls a graft-transmissible signal because branching can be inhibited in Atd27 mutant shoots by grafting to wild-type rootstocks (Fig. 4). Whereas max1 mutant rootstocks retain this inhibitory function, max4 rootstocks do not. This is clear evidence in support of D27 acting upstream of MAX1. On the other hand, the grafting data do not permit the ordering of D27 with respect to MAX4, presumably because the carotenoid-derived substrates of D27 and MAX4 are unable to leave the plastid (Alder et al., 2012). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. However, the majority of the feedback induced by d27 is likely to be local, since d27 rootstocks cause high expression of MAX3 and MAX4 similar to mutant self-grafts even where the scion is the wild type.
The regulation by auxin of a third protein in the SL pathway further supports the hypothesis that SLs act downstream of auxin in the control of shoot branching (Brewer et al., 2009). However, again, very small increases in the expression of PIN1 and IAA5 in d27 mutants (Fig. 6D) indicate enhanced auxin signaling or content, which could be indicative of enhanced auxin transport as observed in Osd27 and several other branching mutants (Lin et al., 2009;Crawford et al., 2010;Domagalska and Leyser, 2011). We have recently modeled long-distance auxin transport in the stem, and these data, in addition to data on auxin transport under high auxin content, demonstrate that wild-type plants do not have reduced auxin transport capacity relative to SL mutants (Brewer et al., 2009;Renton et al., 2012). Rather, in those experiments, the enhanced auxin transport seen in max3 and max4 mutants was more likely to be due to enhanced uptake of the exogenously supplied radiolabeled auxin to the top of the long-distance polar auxin transport stream (Renton et al., 2012).
In summary, d27 represents a new genetic tool for the elucidation of shoot branching mechanisms and their regulation in Arabidopsis. Together with MAX3 and MAX4, D27 acts in the plastid on the production of carlactone, an intermediate in the SL pathway, prior to MAX1. These genes encoding plastid-localized proteins, although expressed somewhat differently at the level of different plant parts, appear to carry conserved properties in terms of the regulation of their expression, particularly by the plant hormone auxin.
Unless otherwise stated, Arabidopsis (Arabidopsis thaliana) plants were grown under fluorescent lamps emitting 100 to 120 mmol photons s 21 m 22 (intensity at the rosette level) in a 6:1:1 mixture of peat, vermiculite, and perlite or in University of California mix with added 60% vermiculite. Plants from the Col-0 background were grown in a 16-h-light/8-h-dark photoperiod and a 22°C light/16°C dark temperature cycle, or in constant 22°C. Ws-4 was initially grown for 2 weeks in 14 h of light/10 h of dark and constant 18°C, in order to compensate for the extreme early flowering in Ws-4 and to match their flowering time with Col-0.
Grafting and Branching Assays
Grafting of Arabidopsis seedlings was performed as described in Brosnan et al. (2007) and as described in Brewer et al. (2009). Treatment of plants with GR24 in Phytatrays was performed as described (Brewer et al., 2009). Treatment of plants grown in hydroponic medium has been described previously .
IAA Treatments
Prebolting Ws-4 plants were treated with a lanolin ring around the top of the hypocotyl containing 2.5% dimethyl sulfoxide and 2.5% ethanol (control) with 10 mM IAA and/or 10 mM NPA (an auxin transport inhibitor). Decapitation involved removing the apical meristem and some small leaves/flowers from the shoot apex of each plant. Hypocotyl tissue was harvested 4 h post treatment. Three replicates were used for each genotype, with approximately 20 plants used per replicate.
RNA was isolated from 7-d-old whole seedlings using the Qiagen RNeasy procedure. cDNA was generated from 1 mg of total RNA using Invitrogen SuperScript III. The At1g03055.1 coding sequence was first amplified using primers MW350 59-AAAAAAGCAGGCTATGAACACTAAGCTATCAC-39 and MW351 59-CAAGAAAGCTGGGTCTAATGCTTCACACCGTAGC-39 (translation initiation codon and stop codon, respectively, in boldface) using a proofreading DNA polymerase (Phusion; New England Biolabs). Approximately 250 pg of the PCR product was then reamplified using Gateway attB adapter primers 59-GGGGACAAGTTTGTACAAAAAAGCAGGCT-39 and 59-GGGGACCACTTTGTACAAGAAAGCTGGGT-39 (overlapping regions with MW350 and MW351, respectively, are underlined). The resulting PCR product was cloned into pDONR207 via Gateway-based recombination, and positive clones were confirmed by sequencing. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. 4). This suggests that d27 may generate a small amount of SL, or a substance that can partly compensate for SL deficiency, and is consistent with the relatively mild branching phenotype of d27 compared with max1, max3, and max4 (Figs. 3C and 4; Supplemental Fig. S3).
In contrast to graft combinations involving d27 with the wild type or max1, reciprocal grafts between d27 and max4 failed to restore branching to wild-type levels in either mutant shoot ( Fig. 4; Sorefan et al., 2003;Hayward et al., 2009). This situation is similar to grafts between the carotenoid cleavage dioxygenase mutants of Arabidopsis (max3 and max4) and pea (Pisum sativum; rms1 and rms5), which, unlike grafts with wild-type partners, are unable to show branching inhibition (Morris et al., 2001;Booker et al., 2005). As discussed below, this is consistent with D27 acting in the plastid on a plastid-localized, nonmobile substrate. Unlike grafts between max3 and max4, max4 rootstocks caused a slight enhancement of branching in d27 shoots and d27 rootstocks caused a slight reduction of branching in max4 shoots. As d27 has an intermediate branching phenotype compared with max4 ( Fig. 4), d27 presumably has elevated graft-transmissible hormone content compared with max4. In summary, these grafting data are consistent with D27 functioning upstream of MAX1 in the biosynthesis of SL.
AtD27 Is Localized to the Plastid
Given the apparent nonmobility of the D27 precursor and the biochemical function of D27, it is likely that the Arabidopsis D27 protein is spatially restricted to the plastid. To determine the likely cellular location of D27, we generated a chimeric construct encoding the full-length D27 protein fused to the N terminus of GFP. Following transient expression in onion (Allium cepa) epidermis, the GFP fluorescence fully overlapped with the red fluorescence from the coexpressed plastid-specific marker (Fig. 5, A-D). No GFP fluorescence was detected beyond the plastid, suggesting that the full-length D27 protein was strictly localized to this organelle. This is in accord with plastid localization of OsD27 (Lin et al., 2009). The D27 protein encodes a predicted N-terminal plastid transit peptide of 47 amino acids (Supplemental Fig. S1). To test whether this presequence is sufficient to direct plastid localization, we generated a translational fusion between the first 50 amino acids of D27 and GFP and subjected onion epidermal cells to transient transformation as before. Again, the fluorescence signals from both the GFP and the plastid-specific marker fully overlapped (Fig. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. Based on our phylogenetic analysis, it is possible that another D27-like protein can perform the isomerization of all-transb-carotene in the absence of functional D27. Although the function of At1g64680 is unknown, the predicted protein has been localized to the chloroplast envelope and/or thylakoids by liquid chromatographytandem mass spectrometry analysis (Ferro et al., 2010), while its closest homolog in rice, encoded by Os08g0114100, is weakly predicted to be plastidic by TargetP software (Emanuelsson et al., 2007). Alternatively, nonenzymic isomerization may provide a small amount of 9-cis-b-carotene substrate for MAX3/ CCD7, allowing only limited levels of SL to be synthesized in the d27 mutant (despite enhanced MAX3 and MAX4 expression). However, the rice d27 mutant does not contain any detectable SL, either by liquid chromatography-mass spectrometry or a bioassay based on Orobanche seed germination (Lin et al., 2009), which may suggest that any residual SLs are below the limit of detection in d27. On the other hand, max1 and max4 mutants, which exhibit a strong branching phenotype, still contain detectable levels of orobanchol . This contradiction means that the basis for the relatively mild branching phenotype of d27 in both rice and Arabidopsis is currently unclear. Therefore, detailed biochemical analyses of the d27 Figure 7. Position of D27 within the SL biosynthesis and signaling pathway. Enzymatic steps are denoted with solid arrows, transport steps with dashed arrows, and signaling steps with thick arrows. AtD14 may be a SL receptor, a hydrolytic enzyme that processes SL into a further active compound, or a combination of both; for a discussion, see Scaffidi et al. (2012). [See online article for color version of this figure. ] mutant, intact or grafted, compared with other mutant and wild-type lines may be useful in evaluation of the levels and profiles of endogenous SLs and their potential correlations with phenotypic responses.
Interestingly, despite the complete rescue of d27 shoots by grafting to wild-type rootstocks, the expression of D27 is not greatest in the rootstock. Indeed, its expression in roots and hypocotyls of 3-week-old plants is greatly and slightly reduced, respectively, compared with shoots (Fig. 6A). This regulation is a point of difference with MAX3 and MAX4, which are both highly expressed in the hypocotyl. In a further contrast, MAX3 is relatively weakly expressed in roots compared with shoots, while MAX4 is again highly expressed. The expression patterns that we observed are fully consistent with meta-profiles derived from multiple microarray experiments (Supplemental Fig. S4). Recently, it was found that two GRAS-type transcription factors, NSP1 and NSP2, regulate D27 expression and SL levels in Medicago and rice . List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. To verify that d27 shoots are similarly responsive to a graft-transmissible signal, we generated reciprocal grafts between wildtype ecotype Columbia (Col-0) and d27 and found that wild-type rootstocks could suppress the increased branching phenotype of d27 shoots (Fig. 4). A mutant rootstock had no effect on the phenotype of a wildtype shoot, showing that D27 expression in the shoot is sufficient to inhibit branching (Fig. 4).
It has been shown previously that MAX1 operates downstream of MAX3 and MAX4 in SL biosynthesis, because a max1 rootstock can restore a wild-type branching pattern to max3 and max4 shoots, but not vice versa (Booker et al., 2005). These findings have also demonstrated that a compound intermediate between MAX3/MAX4 and MAX1 is subject to longdistance transport from the root to the shoot. Likewise, the substrates of MAX3 and MAX4 are nonmobile (or are unable to access MAX3/MAX4 in the plastid of a remote cell), because a max3 rootstock fails to complement a max4 shoot, and vice versa (Booker et al., 2005). To investigate the relative position of D27 within the MAX pathway, we generated reciprocal grafts between d27 and either max1 or max4. A max1 rootstock was able to fully restore the branching phenotype of a d27 shoot, demonstrating that MAX1 operates downstream of D27 (Fig. 4). Interestingly, as for max4 discussed below, the branching of max1 scions grafted to d27 rootstocks was reduced to that of d27 self-grafts (Fig. 4). This suggests that d27 may generate a small amount of SL, or a substance that can partly compensate for SL deficiency, and is consistent with the relatively mild branching phenotype of d27 compared with max1, max3, and max4 (Figs. 3C and 4; Supplemental Fig. S3).
In contrast to graft combinations involving d27 with the wild type or max1, reciprocal grafts between d27 and max4 failed to restore branching to wild-type levels in either mutant shoot ( Fig. 4; Sorefan et al., 2003;Hayward et al., 2009). This situation is similar to grafts between the carotenoid cleavage dioxygenase mutants of Arabidopsis (max3 and max4) and pea (Pisum sativum; rms1 and rms5), which, unlike grafts with wild-type partners, are unable to show branching inhibition (Morris et al., 2001;Booker et al., 2005). As discussed below, this is consistent with D27 acting in the plastid on a plastid-localized, nonmobile substrate. Unlike grafts between max3 and max4, max4 rootstocks caused a slight enhancement of branching in d27 shoots and d27 rootstocks caused a slight reduction of branching in max4 shoots. As d27 has an intermediate branching phenotype compared with max4 ( Fig. 4), d27 presumably has elevated graft-transmissible hormone content compared with max4. List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering
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!!! Q: Continue the text. Add 2 sentences that ends with '(Lin et al., 2009)'. Add a conclusion.
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The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. * Corresponding author; e-mailThe author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christine A. Beveridge ([email protected]).
[C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription.
!!! Answer:
The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones.
Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. * Corresponding author; e-mailThe author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christine A. Beveridge ([email protected]).
[C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp. 112.196253 response (Bennett et al., 2006;Brewer et al., 2009;Hayward et al., 2009). Based on impaired SL response in corresponding mutants of Arabidopsis and rice (Oryza sativa), two proteins have been identified as necessary for this process: the F-box protein AtMAX2/ OsD3 (Gomez-Roldan et al., 2008;Umehara et al., 2008) and the a/b-fold hydrolase family protein AtD14/OsD14 (Arite et al., 2009;Waters et al., 2012). Relative to wild-type plants, the SL response mutants produce elevated amounts of SL, presumably due to impaired feedback on SL biosynthesis (Umehara et al., 2008;Arite et al., 2009;Kohlen et al., 2011). Accordingly, MAX3 and MAX4 gene expression is greatly enhanced in max2 mutants . In rice, a plastid-targeted protein, DWARF27 (D27), is required for SL biosynthesis (Lin et al., 2009). As with other SL mutants in rice, the d27 mutant has a highly tillered and dwarf habit, although the phenotype of d27 is intermediate between the wild type and other characterized SL-related mutants. Like MAX3 and MAX4 SL biosynthesis genes, D27 is expressed in the vasculature. D27 also has strong expression in axillary buds (Lin et al., 2009). Recent in vitro evidence suggests that D27 is a b-carotene isomerase that converts all-trans-b-carotene into 9-cis-b-carotene, which is then further processed by CCD7 and CCD8 into a SL precursor termed carlactone (Alder et al., 2012). If this biochemical activity is also the case in planta, it is likely that D27 acts upstream of MAX1 in the control of the biosynthesis of a mobile shoot-branching signal. The availability of an equivalent d27 mutant in a species amenable to grafting would allow a more direct test of this hypothesis. Moreover, it will allow an extended genetic, biochemical, and physiological analysis of SL biosynthesis and its regulation. In this survey, we identify the Arabidopsis ortholog of OsD27 and characterize it with respect to its SL response, expression profile, and regulation and use grafting to place it within the SL biosynthesis pathway.
At1g03055 Is Orthologous to OsD27
The Arabidopsis genome encodes three proteins with superficial similarity to rice OsD27. To identify the likely ortholog of OsD27, we performed a phylogenetic analysis of D27-like proteins from land plants, green algae, and cyanobacteria. The eukaryotic sequences broadly grouped into three distinct clades, two of which (clades 1 and 2) only contained representatives from land plants, while the third (clade 3) also contained members from chlorophyte algae and diatoms (Fig. 1). List of protein sequences used in phylogeny reconstruction.
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Domain: Biology Environmental Science Medicine Engineering<|endoftext|>This below document has 2 sentences that end with 'System (BioTek, Winooski, VT, USA)',
2 sentences that end with 'USA) following the manufacturer's protocol',
2 sentences that end with 'USA), following the manufacturer's protocol'. It has approximately 711 words, 25 sentences, and 7 paragraph(s).
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. Briefly, this protocol consists of increasing the temperature over 24 h at a rate of 1 • C in 5 h. This protocol maintains the thermal stress temperature (19 ± 1 • C) for 5 days. This high temperature protocol was selected considering heat waves observed in the summer season on the Chilean coast in recent years [27]. The control group was maintained at the control temperature (14 ± 1 • C) during the assay. At the end of the experiment, six individuals per group (two tanks per group, with three animals sampled per tank, total of N = 12 sampled fish) were netted and sampled. Blood samples were collected via caudal puncture using heparinized tubes, immediately centrifuged at 5000× g for 10 min for serum obtention and stored at −80 • C until analysis. After blood sampling, fish were euthanized (overdose of anesthetic 3-aminobenzoic acid ethyl ester, 300 mg/L). Fish livers were collected and stored for RNA extraction in RNAsave solution (Biological Industries, Cromwell, CT, USA) or immediately frozen in liquid nitrogen and stored at −80 • C until analysis for oxidative damage evaluation.
AST, ALT and AP Evaluation
The plasmatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AP) were determined using commercially available kits from Valtek (Santiago, Chile) following the manufacturer's instruction. Briefly, these kits determined the enzymatic activity (IU/L) via the generation of colorimetric products from glutamate (colorimetric product: 450 nm), pyruvate (535 nm), and p-nitrophenol (405 nm) for AST, ALT and AP activity, respectively.
Oxidative Stress Assays in Liver
To determine the oxidative damage in the liver of red cusk-eel in response to hightemperature stress, DNA oxidative damage, protein carbonylation, and lipid peroxidation were evaluated using commercially available kits. The DNA oxidative damage assay was performed with genomic DNA (gDNA) purified from 25 mg of the liver using the DNAzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol and quantified with the Epoch Spectrophotometer System (BioTek, Winooski, VT, USA). Then, the apurinic/apyrimidinic sites (AP sites) were determined in the gDNA using the OxiSelect Oxidative DNA Damage Quantification Kit (Cell Biolabs, CA, USA) following the manufacturer's protocol. The protein carbonylation assay was performed using total protein extracted from 100 mg of the liver in 1 mL of lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% NP-40, solubilized at 4 • C after 12,000× g centrifugation. Proteins were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Batavia, IL, USA). Then, carbonylated protein content was quantified using the OxiSelect Protein Carbonyl Spectrophotometric Assay (Cell Biolabs, San Diego, CA, USA) following the manufacturer's protocol. The lipid peroxidation determination in the liver was performed using the OxiSelect HNE Adduct Competitive ELISA Kit (Cell Biolabs, San Diego, CA, USA), following the manufacturer's protocol. This kit determines lipid peroxidation through the quantification of hydroxynonenal (HNE) protein adducts in the extracted proteins of the liver.
Liver RNA Extraction, Library Preparation and Illumina Sequencing
Total RNA was extracted from the liver stored in RNAsave solution (Biological Industries, Cromwell, CT, USA) using the TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA) protocol. Total RNA was quantified by fluorometry with the Qubit ® RNA quantitation assay (Invitrogen, Carlsbad, CA, USA) and purity was determined according to 260/280 ratio using the Epoch Spectrophotometer System (BioTek, Winooski, VT, USA). The RNA integrity was measured according to RNA Quality Measurement Number (RQN) through a Fragment Analyzer with the Standard Sensitivity RNA Analysis kit (Advanced Analytical Technologies, Fiorenzuola, Italy), selecting samples with RQN ≥ 8. The cDNA libraries construction were performed with 1 µg of total RNA per sample using the Illumina ® TruSeq RNA Sample Prep Kit v2 (Illumina ® , San Diego, CA, USA), following the manufacturer's protocol. The sizes of the mRNA libraries were determined through a Fragment Analyzer using the NGS Fragment Analysis kit (Advanced Analytical Technologies) and quantified by qPCR using the Kapa Library Quantification kit (Roche, Little Falls, NJ, USA). The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
==
Domain: Biology Environmental Science
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@@@ Instruction: Add 2 sentences that ends with 'downregulated metabolites (Figure 2B)'. Add a concluding sentence.
===
Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. A total of 31 anthocyanins were identified in all of the samples. Then, the |log2(fold-change)| ≥ 1 and p value < 0.05 was used to determine significantly metabolites between groups (Figure 2B). In group LFC vs. JFC, 23 upregulated metabolites and 8 downregulated metabolites were found to be significantly different (Figure 2B). In LFC vs. In BSH vs. Counting the differential metabolites compared with both LFC and JFC for each individual plant, a Venn diagram was used to show that there were 6 differential metabolites in the comparison group compared with JFC and 24 common differential metabolites compared with LFC (Figure 2C,D). Thirty-one common detected metabolites were used to draw clustering heatmap by TBtools ( [URL] on 13 September 2023). The contents of cyanidin and delphinidin could be mainly responsible for the leaves color of Toona sinensis (Figure 2E).
Transcriptome Analysis of Five Toona sinensis Clones
Fifteen cDNA libraries (each clone with three biological replicates) were sequenced using the Illumina HiSeq platform. After removing low-quality reads, each sample yielded between 42.16 M to 52.03 M clean reads (Supplementary Figure S1). The Q30 base exceeded 92.56% and the guanine-cytosine (GC) content of each sample was greater than 42.27%, indicating acceptable sequencing quality. After filtration, the clean data reached 6.32-7.80Gb for each sample. The genome of Toona sinensis var. Heiyouchun was utilized, which marked a significant was the first chromosome-level genome assembled of Toona, as our reference genome [36]. Furthermore, comparison of all clean reads with this reference genome and subsequently performed gene annotations, the results revealed a high rate of similarity, ranging from 92.26% to 94.03%. These results validate the reliability of our data. Novel genes were defined as unigenes found in the sequencing results but not included in the reference genome (or reference gene set) after reconstructing the transcripts using StringTie v1.3.4dsoftware. Comparison identified 9127 novel genes. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
@@@ Answer:
Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. A total of 31 anthocyanins were identified in all of the samples. Then, the |log2(fold-change)| ≥ 1 and p value < 0.05 was used to determine significantly metabolites between groups (Figure 2B). In group LFC vs. JFC, 23 upregulated metabolites and 8 downregulated metabolites were found to be significantly different (Figure 2B). In LFC vs. BSH, there were 24 upregulated metabolites and 6 downregulated metabolites (Figure 2B). In BSH vs. JFC, there were 10 upregulated metabolites and 11 downregulated metabolites (Figure 2B). Counting the differential metabolites compared with both LFC and JFC for each individual plant, a Venn diagram was used to show that there were 6 differential metabolites in the comparison group compared with JFC and 24 common differential metabolites compared with LFC (Figure 2C,D). Thirty-one common detected metabolites were used to draw clustering heatmap by TBtools ( [URL] on 13 September 2023). The contents of cyanidin and delphinidin could be mainly responsible for the leaves color of Toona sinensis (Figure 2E).
Transcriptome Analysis of Five Toona sinensis Clones
Fifteen cDNA libraries (each clone with three biological replicates) were sequenced using the Illumina HiSeq platform. After removing low-quality reads, each sample yielded between 42.16 M to 52.03 M clean reads (Supplementary Figure S1). The Q30 base exceeded 92.56% and the guanine-cytosine (GC) content of each sample was greater than 42.27%, indicating acceptable sequencing quality. After filtration, the clean data reached 6.32-7.80Gb for each sample. The genome of Toona sinensis var. Heiyouchun was utilized, which marked a significant was the first chromosome-level genome assembled of Toona, as our reference genome [36]. Furthermore, comparison of all clean reads with this reference genome and subsequently performed gene annotations, the results revealed a high rate of similarity, ranging from 92.26% to 94.03%. These results validate the reliability of our data. Novel genes were defined as unigenes found in the sequencing results but not included in the reference genome (or reference gene set) after reconstructing the transcripts using StringTie v1.3.4dsoftware. Comparison identified 9127 novel genes. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
==
Domain: Biology Environmental Science
|
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### Question: Context: Analysis of Anthocyanidin Biosynthetic Pathway Genes and Anthocyanin Derivates in JFC, BSH, and LFC
According to transcriptome and metabolites studies, anthocyanin biosynthesis may be responsible for the distinct red leaf colorations of Toona. Therefore, it is necessary to investigate the mechanisms that underlie this process. Genes encoding anthocyanin biosynthetic enzymes were found to be associated with different compositions of the compounds in the anthocyanin biosynthesis pathways. We constructed a pathway diagram displaying the expression heat map of structural genes and compounds for red, light-red, and green leaves (Figure 4). The PAL gene catalyzed the conversion of L-Phenylalanine to cinnamic acid and was highly expressed in JFC. Among the 4CL genes, two exhibited a gradual decrease in expression from red to green leaves across JFC, BSH, and LFC. CHS, the first key enzyme in the flavonoid pathway, played a pivotal role in catalyzing the synthesis of chalcone. Three CHS genes were differentially expressed in these clones. Two of these genes were significantly up-regulated in BSH and JFC, compared with LFC, whereas one was more highly expressed in LFC than in BSH and JFC. Two CHI genes responsible for converting naringenin chalcone to naringenin exhibited higher expression levels in BSH and lower expression in LFC. The accumulation of anthocyanin was positively correlated with the expression level of these genes. The study found that there was a gradual decrease in the content of naringenin metabolite in JFC, BSH, and LFC. Moreover, the study found that the FLS, DFR, and ANS genes play a crucial role in transforming naringenin into delphinidin, cyanidin, and pelargonidin, respectively, with their expression levels being up-regulated in JFC and down-regulated in LFC (Figure 4). UFGT is a critical enzyme involved in the biosynthesis of stable anthocyanins from unstable anthocyanin glycosides. The study identified two UFGT genes whose expression levels were significantly higher in JFC than in BSH, with the lowest levels observed in LFC, resulting in the highest accumulation of different anthocyanin compounds in JFC compared to LFC. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
===
Append an analysis of the words and related contexts for this document. Add an introductory paragraph.
### Assistant:
Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The DEGs were counted with an absolute |log2fold-change| ≥ 1 and a false discovery rate (FDR) ≤ 0.05 in each compared groups. According to five clones of Toona leaves, 10 pairs of comparisons were performed and the number of up-regulated and down-regulated differential genes was counted in all groups (Figure 3B). Across all compared groups, a total of 310 DEGs were co-detected (Figure 3C). The KEGG-based enrichment analysis revealed that all DEGs compared to LFC were enriched into 140 KEGG pathways. These pathways included flavonoid biosynthesis, phenylpropanoid biosynthesis, and anthocyanin biosynthesis, all of which were significantly enriched (Figure 3D). These terms have previously been associated with anthocyanin production.
Analysis of Anthocyanidin Biosynthetic Pathway Genes and Anthocyanin Derivates in JFC, BSH, and LFC
According to transcriptome and metabolites studies, anthocyanin biosynthesis may be responsible for the distinct red leaf colorations of Toona. Therefore, it is necessary to investigate the mechanisms that underlie this process. Genes encoding anthocyanin biosynthetic enzymes were found to be associated with different compositions of the compounds in the anthocyanin biosynthesis pathways. We constructed a pathway diagram displaying the expression heat map of structural genes and compounds for red, light-red, and green leaves (Figure 4). The PAL gene catalyzed the conversion of L-Phenylalanine to cinnamic acid and was highly expressed in JFC. Among the 4CL genes, two exhibited a gradual decrease in expression from red to green leaves across JFC, BSH, and LFC. CHS, the first key enzyme in the flavonoid pathway, played a pivotal role in catalyzing the synthesis of chalcone. Three CHS genes were differentially expressed in these clones. Two of these genes were significantly up-regulated in BSH and JFC, compared with LFC, whereas one was more highly expressed in LFC than in BSH and JFC. Two CHI genes responsible for converting naringenin chalcone to naringenin exhibited higher expression levels in BSH and lower expression in LFC. The accumulation of anthocyanin was positively correlated with the expression level of these genes. The study found that there was a gradual decrease in the content of naringenin metabolite in JFC, BSH, and LFC. Moreover, the study found that the FLS, DFR, and ANS genes play a crucial role in transforming naringenin into delphinidin, cyanidin, and pelargonidin, respectively, with their expression levels being up-regulated in JFC and down-regulated in LFC (Figure 4). UFGT is a critical enzyme involved in the biosynthesis of stable anthocyanins from unstable anthocyanin glycosides. The study identified two UFGT genes whose expression levels were significantly higher in JFC than in BSH, with the lowest levels observed in LFC, resulting in the highest accumulation of different anthocyanin compounds in JFC compared to LFC. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
===
#######
## What is the function of phenylpropanoid in the body? what are some examples of the acids? what are the uses of the alcohols?
The phenylpropanoids are a diverse family of organic compounds that are synthesized by plants from the amino acids phenylalanine and tyrosine. Their name is derived from the six-carbon, aromatic phenyl group and the three-carbon propene tail of coumaric acid, which is the central intermediate in phenylpropanoid biosynthesis
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What is the biosynthesis of Flavonoids? Can you tell me what else happened following the synthesis? Did anything else happen?
## Flavonoids are synthesized by the phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA. This can be combined with malonyl-CoA to yield the true backbone of flavonoids, a group of compounds called chalcones, which contain two phenyl rings
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## What is the active site of Phenylalanine ammonia-lyase? What is the function of the active site? What are some of the reactions it participates in? What modifications are made to the active site?
The enzyme phenylalanine ammonia lyase (EC 4.3.1.24) catalyzes the conversion of L-phenylalanine to ammonia and trans-cinnamic acid.: L-phenylalanine = trans-cinnamate + NH3 Phenylalanine ammonia lyase (PAL) is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds such as flavonoids, phenylpropanoids, and lignin in plants. Phenylalanine ammonia lyase is found widely in plants, as well as some bacteria, yeast, and fungi, with isoenzymes existing within many different species
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What is known about pelargonidin? which plant has it? what else?
## Pelargonidin is an anthocyanidin, a type of plant pigment producing a characteristic orange color used in food and industrial dyes. Pelargonidin Pelargonidin is an anthocyanidin, a type of plant pigment producing a characteristic orange color used in food and industrial dyes
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## What is the function of Anthocyanin 6"-O-malonyltransferase?
Anthocyanin 6"-O-malonyltransferase In enzymology, an anthocyanin 6"-O-malonyltransferase () is an enzyme that catalyzes the chemical reaction Thus, the two substrates of this enzyme are malonyl-CoA and anthocyanidin 3-O-beta-D-glucoside, whereas its two products are CoA and anthocyanidin 3-O-(6-O-malonyl-beta-D-glucoside). This enzyme belongs to the family of transferases, specifically those acyltransferases transferring groups other than aminoacyl groups
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What are the four steps of doxorubicin biosynthesis? Why is it similar?
## Doxorubicin (DXR) is a 14-hydroxylated version of daunorubicin, the immediate precursor of DXR in its biosynthetic pathway. Daunorubicin is more abundantly found as a natural product because it is produced by a number of different wild type strains of streptomyces
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* a n a l y s i s ( analysis )
Defintion: 'analysis' means an investigation of the component parts of a whole and their relations in making up the whole
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* comparison (spelling: c o m p a r i s o n )
* comparing (spelling: c o m p a r i n g )
Defintion: 'comparison' means the act of examining resemblances. Example usage: they made a comparison of noise levels; the fractions selected for comparison must require pupils to consider both numerator and denominator; Toona leaves, 10 pairs of comparison were performed and the
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analysis, comparisons which are types of investigation. 'investigation' means the work of inquiring into something thoroughly and systematically
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* associate (spelling: a s s o c i a t e )
* tie in (spelling: t i e i n )
* relate (spelling: r e l a t e )
* link (spelling: l i n k )
* colligate (spelling: c o l l i g a t e )
* link up (spelling: l i n k u p )
* connect (spelling: c o n n e c t )
Defintion: 'to associate' means make a logical or causal connection, as in, 'terms have previously been associated with anthocyanin ...'
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* correlate (spelling: c o r r e l a t e )
Defintion: 'to correlate' means bring into a mutual, complementary, or reciprocal relation, as in, 'anthocyanin was positively correlated with the expression level ...'
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* enrich (spelling: e n r i c h )
Defintion: 'to enrich' means make wealthy or richer, as in, 'DEGs compared to LFC were enriched into 140 KEGG pathways. ...'
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* catalyze (spelling: c a t a l y z e )
* catalyse (spelling: c a t a l y s e )
Defintion: 'to catalyze' means change by catalysis or cause to catalyze, as in, 'The PAL gene catalyzed the conversion of ...'
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* construct (spelling: c o n s t r u c t )
* build (spelling: b u i l d )
* make (spelling: m a k e )
Defintion: 'to construct' means make by combining materials and parts, as in, 'We constructed a pathway diagram ...'
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* leave (spelling: l e a v e )
* result (spelling: r e s u l t )
* lead (spelling: l e a d )
Defintion: 'to leave' means have as a result or residue, as in, 'levels observed in LFC, resulting in the highest accumulation ...'
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* convert (spelling: c o n v e r t )
* change over (spelling: c h a n g e o v e r )
Defintion: 'to convert' means change from one system to another or to a new plan or policy, as in, 'CHI genes responsible for converting naringenin chalcone to ...'
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* t r a n s f o r m ( transform )
* t r a n s m u t e ( transmute )
* t r a n s u b s t a n t i a t e ( transubstantiate )
Defintion: 'to transform' means change or alter in form, appearance, or nature, as in, 'play a crucial role in transforming naringenin into ...'
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* e n c o d e ( encode )
Defintion: 'to encode' means convert information into code, as in, 'Genes encoding anthocyanin biosynthetic ...'
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* exhibit (spelling: e x h i b i t )
Defintion: 'to exhibit' means show an attribute, property, knowledge, or skill, as in, 'Among the 4CL genes, two exhibited a gradual decrease in ...'
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* imply (spelling: i m p l y )
* involve (spelling: i n v o l v e )
Defintion: 'to imply' means have as a necessary feature, as in, 'UFGT is a critical enzyme involved in the biosynthesis of ...'
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* r e v e a l ( reveal )
Defintion: 'to reveal' means disclose directly or through prophets, as in, 'enrichment analysis revealed that all DEGs compared to ...'
==
Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. The effect of high-temperature stress on the liver, a key organ for fish metabolism, is unknown. In this study we determined for the first time the effects of high-temperature stress on the liver of red cusk-eel. The results showed that high-temperature stress increased hepatic enzyme activity in the plasma of stressed fish. Additionally, this stressor generated oxidative damage in liver, and generated a transcriptional response with 1239 down-regulated and 1339 up-regulated transcripts associated with several processes, including unfolded protein response, heat shock response and oxidative stress, among others. Together, these results indicate that high-temperature stress generates a relevant impact on liver, with should be considered for the aquaculture and fisheries industry of this species under a climate change scenario. Abstract Environmental stressors, such as temperature, are relevant factors that could generate a negative effect on several tissues in fish. A key fish species for Chilean aquaculture diversification is the red cusk-eel (Genypterus chilensis), a native fish for which knowledge on environmental stressors effects is limited. This study evaluated the effects of high-temperature stress on the liver of red cusk-eel in control (14 °C) and high-temperature (19 °C) groups using multiple approaches: determination of plasmatic hepatic enzymes (ALT, AST, and AP), oxidative damage evaluation (AP sites, lipid peroxidation, and carbonylated proteins), and RNA-seq analysis. High-temperature stress generated a significant increase in hepatic enzyme activity in plasma. In the liver, a transcriptional regulation was observed, with 1239 down-regulated and 1339 up-regulated transcripts. Additionally, high-temperature stress generated oxidative stress in the liver, with oxidative damage and transcriptional modulation of the antioxidant response. Furthermore, an unfolded protein response was observed, with several pathways enriched, as well as a heat shock response, with several heat shock proteins up regulated, suggesting candidate biomarkers (i.e., serpinh1) for thermal stress evaluation in this species. The present study shows that high-temperature stress generated a major effect on the liver of red cusk-eel, knowledge to consider for the aquaculture and fisheries of this species.
Introduction
Environmental factors are important for the physiology of fish, particularly those associated with water conditions. Among these, temperature, pH, and dissolved oxygen (DO) are crucial for the homeostasis of the fish in marine environments, and changes in these factors could lead to generating a stressful status for the animal [1]. It has been reported that an increase in water temperature could lead to stress and negative effects on marine fish, including salmonids [2,3], catfish [4], Atlantic cod [5], and gilthead seabream [6]. Understanding the effects of water temperature on fish is crucial in the actual scenario of climate change, considering the average rising sea temperature per decade [7]. Additionally, the effect of climate change on sea temperatures is expected to influence relevant phenomena of the Pacific coast, such as El Niño-Southern Oscillation (ENSO), increasing their intensity and frequency [8], which is relevant for marine species of the South Pacific coast, considering the increase in water temperatures associated with this phenomena. Therefore, it is important to understand the effect of an increase in temperature and how it affects the stress response in marine fish with aquaculture potential.
Chile is a relevant country in seafood production, with an important fishery and aquaculture industry. Additionally, the Chilean aquaculture sector is recognized for its important salmon and mussel industries [9]. However, in the last decade, an important effort has been made by the public private association to diversify Chilean aquaculture with native fish of commercial value. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
==
Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. In this study, we identified a total of 11 MADS-box genes, 33 MYBs, 42 bHLHs, 36 bZIPs, and 15 WD40 genes in Toona. The RNA expression levels in JFC, BSH, and LFC was compared, which revealed that 12 bHLH, 10 bZIP, 12 MYB, 2 MADS-box, and 1 WD40 were significantly highly expressed in JFC, lowest in LFC (Figure 6A). Furthermore, the heatmap showed that all highly correlated TFs with anthocyanin derivates belonged to these groups.
Expression Profiles of TFs and Plant Hormone Genes Related to Anthocyanin Biosynthesis
Previous studies have demonstrated that the biosynthesis of anthocyanins requires the involvement of transcription factors, such as MADS-box, MYB, bHLH, and bZIP. In this study, we identified a total of 11 MADS-box genes, 33 MYBs, 42 bHLHs, 36 bZIPs, and 15 WD40 genes in Toona. The RNA expression levels in JFC, BSH, and LFC was compared, which revealed that 12 bHLH, 10 bZIP, 12 MYB, 2 MADS-box, and 1 WD40 were significantly highly expressed in JFC, lowest in LFC (Figure 6A). Furthermore, the heatmap showed that all highly correlated TFs with anthocyanin derivates belonged to these groups. Studies suggest that plant hormones have a crucial function in regulating anthocyanin concentrations in leaves and fruits via direct or indirect regulation of transcription factors responsible for anthocyanin biosynthesis. The hormones capable of activating or inhibiting the structural genes involved in the biosynthesis pathway include auxin, gibberellins, cytokinin, brassinosteroids, jasmonic acid, salicylic acid, and abscisic acid. Furthermore, we identified several differentially expressed genes that participate in plant Studies suggest that plant hormones have a crucial function in regulating anthocyanin concentrations in leaves and fruits via direct or indirect regulation of transcription factors responsible for anthocyanin biosynthesis. The hormones capable of activating or inhibiting the structural genes involved in the biosynthesis pathway include auxin, gibberellins, cytokinin, brassinosteroids, jasmonic acid, salicylic acid, and abscisic acid. Furthermore, we identified several differentially expressed genes that participate in plant hormone signal transduction, including 8 Auxin-related genes, 9 CTK-related genes, 10 GA-related genes, 23 BR-related genes, 7 ABA-related genes, 11 ethlye-responsive genes, 6 JA-related genes, and 3SA-related genes. Among these genes, the following exhibited a positive correlation coefficient greater than 0.8 with anthocyanin compounds: 2 Auxinrelated genes (Maker00008503 and Maker00022256), 1 CTK-related gene (Maker00000350), 1 GA-related gene (Maker000117930), 2 BR-related genes (Maker00026791 and novel.6183), 1 Ethylene-responsive genes (Marker00033233), and 2 SA-related genes (Maker00020104 and Maker00032838) (Figure 6B). This indicates that these genes may positively regulate anthocyanin biosynthesis either directly or indirectly.
Expression Analysis of Genes through qRT-PCR
To validate the RNA-seq results, we utilized qRT-PCR to evaluate the expression of 8 structural genes and 4 TFs involved in the anthocyanin biosynthesis pathway. Our observations revealed that bHLH162, CPC, and SOC1 TFs were significantly upregulated in JFC and exhibited the lowest expression levels in LFC (Figure 7A). Additionally, there was a strong correlation between the RNA-seq and the characteristic leaf color of these samples. The qRT-PCR outcomes of structural genes were also significantly correlated with RNA-seq (Figure 7B). Moreover, our data suggested that the expression levels of bHLH162 (Maker00021798, 2.99 times higher in JFC than in LFC), CPC (Maker00014746, 10.48 times), and SOC1 (Maker00029632, 6.67 times) were synchronized with DFR (Marker00011618, 1.84 times), ANS (Marker00000819, 2.95 times), and UFGT-1 (Marker00029333, 3.07 times), indicating that they may be involved in regulating anthocyanin production. These findings demonstrated that transcription data accurately reflect gene expression in the anthocyanin biosynthesis of Toona sinesis.
Expression Analysis of Genes through qRT-PCR
To validate the RNA-seq results, we utilized qRT-PCR to evaluate the expression of 8 structural genes and 4 TFs involved in the anthocyanin biosynthesis pathway. Our observations revealed that bHLH162, CPC, and SOC1 TFs were significantly upregulated in JFC and exhibited the lowest expression levels in LFC (Figure 7A). Additionally, there was a strong correlation between the RNA-seq and the characteristic leaf color of these samples. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
==
Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. The raw data are available from NCBI under BioProject , and lipid peroxidation in terms of HNE adducts (C) in control and stress groups. Bars represent the mean ± SEM. Significant differences between the control and stress groups are indicated by asterisks; * (p < 0.05), ** (p < 0.01) and **** (p < 0.0001).
Differentially Expressed Transcripts in Hepatic Response to High-Temperature Stress
To understand the complexity of the stress response in the liver of G. chilensis, we performed RNA-seq analysis on the liver of each experimental group. The sequencing The raw data are available from NCBI under BioProject PRJNA835467 with BioSamples accession number SAMN28102858, SAMN28102859, SAMN28102860, SAMN28102861, SAMN28102862, SAMN28102863, SAMN28102864, SAMN28102865, SAMN28102866, and SAMN28102867. After trimming by quality, adapters, and size, we obtained an average of 58,018,122 high-quality filtered paired-end reads per library (Table 2). These reads were mapped to the G. chilensis reference transcriptome (NCBI accession number SRS614525), obtaining an average of 85.2% mapped reads (Table 2). Expression values were used for normalization and differential expression analysis with the R package DESeq2 (version 1.2.10) [29], obtaining a total of 2578 differentially expressed transcripts between control and stressed groups. Of these transcripts, 1239 were down-regulated (Table S1) and 1339 were up-regulated (Table S2) in the stressed group (Figure 3 and Figure S1), evidencing five clusters of transcripts with different patterns of expression for the control and thermal stress groups ( Figure S2). SAMN28102865, SAMN28102866, and SAMN28102867. After trimming by quality, adapters, and size, we obtained an average of 58,018,122 high-quality filtered paired-end reads per library (Table 2). These reads were mapped to the G. chilensis reference transcriptome (NCBI accession number SRS614525), obtaining an average of 85.2% mapped reads (Table 2). Expression values were used for normalization and differential expression analysis with the R package DESeq2 (version 1.2.10) [29], obtaining a total of 2578 differentially expressed transcripts between control and stressed groups. Of these transcripts, 1239 were down-regulated (Table S1) and 1339 were up-regulated (Table S2) in the stressed group (Figures 3 and S1), evidencing five clusters of transcripts with different patterns of expression for the control and thermal stress groups ( Figure S2).
GO Enrichment and Pathway Analysis in the Liver
The differentially expressed transcripts were used for an enrichment analysis using GO terms to determine the overrepresented processes in response to high-temperature stress in the up-and down-regulated transcripts of the liver. The enrichment analysis showed 38 enriched processes for up-regulated transcripts (Table 3), including 18 biological processes (BP), 16 cellular components (CC), and 4 molecular functions (MF). Several of the enriched terms were related to protein metabolism, including protein folding (GO:0006457), protein transport (GO:0015031), protein localization (GO:0008104), protein retention in ER lumen (GO:0006621), and unfolded protein binding (GO:0051082). Additionally, the BP term response to heat (GO:0009408) was also enriched in the up-regulated transcripts in the high-temperature stress group. No enriched GO terms were identified in the downregulated transcripts (FDR < 0.05). The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science<|endoftext|>High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. SAMN28102865, SAMN28102866, and SAMN28102867. After trimming by quality, adapters, and size, we obtained an average of 58,018,122 high-quality filtered paired-end reads per library (Table 2). These reads were mapped to the G. chilensis reference transcriptome (NCBI accession number SRS614525), obtaining an average of 85.2% mapped reads (Table 2). Expression values were used for normalization and differential expression analysis with the R package DESeq2 (version 1.2.10) [29], obtaining a total of 2578 differentially expressed transcripts between control and stressed groups. Of these transcripts, 1239 were down-regulated (Table S1) and 1339 were up-regulated (Table S2) in the stressed group (Figures 3 and S1), evidencing five clusters of transcripts with different patterns of expression for the control and thermal stress groups ( Figure S2).
GO Enrichment and Pathway Analysis in the Liver
The differentially expressed transcripts were used for an enrichment analysis using GO terms to determine the overrepresented processes in response to high-temperature stress in the up-and down-regulated transcripts of the liver. The enrichment analysis showed 38 enriched processes for up-regulated transcripts (Table 3), including 18 biological processes (BP), 16 cellular components (CC), and 4 molecular functions (MF). Several of the enriched terms were related to protein metabolism, including protein folding (GO:0006457), protein transport (GO:0015031), protein localization (GO:0008104), protein retention in ER lumen (GO:0006621), and unfolded protein binding (GO:0051082). Additionally, the BP term response to heat (GO:0009408) was also enriched in the up-regulated transcripts in the high-temperature stress group. No enriched GO terms were identified in the downregulated transcripts (FDR < 0.05). In the KEGG pathway analysis, we identified several pathways represented in the down-regulated and up-regulated transcripts, including apoptosis, cell cycle, MAPK signaling pathway, autophagy, protein processing in endoplasmic reticulum, and ubiquitinmediated proteolysis. Among these pathways, protein processing in endoplasmic reticulum was one of the most represented on the differentially expressed transcripts (Figure 4), with 31 up-regulated and three down-regulated transcripts represented in this pathway, including several heat shock proteins; heat shock protein 40 (hsp40, also known as dnaJ homolog subfamily B member 1), heat shock protein 70 (hsp70) and heat shock protein 90 (hsp90).
(casp3) and complement C3-like (c3) ( Table 1). All of the selected differentially expressed transcripts on RNA-seq were validated by qPCR, confirming the results of the RNA-seq analysis ( Figure 5), with a Pearson's correlation coefficient of r = 0.847, confirming the significant differential expression of all tested transcripts by qPCR between the control and high-temperature group ( Figure S3). Figure 5. qPCR validation of selected differentially expressed transcripts in the liver of G. chilensis in response to high-temperature stress. The transcript expression levels were normalized with the geometric means of actb and taf12. The differential expression levels according to qPCR (black bars) and RNA-seq (gray bars) for these selected genes are expressed as log2 fold changes. The log2FC represents the expression change in the stress group compared with the control group. Results are expressed as the mean ± standard error. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The colors of these varieties are: brilliant-red buds (JFC), pink buds (FHXN), light-red with a little green buds (BSH), dark-purple buds (HBG), and green buds (LFC). Fresh buds from five different clones were collected, with three replicates taken for each clone, each replicate sourced from different trees. Buds measuring 10-15 cm in length and weighing approximately 5-10 g were collected for each sample and subsequently underwent freeze-drying for RNA-seq and metabolites measures.
Sample Preparation and Extraction
The sample underwent freeze-drying, followed by grinding into a powder (30 Hz, 1.5 min), and subsequently, it was stored at −80 • C until required. Subsequently, 50 mg of the powder was precisely weighed and subjected to extraction using 0.5 mL of methanol/ water/hydrochloric acid (500:500:1, v/v/v). The resulting extract was vigorously vortexed for 5 min, followed by ultrasonication for another 5 min, and then centrifuged at 12,000× g at 4 • C for 3 min. The residue underwent a repeat extraction using the same procedure and conditions. The supernatants obtained were collected and filtered through a 0.22 µm membrane filter (Anpel) prior to LC-MS/MS analysis.
ESI-MS/MS Conditions
Linear ion trap (LIT) and triple quadrupole (QQQ) scans were obtained using a triple quadrupole-linear ion trap mass spectrometer, the AB Scoex Qtrap ® 6500+ LC-MS/MS System (Sciex, Framingham, MA, USA), equipped with an ESI Turbo Ion-Spray interface. The instrument operated in positive ion mode and was controlled by Analyst 1.6.3software (Sciex). The ESI source operated with the following parameters: Ion Source: ESI+, Source Temperature: 550 • C, Ion Spray Voltage (IS): 5500 V, Curtain Gas (CUR): Set at 35 psi. Anthocyanins were subjected to analysis using scheduled multiple reaction monitoring (MRM). Data acquisition was conducted through Analyst 1.6.3software (Sciex). Quantification of all metabolites was performed using Multiquant 3.0.3software (Sciex). Further optimization of declustering potentials (DP) and collision energies (CE) for individual MRM transitions was carried out. A specific set of MRM transitions was monitored for each period, corresponding to the elution of metabolites during that timeframe.
Identification of Differentially Accumulated Metabolites
Significantly regulated metabolites between groups using the identification criterion of the absolute |log 2 (fold-change)| ≥ 1 and p value < 0.05, based on the Student's t-test. Identified metabolites using the Kyoto Encyclopedia of Gene and Genomes (KEGG) compound database available at [URL] on 20 June 2022. Next, we mapped the annotated metabolites to KEGG Pathway database found at [URL] on 20 June 2022. The pathways that significantly regulated metabolites mapped to were integrated into MSEA (metabolite sets enrichment analysis) and evaluated for significance by checking the hypergeometric test's p-values.
RNA Sequencing
Total RNA was extracted from frozen leaves utilizing the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to evalute the quality of the gathered RNAs by examining their integrity. Subsequently, Poly (A) mRNA was enriched from total RNA using Oligo (dT) magnetic beads. To facilitate sequencing, Poly (A) mRNA was fragmented by an RNA fragmentation kit (Ambion, Austin, TX, USA). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Among the 4CL genes, two exhibited a gradual decrease in expression from red to green leaves across JFC, BSH, and LFC. CHS, the first key enzyme in the flavonoid pathway, played a pivotal role in catalyzing the synthesis of chalcone. Three CHS genes were differentially expressed in these clones. Two of these genes were significantly up-regulated in BSH and JFC, compared with LFC, whereas one was more highly expressed in LFC than in BSH and JFC. Two CHI genes responsible for converting naringenin chalcone to naringenin exhibited higher expression levels in BSH and lower expression in LFC. The accumulation of anthocyanin was positively correlated with the expression level of these genes. The study found that there was a gradual decrease in the content of naringenin metabolite in JFC, BSH, and LFC. Moreover, the study found that the FLS, DFR, and ANS genes play a crucial role in transforming naringenin into delphinidin, cyanidin, and pelargonidin, respectively, with their expression levels being up-regulated in JFC and down-regulated in LFC (Figure 4). UFGT is a critical enzyme involved in the biosynthesis of stable anthocyanins from unstable anthocyanin glycosides. The study identified two UFGT genes whose expression levels were significantly higher in JFC than in BSH, with the lowest levels observed in LFC, resulting in the highest accumulation of different anthocyanin compounds in JFC compared to LFC. These results revealed that the expression of genes in anthocyanin biosynthesis was consistent with accumulation of anthocyanins.
Correlation Analysis of Differentially Expressed Transcripts and Anthocyanin Compounds to Identify Key TFs and Plant Hormones Genes
To achieve the regulatory network between regulatory genes, including transcription factors and plant hormone-related genes, and anthocyanin metabolites, an association analysis was conducted between transcripts and metabolome (Supplementary Figure S2). The major transcription factors and plant hormone-related genes were then identified using differentially expressed genes and metabolites that showed significant differences between JFC (red) and LFC (green). A conjoint analysis was conducted to establish relationships between 510 genes and 23 metabolites with a positive correlation coefficient greater than 0.8 within JFC and LFC. Among these genes and metabolites, 23 TFs and
Correlation Analysis of Differentially Expressed Transcripts and Anthocyanin Compounds to Identify Key TFs and Plant Hormones Genes
To achieve the regulatory network between regulatory genes, including transcription factors and plant hormone-related genes, and anthocyanin metabolites, an association analysis was conducted between transcripts and metabolome (Supplementary Figure S2). The major transcription factors and plant hormone-related genes were then identified using differentially expressed genes and metabolites that showed significant differences between JFC (red) and LFC (green). A conjoint analysis was conducted to establish relationships between 510 genes and 23 metabolites with a positive correlation coefficient greater than 0.8 within JFC and LFC. Among these genes and metabolites, 23 TFs and phytohormonerelated genes were identified and showed strong correlations with 13 anthocyanin derivates (Table 1). Using Cytoscape 3.10.0,a regulatory network was constructed between these 23 genes and 13 anthocyanin derivatives (Figure 5A). The top 3 TFs associated with the biosynthesis of anthocyanins belonged to AGL9 (MADS-box), SOC1 (MADS-box), MYB (CPC) (Table 1). Furthermore, among the top five compounds, Cyanidin-3-O-arabinoside and Cyanidin-3-O-galactoside emerged as the most significantly different anthocyanin derivatives in Toona sinensis (Table 1). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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This below document has 2 sentences that start with ' Additionally, the', 2 sentences that end with 'in this species'. It has approximately 946 words, 23 sentences, and 5 paragraph(s).
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. This response was also observed at the transcriptional level, with the up-regulation of GPx genes in several tissues of teleost fish under thermal stress, including black porgy (Acanthopagrus schlegeli) [57] and pufferfish [58], as well as in red cusk-eel eggs under thermal stress [23], where gpx1 was increased, an effect not observed for this species in skeletal muscle [24]. Additionally, the effect of high-temperature stress on the liver related to DNA damage was also observed at the transcriptional level, with the up-regulation of genes involved in DNA mismatch repair (msh2 and msh3), concordant with the previously reported effect of thermal stress on zebrafish (Danio rerio) [59] and American lobster [60]. However, the thermal stress response associated with oxidative stress in teleost fish could vary according to species and tissues, as observed in sheepshead minnow (Cyprinodon variegatus), where a limited effect on antioxidant enzymes and no lipid peroxidation were present [61], in contrast to the variable lipid peroxidation observed in Senegalese sole [55]. We previously observed in red cusk-eel that the impact of high-temperature stress on oxidative damage could vary according to tissue, with lipid peroxidation and DNA damage observed for skeletal muscle [24], but no oxidative damage was observed in the ovaries under high-temperature stress [23], showing that the liver is a sensitive organ under thermal stress in red cusk-eel under an ENSO temperature increase scenario.
High-Temperature Stress in Hepatic Protein Processing and Folding
Temperature can modulate several cellular processes through gene expression regulation [2]. Here, we used RNA-seq analysis to evaluate, for the first time, the effect of thermal stress on the liver of red cusk-eel, considering that no studies have previously evaluated the temperature effect on the liver in any species of the Genypterus genus. We observed a higher hepatic transcriptional response to high-temperature stress compared to other types of stressors, such as handling stress, previously observed for red cusk-eel (4.6 times the differentially expressed transcripts) [25], evidencing that a high temperature could have a higher transcriptional impact on the liver than others stressors in this species. At the cellular level, the function of endoplasmic reticulum is key to protein synthesis, folding, and exporting [62]. However, external processes such as thermal stress could generate alterations in homeostasis, affecting normal protein folding, which leads to endoplasmic reticulum (ER) stress. To alleviate this stress, the unfolded protein response molecular mechanism is activated in cells [63]. This ER stress and subsequent unfolded protein response is concordant with our results in the liver, where we observed enriched processes associated with this type of response in the up-regulated genes in the stress group, including protein folding, protein transport, protein localization, protein retention in ER lumen, and unfolded protein binding. In this sense, we found several genes associated with protein processing and folding pathways, including transcripts involved in: protein export, such as signal recognition particle 14 and 19 kDa (srp14 and srp19) and signal peptidase complex subunit 2-like (spcs2) [64]; transcripts associated with protein processing in endoplasmic reticulum (Figure 4), with part of this pathway down-regulated, including TNF receptorassociated factor 2-like (traf2), and mainly an up-regulation of this pathway, including ubiquilin-4 (ubqln4), cytoskeleton-associated protein 4 (ckap4); ribosome-binding protein 1 (rrbp1, also known as p180); and glucosidase 2 subunit beta (prkcsh). Others include transcripts associated with the unfolded protein response, including eukaryotic translation initiation factor 2 alpha kinase 1 (eik2ak1, also known as hri), 3 (eik2ak3, also known as perk) and 5 (eik2ak5), cyclic AMP-dependent transcription factor ATF-4-like (atf4) and DNA damage-inducible transcript 3 -like (ddit4, also known as chop). The unfolded protein response could be initiated by EIK2AK3 kinase activation of eIF2α, leading to ribosome inhibition and attenuating protein synthesis [65]. Additionally, the ATF4 gene was activated, which would lead to DDIT4 gene regulation and an antioxidant response in cells [66], evidencing that thermal stress in the liver of red cusk-eel modulates the protein processing and generates an unfolded protein response that is not able to control the oxidative stress and damage in this tissue. This is concordant with the results observed in previous studies where unfolded protein response genes were activated under thermal stress in mammals [67,68] and fish, e.g., in the liver of Tambaqui (Colossoma macropomum) [69] and gilthead sea bream (Sparus aurata) [70].
Heat Shock Protein as Thermal Stress Biomarkers
The cellular response to thermal stress is a key process to preserve protein integrity; this is known as the heat shock response, which includes heat shock proteins (Hsps) to re-fold the proteins damaged by temperature [71]. This response was observed in the liver of red cusk-eel with the enrichment of the term response to heat (GO:0009408), highlighting several heat shock proteins differentially expressed in response to high-temperature stress, including hsp40, hsp60, hsp70, hsp90, and serpinh1. The up-regulation of these Hsps was previously observed in other fish under thermal stress, with an increase in the liver of three-spined stickleback (Gasterosteus aculeatus) (hsp60, hsp70, and hsp90) [72], Atlantic salmon (Salmo salar) (hsp70) [73,74], Atlantic cod (Gadus morhua) (hsp70 and hsp90) [74], and Wuchang bream (Megalobrama amblycephala) (hsp60, hsp70, and hsp90) [75]. The hsp60 and hsp70 expression levels were also regulated under thermal stress in the postovulatory eggs and skeletal muscle of red cusk-eel [23,24], showing that these genes could be valuable biomarkers of thermal stress in this species. However, the most up-regulated gene in the liver under high-temperature stress was serpinh1 (5.7log 2 Fold Change, Table S2). The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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@@@ User: Continue the text. Add 2 sentences that ends with 'BLAST (Diamond version 2.0.9)software'. Add an introductory paragraph.
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Transcriptome Analysis of Five Toona sinensis Clones
Fifteen cDNA libraries (each clone with three biological replicates) were sequenced using the Illumina HiSeq platform. After removing low-quality reads, each sample yielded between 42.16 M to 52.03 M clean reads (Supplementary Figure S1). The Q30 base exceeded 92.56% and the guanine-cytosine (GC) content of each sample was greater than 42.27%, indicating acceptable sequencing quality. After filtration, the clean data reached 6.32-7.80Gb for each sample. The genome of Toona sinensis var. Heiyouchun was utilized, which marked a significant was ...
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The contents of cyanidin and delphinidin could be mainly responsible for the leaves color of Toona sinensis (Figure 2E).
Transcriptome Analysis of Five Toona sinensis Clones
Fifteen cDNA libraries (each clone with three biological replicates) were sequenced using the Illumina HiSeq platform. After removing low-quality reads, each sample yielded between 42.16 M to 52.03 M clean reads (Supplementary Figure S1). The Q30 base exceeded 92.56% and the guanine-cytosine (GC) content of each sample was greater than 42.27%, indicating acceptable sequencing quality. After filtration, the clean data reached 6.32-7.80Gb for each sample. The genome of Toona sinensis var. Heiyouchun was utilized, which marked a significant was the first chromosome-level genome assembled of Toona, as our reference genome [36]. Furthermore, comparison of all clean reads with this reference genome and subsequently performed gene annotations, the results revealed a high rate of similarity, ranging from 92.26% to 94.03%. These results validate the reliability of our data. Novel genes were defined as unigenes found in the sequencing results but not included in the reference genome (or reference gene set) after reconstructing the transcripts using StringTie v1.3.4dsoftware. Comparison identified 9127 novel genes. The PCA results of transcriptome showed significant differences among five clones (Figure 3A). To investigate the function of unigenes in Toona, we conducted a comparative analy sis with various databases including KEGG, NR, Swiss-Prot, GO, KOG, and Trembl usin BLAST (Diamond version 2.0.9)software. To obtain annotation information on unigene amino acid sequences were derived from the unigene sequences and compared agains the Pfam database using HMMER V3.2 ( [URL] on 10 June 2022) sof ware. Interestingly, when compared to data in the NR database, it was observed that gen sequences from Toona sinensis exhibited a greater degree in similarity to Citrus clementin To investigate the function of unigenes in Toona, we conducted a comparative analysis with various databases including KEGG, NR, Swiss-Prot, GO, KOG, and Trembl using BLAST (Diamond version 2.0.9)software. To obtain annotation information on unigenes, amino acid sequences were derived from the unigene sequences and compared against the Pfam database using HMMER V3.2 ( [URL] on 10 June 2022) software. Interestingly, when compared to data in the NR database, it was observed that gene sequences from Toona sinensis exhibited a greater degree in similarity to Citrus clementina (30.91%), followed by Citrus sinensis (30.47%) as depicted in Supplementary Figure S1B. The function terms of unigenes were annotated in the KOG and GO database, and the function groups were showed in Supplementary Figure S1C,D.
Based on the reference transcriptome, 40,421 genes were assembled from clean reads, which included 31,647 genes annotation in reference genome and 9127 novel transcripts. In addition, 23,909, 16,532, 25,617, 15,566, 32,348, and 25,575 unigenes were annotated by KEGG, NR, SWISSPORT, THEM, GO, and KOG database. There were 14,153 unigenes annotated by all of the databases. A comparison with six public databases (KEGG, NR, SwissProt, Tremble, GO and KOG) annotated a total of 13,295 differentially expressed genes (DEGs) and predicated their potential functions. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. According to DESeq, genes were considered substantially differentially expressed if their adjusted p-value was less than 0.05. We used the top GO R package v.1, which is based on the Kolmogorov-Smirnov test, to carry out GO enrichment analysis of the differentially expressed genes. We performed pathway analysis utilizing the KEGG database ( [URL] on 10 June 2022) to investigate relevant pathways of substantially differentially expressed genes [56][57][58].
Correlation Analysis of Transcriptome and Metabolome
To integrate transcriptome and metabolome datasets, Pearson correlation coefficients were utilized. Gene-metabolite coefficients were obtained by calculating the average expression levels of transcripts and metabolite contents. The fold changes for both transcriptome and metabolome data in each group were also computed. A correlation was considered significant if the Pearson correlation coefficients exceed 0.8 and p-values were less than 0.05 (Table 1). Significant positive correlations between transcription factors (TFs) and anthocyanin derivatives in groups JFC and LFC were detected and visually presented utilizing Cytoscape 3.10.0.
Verification of RNA-Seq Data by qRT-PCR
Total RNA was extracted from Toona sinesis leaves and reverse-transcribed using the Quantscript Reverse Transcriptase Kit. Each clone had three biological replicates, and each sample had three technical replicates. The obtained cDNA served as a template for determining gene expression levels, employing specific primers for genes linked to anthocyanin biosynthesis as well as ACTIN gene (used as an internal control). The primers used in the qRT-PCR analysis were shown in Table 2. The melt curve of ACTIN in the qPCR products was presented in Supplementary Figure S3. The reaction system contained 5 µL 2 × Q3 SYBR qPCR Master Mix-Universal (TOLOBIO), 1 µL cDNA template, 0.5 µL of each forward and reverse primer, and 3 µL of RNase-free water.qRT-PCR was performed using Applied Biosystems 7500 Fast Real-Time PCR System.
Conclusions
In this study, an integrative analysis of the transcriptome and metabolome of five distinct Toona sinensis clones were performed to explore the related genes and metabolites associated with the anthocyanin biosynthesis. Furthermore, our analyses found that the red leaves 'JFC' contained the highest content of stable cyanidin, delphinidin, and peonidin, especially Cyanidin-3-O-arabinoside. The integrated analysis also identified the major transcription factor SOC1, CPC, and bHLH162. Moreover, the regulatory network construction of TFs, metabolic, structural genes reveals the underlying mechanisms of the anthocyanin biosynthesis pathway in Toona sinensis. Overall, this study provides a foundation for future research aimed at manipulating anthocyanin biosynthesis to improve plant coloration or to derive human health benefits.
Figure 1 .
Figure 1. Morphological observation and anthocyanin content of leaves from different Toona clones (JFC, FHXN, BSH, HBG, and LYC).(A) The morphological characteristics of leaves from the five Toona clones.(B) The color of anthocyanin present in the leaves of these clones.(C) The quantification of anthocyanin content in each of the Toona clones. Data are shown as mean ± standard deviation, n = 3. Bars with different letters are significantly different at p < 0.05.
Figure 1 .
Figure 1. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. chilensis showed a variable tissue response under handling stress, with altered metabolic status in the liver, a modulation of the immune response in the head kidney, and an induction of atrophy in skeletal muscle through coding and noncoding regulation [26]. Additionally, it has been observed that thermal stress could induce muscle atrophy in this species [24], as well as oxidative damage in eggs, with a minor effect on the ovary [23]. One of the most important organs for fish metabolism is the liver, which is directly involved in stress response by metabolizing and liberating stored energy to respond to stress [17]. Nevertheless, the liver response to thermal stress in Genypterus species has not been previously studied, nor has the impact of this stressor on the oxidative status of this tissue. It is important to consider that sea temperatures will increase through sea heat waves due to the effect of ENSO under a climate change scenario, which will affect the Chilean coast associated with the geographic range of G. chilensis. Therefore, the objective of this study was to evaluate the effect of high-temperature stress on the liver of G. chilensis in terms of the transcriptomic and oxidative stress status to determine the negative impact of this type of stressor on liver.
Ethics Statement
All procedures with the red cusk-eel individuals and all scientific activities adhered to animal welfare procedures and were approved by the bioethical committees of the Universidad Andres Bello (007/2018) and the National Commission for Scientific and Technological Research (CONICYT) of the Chilean government.
Fish Sampling and Experimental Design
In this study, we used reproductively immature red cusk-eel juveniles (G. chilensis) of 12 months of age (average weight of 665 ± 52.7 g; average length of 60 ± 4.8 cm), collected from the Centro de Investigación Marina de Quintay (CIMARQ), maintained under natural photoperiod conditions (L:D 12:12), and controlled temperature (14 ± 1 • C), and fed daily with commercial pellet food. Fish were separated into control and stress groups, with the stress group subjected to a standardized thermal stress protocol proven to generate stress in red cusk-eel [24]. Briefly, this protocol consists of increasing the temperature over 24 h at a rate of 1 • C in 5 h. This protocol maintains the thermal stress temperature (19 ± 1 • C) for 5 days. This high temperature protocol was selected considering heat waves observed in the summer season on the Chilean coast in recent years [27]. The control group was maintained at the control temperature (14 ± 1 • C) during the assay. At the end of the experiment, six individuals per group (two tanks per group, with three animals sampled per tank, total of N = 12 sampled fish) were netted and sampled. Blood samples were collected via caudal puncture using heparinized tubes, immediately centrifuged at 5000× g for 10 min for serum obtention and stored at −80 • C until analysis. After blood sampling, fish were euthanized (overdose of anesthetic 3-aminobenzoic acid ethyl ester, 300 mg/L). Fish livers were collected and stored for RNA extraction in RNAsave solution (Biological Industries, Cromwell, CT, USA) or immediately frozen in liquid nitrogen and stored at −80 • C until analysis for oxidative damage evaluation.
AST, ALT and AP Evaluation
The plasmatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AP) were determined using commercially available kits from Valtek (Santiago, Chile) following the manufacturer's instruction. Briefly, these kits determined the enzymatic activity (IU/L) via the generation of colorimetric products from glutamate (colorimetric product: 450 nm), pyruvate (535 nm), and p-nitrophenol (405 nm) for AST, ALT and AP activity, respectively.
Oxidative Stress Assays in Liver
To determine the oxidative damage in the liver of red cusk-eel in response to hightemperature stress, DNA oxidative damage, protein carbonylation, and lipid peroxidation were evaluated using commercially available kits. The DNA oxidative damage assay was performed with genomic DNA (gDNA) purified from 25 mg of the liver using the DNAzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol and quantified with the Epoch Spectrophotometer System (BioTek, Winooski, VT, USA). The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The PCA results of transcriptome showed significant differences among five clones (Figure 3A). To investigate the function of unigenes in Toona, we conducted a comparative analy sis with various databases including KEGG, NR, Swiss-Prot, GO, KOG, and Trembl usin BLAST (Diamond version 2.0.9)software. To obtain annotation information on unigene amino acid sequences were derived from the unigene sequences and compared agains the Pfam database using HMMER V3.2 ( [URL] on 10 June 2022) sof ware. Interestingly, when compared to data in the NR database, it was observed that gen sequences from Toona sinensis exhibited a greater degree in similarity to Citrus clementin To investigate the function of unigenes in Toona, we conducted a comparative analysis with various databases including KEGG, NR, Swiss-Prot, GO, KOG, and Trembl using BLAST (Diamond version 2.0.9)software. To obtain annotation information on unigenes, amino acid sequences were derived from the unigene sequences and compared against the Pfam database using HMMER V3.2 ( [URL] on 10 June 2022) software. Interestingly, when compared to data in the NR database, it was observed that gene sequences from Toona sinensis exhibited a greater degree in similarity to Citrus clementina (30.91%), followed by Citrus sinensis (30.47%) as depicted in Supplementary Figure S1B. The function terms of unigenes were annotated in the KOG and GO database, and the function groups were showed in Supplementary Figure S1C,D.
Based on the reference transcriptome, 40,421 genes were assembled from clean reads, which included 31,647 genes annotation in reference genome and 9127 novel transcripts. In addition, 23,909, 16,532, 25,617, 15,566, 32,348, and 25,575 unigenes were annotated by KEGG, NR, SWISSPORT, THEM, GO, and KOG database. There were 14,153 unigenes annotated by all of the databases. A comparison with six public databases (KEGG, NR, SwissProt, Tremble, GO and KOG) annotated a total of 13,295 differentially expressed genes (DEGs) and predicated their potential functions. The DEGs were counted with an absolute |log2fold-change| ≥ 1 and a false discovery rate (FDR) ≤ 0.05 in each compared groups. According to five clones of Toona leaves, 10 pairs of comparisons were performed and the number of up-regulated and down-regulated differential genes was counted in all groups (Figure 3B). Across all compared groups, a total of 310 DEGs were co-detected (Figure 3C). The KEGG-based enrichment analysis revealed that all DEGs compared to LFC were enriched into 140 KEGG pathways. These pathways included flavonoid biosynthesis, phenylpropanoid biosynthesis, and anthocyanin biosynthesis, all of which were significantly enriched (Figure 3D). These terms have previously been associated with anthocyanin production.
Analysis of Anthocyanidin Biosynthetic Pathway Genes and Anthocyanin Derivates in JFC, BSH, and LFC
According to transcriptome and metabolites studies, anthocyanin biosynthesis may be responsible for the distinct red leaf colorations of Toona. Therefore, it is necessary to investigate the mechanisms that underlie this process. Genes encoding anthocyanin biosynthetic enzymes were found to be associated with different compositions of the compounds in the anthocyanin biosynthesis pathways. We constructed a pathway diagram displaying the expression heat map of structural genes and compounds for red, light-red, and green leaves (Figure 4). The PAL gene catalyzed the conversion of L-Phenylalanine to cinnamic acid and was highly expressed in JFC. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Its tender young buds exhibit a diverse range of colors, primarily determined by the presence and composition of anthocyanins and flavonoids. However, the lying mechanisms of anthocyanin biosynthesis in Toona sinensis have been rarely reported. To explore the related genes and metabolites associated with composition of leaf color, we conducted an analysis of the transcriptome and metabolome of five distinct Toona clones. The results showed that differentially showed genes and metabolites implied in anthocyanin biosynthesis pathway were mainly enriched. A conjoint analysis of transcripts and metabolites was carried out in JFC (red) and LFC (green), resulting in the recognition of 510 genes and 23 anthocyanin-related metabolites with a positive correlation coefficient greater than 0.8. Among these genes and metabolites, 23 transcription factors and phytohormone-related genes showed strong coefficients with 13 anthocyanin derivates, which mainly belonged to the stable types of delphinidin, cyanidin, peonidin. The core derivative was found to be Cyanidin-3-O-arabinoside, which was present in JFC at 520.93 times the abundance compared to LFC. Additionally, the regulatory network and relative expression levels of genes revealed that the structural genes DFR, ANS, and UFGT1 might be directly or indirectly regulated by the transcription factors SOC1 (MADS-box), CPC (MYB), and bHLH162 (bHLH) to control the accumulation of anthocyanin. The expression of these genes was significantly higher in red clones compared to green clones. Furthermore, RNA-seq results accurately reflected the true expression levels of genes. Overall, this study provides a foundation for future research aimed at manipulating anthocyanin biosynthesis to improve plant coloration or to derive human health benefits.
Introduction
As the national economy grows rapidly, and living standards continue to improve, consumers today demand more than just food and clothing. They desire a healthy diet that also helps prevent diseases. Food that is high in nutrition and has antioxidant properties is particularly appealing [1]. Chinese Toon (Toona sinensis), a tree with both medicinal and edible uses, contains rich chemical substances with functions such as antifungal, antiglycation, or anti-tumor activities. Flavonoid compounds, especially anthocyanin, have been identified as the major bioactive constituents in Toona [2][3][4] which are considered to be of the most beneficial plant-derived antioxidants and have led to increased efforts to analyze the genetic controls of the mechanism of flavonoids synthesis [5,6].
Anthocyanins belong to the flavonoid class of phenolic compounds. Anthocyanins are water-soluble pigments present in plants that contribute to the colors of leaves, flowers, and fruit, which are also crucial for plant growth and beneficial for human health due to their ability to scavenge active oxygens [7]. More than 600 distinct anthocyanin types have been identified so far through previous research [8]. The most prevalent anthocyanin pigments, which generate purple, blue, and red colors, are cyanidin (Cy), delphinidin (Dp), pelargonidin (Pg), peonidin (Pn), petunidin (Pt), and malvidin (Mv) [9]. Anthocyanin biosynthesis and regulation is a complex process influenced by various internal and external factors [10,11]. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>This below document has
* 2 sentences that start with 'qPCR validation of selected differentially',
* 2 sentences that start with 'chilensis in response to high-temperature',
* 2 sentences that start with 'The transcript expression levels were',
* 2 sentences that start with 'The differential expression levels according',
* 2 sentences that end with 'in the liver of G',
* 2 sentences that end with 'to high-temperature stress',
* 2 sentences that end with 'means of actb and taf12'. It has approximately 536 words, 24 sentences, and 4 paragraph(s).
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. In the KEGG pathway analysis, we identified several pathways represented in the down-regulated and up-regulated transcripts, including apoptosis, cell cycle, MAPK signaling pathway, autophagy, protein processing in endoplasmic reticulum, and ubiquitinmediated proteolysis. Among these pathways, protein processing in endoplasmic reticulum was one of the most represented on the differentially expressed transcripts (Figure 4), with 31 up-regulated and three down-regulated transcripts represented in this pathway, including several heat shock proteins; heat shock protein 40 (hsp40, also known as dnaJ homolog subfamily B member 1), heat shock protein 70 (hsp70) and heat shock protein 90 (hsp90).
(casp3) and complement C3-like (c3) ( Table 1). All of the selected differentially expressed transcripts on RNA-seq were validated by qPCR, confirming the results of the RNA-seq analysis ( Figure 5), with a Pearson's correlation coefficient of r = 0.847, confirming the significant differential expression of all tested transcripts by qPCR between the control and high-temperature group ( Figure S3). Figure 5. qPCR validation of selected differentially expressed transcripts in the liver of G. chilensis in response to high-temperature stress. The transcript expression levels were normalized with the geometric means of actb and taf12. The differential expression levels according to qPCR (black bars) and RNA-seq (gray bars) for these selected genes are expressed as log2 fold changes. The log2FC represents the expression change in the stress group compared with the control group. Results are expressed as the mean ± standard error. Significant differences in the validated qPCR data between control and stress groups are indicated by asterisks in the log2 Fold qPCR bars; (* pvalue < 0.05).
Discussion
Stress in fish is a relevant issue for aquaculture species and native populations of fish [15]. The environmental stress factor associated with water parameters represents a key issue in fish, especially considering the variations in the environment in the shortand long-term associated with global warming, climate change, ENSO, and pollution of the oceans, which could lead to modification of sea temperature, pH, and DO level, as well as increased levels of pollutants, including microplastics and toxic compounds, which could generate stress in teleost fish [7,8,[34][35][36]. These environmental stressors could also affect the red cusk-eel, a relevant species for Chilean fisheries and aquaculture diversification. However, studies aimed at understanding the effect of stressors in Genypterus species are limited [25,26,37,38], with no information about the impact of thermal stress on the liver for Genypterus species. In this sense, thermal stress has been previously studied in other tissues of Genypterus species, including the skeletal muscle, ovary, and post-ovulatory eggs [23,24]. Therefore, to understand the effect of high- Figure 5. qPCR validation of selected differentially expressed transcripts in the liver of G. chilensis in response to high-temperature stress. The transcript expression levels were normalized with the geometric means of actb and taf12. The differential expression levels according to qPCR (black bars) and RNA-seq (gray bars) for these selected genes are expressed as log 2 fold changes. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in colored leaves of different clones compared to green leaves revealed significant enrichment of genes involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and the anthocyanin biosynthesis pathway. This finding supports previous studies demonstrating the involvement of multiple enzymes encoded by early biosynthesis genes and anthocyanin biosynthesis genes in anthocyanin biosynthesis. The study indicates that the biosynthesis of naringenin is a crucial step in the flavonoids metabolic pathway and plays a decisive role in the synthesis of anthocyanin compounds. The PAL, C4H, 4CL, CHS, and CHI are key genes in the flavonoids metabolic pathway and play a decisive role in determining naringenin production. The study found that JFC had a higher naringenin content than BSH and LFC, but only two 4CL genes (Maker00007314, novel.5717)were expressed at high levels in JFC and low levels in LFC. The expression of 4CL genes was highly positively correlated with naringenin content, indicating that the number and function of genes in the naringenin pathway may affect naringenin content. During the biosynthesis of anthocyanins, DFR catalyzes the reduction reaction of flavonoid-3 ,5 -hydroxylase to form anthocyanins, which was confirmed as a key step for regulating anthocyanin types. ANS converts anthocyanin precursors to anthocyanin glycosides. UFGT can catalyze the conversion of unstable anthocyanidins into stable anthocyanins. The absence of these genes directly affects anthocyanins biosynthesis, leading to pigment loss [38,39]. Down-regulation of the apple DFR gene can inhibit both cyanidin and procyanidin accumulation. In Vitis vinifera, the silencing of the DFR gene leads to the absence of anthocyanins [40]. In Duchesnea indica, a plant belonging to the Rosaceae family, decreased expression of the ANS gene results in white-colored fruits [41]. Conversely, overexpressing the SmANS gene in Salvia miltiorrhiza can increase anthocyanin content but decrease the biosynthesis of salvianolic acid [42]. In our study, we found significantly increased expression of the DFR gene (Marker00011618), ANS gene (Marker00000819), UFGT (Marker 00029333 and novel.8170) in JFC and BSH compared to LFC, which may explain the red color leaves in JFC and the green color leaves in LFC. Difference in gene sequence and promoter regions of these structural genes may be the primary factors contributing to difference in gene function and expression, ultimately impacting anthocyanin biosynthesis in different clones.
The MBW protein complex was proven to activate the expression of structural genes involved in anthocyanin production. In addition to the MBW complex, other TFs such as bZIP [32,43], NAC [44], WRKY [45,46], MADS-box [47,48], and zinc finger [49,50] proteins have also shown to regulate anthocyanin biosynthesis in plants. In Toona, we found that MADS-box, MYB, and C2H2 possessed top five TFs related to anthocyanin derivates according to the joint analysis between transcriptome and metabolome. The gene-gene regulatory network also showed that the SOC1, CPC, and bHLH162 were the major TFs regulating the anthocyanin biosynthesis structural genes. SOC1 encodes a MADS box transcription factor and is involved in the regulation of flowering in response to temperature or light in various plants species, such as Arabidopsis [51], Oryza sativa L. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Consequently, a regulatory network was constructed between these top transcription factors and structural regulatory genes involved in anthocyanin biosynthesis (Figure 5B). These findings suggest that MADS-box, MYB, and bHLH transcription factors may regulate DFR, ANS, UFGT, and other structural genes involved in the biosynthesis of anthocyanin in Toona sinensis by either affecting or participating in the regulation of structural gene.
Int. J. Mol. Sci.2023, 24, x FOR PEER REVIEW 9 of 22 phytohormone-related genes were identified and showed strong correlations with 13 anthocyanin derivates (Table 1). Using Cytoscape 3.10.0,a regulatory network was constructed between these 23 genes and 13 anthocyanin derivatives (Figure 5A). The top 3 TFs associated with the biosynthesis of anthocyanins belonged to AGL9 (MADS-box), SOC1 (MADS-box), MYB (CPC) (Table 1). Furthermore, among the top five compounds, Cyanidin-3-O-arabinoside and Cyanidin-3-O-galactoside emerged as the most significantly different anthocyanin derivatives in Toona sinensis (Table 1). Consequently, a regulatory network was constructed between these top transcription factors and structural regulatory genes involved in anthocyanin biosynthesis (Figure 5B). These findings suggest that MADS-box, MYB, and bHLH transcription factors may regulate DFR, ANS, UFGT, and other structural genes involved in the biosynthesis of anthocyanin in Toona sinensis by either affecting or participating in the regulation of structural gene.
Expression Profiles of TFs and Plant Hormone Genes Related to Anthocyanin Biosynthesis
Previous studies have demonstrated that the biosynthesis of anthocyanins requires the involvement of transcription factors, such as MADS-box, MYB, bHLH, and bZIP. In this study, we identified a total of 11 MADS-box genes, 33 MYBs, 42 bHLHs, 36 bZIPs, and 15 WD40 genes in Toona. The RNA expression levels in JFC, BSH, and LFC was compared, which revealed that 12 bHLH, 10 bZIP, 12 MYB, 2 MADS-box, and 1 WD40 were significantly highly expressed in JFC, lowest in LFC (Figure 6A). Furthermore, the heatmap showed that all highly correlated TFs with anthocyanin derivates belonged to these groups.
Expression Profiles of TFs and Plant Hormone Genes Related to Anthocyanin Biosynthesis
Previous studies have demonstrated that the biosynthesis of anthocyanins requires the involvement of transcription factors, such as MADS-box, MYB, bHLH, and bZIP. In this study, we identified a total of 11 MADS-box genes, 33 MYBs, 42 bHLHs, 36 bZIPs, and 15 WD40 genes in Toona. The RNA expression levels in JFC, BSH, and LFC was compared, which revealed that 12 bHLH, 10 bZIP, 12 MYB, 2 MADS-box, and 1 WD40 were significantly highly expressed in JFC, lowest in LFC (Figure 6A). Furthermore, the heatmap showed that all highly correlated TFs with anthocyanin derivates belonged to these groups. Studies suggest that plant hormones have a crucial function in regulating anthocyanin concentrations in leaves and fruits via direct or indirect regulation of transcription factors responsible for anthocyanin biosynthesis. The hormones capable of activating or inhibiting the structural genes involved in the biosynthesis pathway include auxin, gibberellins, cytokinin, brassinosteroids, jasmonic acid, salicylic acid, and abscisic acid. Furthermore, we identified several differentially expressed genes that participate in plant Studies suggest that plant hormones have a crucial function in regulating anthocyanin concentrations in leaves and fruits via direct or indirect regulation of transcription factors responsible for anthocyanin biosynthesis. The hormones capable of activating or inhibiting the structural genes involved in the biosynthesis pathway include auxin, gibberellins, cytokinin, brassinosteroids, jasmonic acid, salicylic acid, and abscisic acid. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. [52], Gossypium hissytum [53]. In Toona sinensis, the young leaf color is highly sensitive to temperature, indicating a critical role of SOC1 in temperature or light-induced anthocyanin biosynthesis. These transcription factors might regulate anthocyanin biosynthesis process by directly active or repress gene expression by binding to the promoter regions, or indirectly though protein-protein interactions in model plants and other fruits.
Plant hormones can affect anthocyanin biosynthesis through various mechanisms. Previous studies have reported that overexpression of MdIAA26 in apple calli and Arabidopsis promotes the accumulation of anthocyanin, while auxin inhibit it by degrading the MdIAA26 protein [54]. Exogenous ethylene treatment increased anthocyanin accumulation in grape skins and induced the expression of structural genes (VvPAL, Vv4CH, VvCHS, VvCHI, VvF3H, and VvUFGT) and regulatory genes (VvMYBA1, VvMYBA2, and VvMYBA3) related to anthocyanin biosynthesis [55]. Our study identified nine hormone-related genes with a high correlation to regulate anthocyanins biosynthesis, which are highly expressed in JFC and play a significant role in the controlling leaf color variation in Toona. Leaf color is an important trait that is susceptible to both endogenous and exogenous influences. Variations in hormone signal transduction gene expression reflect the essential mechanisms by which plant hormones affect the anthocyanin biosynthesis pathway. These findings enhance our understanding of how anthocyanin biosynthesis works in Toona and how it can be controlled, with implications for breeding new Toona cultivars. Identifying and understanding the beneficial metabolic constituents and regulatory networks of Toona can facilitate the development and utilization of anthocyanin-enriched varieties.
Plant Materials
The study selected five Chinese toon clones, JinFuChun (JFC), BaShanHong (BSH), LvFuChun (LFC), FenHongXinNiang (FHXN), and HeBeiGu (HBG), which cultivated in Research Institution of Subtropical Forestry, Chinese Academy of Forestry. The colors of these varieties are: brilliant-red buds (JFC), pink buds (FHXN), light-red with a little green buds (BSH), dark-purple buds (HBG), and green buds (LFC). Fresh buds from five different clones were collected, with three replicates taken for each clone, each replicate sourced from different trees. Buds measuring 10-15 cm in length and weighing approximately 5-10 g were collected for each sample and subsequently underwent freeze-drying for RNA-seq and metabolites measures.
Sample Preparation and Extraction
The sample underwent freeze-drying, followed by grinding into a powder (30 Hz, 1.5 min), and subsequently, it was stored at −80 • C until required. Subsequently, 50 mg of the powder was precisely weighed and subjected to extraction using 0.5 mL of methanol/ water/hydrochloric acid (500:500:1, v/v/v). The resulting extract was vigorously vortexed for 5 min, followed by ultrasonication for another 5 min, and then centrifuged at 12,000× g at 4 • C for 3 min. The residue underwent a repeat extraction using the same procedure and conditions. The supernatants obtained were collected and filtered through a 0.22 µm membrane filter (Anpel) prior to LC-MS/MS analysis.
ESI-MS/MS Conditions
Linear ion trap (LIT) and triple quadrupole (QQQ) scans were obtained using a triple quadrupole-linear ion trap mass spectrometer, the AB Scoex Qtrap ® 6500+ LC-MS/MS System (Sciex, Framingham, MA, USA), equipped with an ESI Turbo Ion-Spray interface. The instrument operated in positive ion mode and was controlled by Analyst 1.6.3software (Sciex). The ESI source operated with the following parameters: Ion Source: ESI+, Source Temperature: 550 • C, Ion Spray Voltage (IS): 5500 V, Curtain Gas (CUR): Set at 35 psi. Anthocyanins were subjected to analysis using scheduled multiple reaction monitoring (MRM). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Overall, this study provides a foundation for future research aimed at manipulating anthocyanin biosynthesis to improve plant coloration or to derive human health benefits.
Introduction
As the national economy grows rapidly, and living standards continue to improve, consumers today demand more than just food and clothing. They desire a healthy diet that also helps prevent diseases. Food that is high in nutrition and has antioxidant properties is particularly appealing [1]. Chinese Toon (Toona sinensis), a tree with both medicinal and edible uses, contains rich chemical substances with functions such as antifungal, antiglycation, or anti-tumor activities. Flavonoid compounds, especially anthocyanin, have been identified as the major bioactive constituents in Toona [2][3][4] which are considered to be of the most beneficial plant-derived antioxidants and have led to increased efforts to analyze the genetic controls of the mechanism of flavonoids synthesis [5,6].
Anthocyanins belong to the flavonoid class of phenolic compounds. Anthocyanins are water-soluble pigments present in plants that contribute to the colors of leaves, flowers, and fruit, which are also crucial for plant growth and beneficial for human health due to their ability to scavenge active oxygens [7]. More than 600 distinct anthocyanin types have been identified so far through previous research [8]. The most prevalent anthocyanin pigments, which generate purple, blue, and red colors, are cyanidin (Cy), delphinidin (Dp), pelargonidin (Pg), peonidin (Pn), petunidin (Pt), and malvidin (Mv) [9]. Anthocyanin biosynthesis and regulation is a complex process influenced by various internal and external factors [10,11].
The investigation of anthocyanin-regulating genes and associated flavonoid metabolites is critical to elucidate the mechanism that governs the biological synthesis of anthocyanins. To study gene expression and metabolic changes in different cultivars, researchers mainly rely on transcriptomic and metabolic profiling, which complement each other [34,35]. In this study, we conducted a dual-level exploration of genes and metabolites that control the pigmentation of Toona sinensis leaves using transcriptome and metabolomics approaches. Specifically, we analyzed the transcriptome and metabolome data of five Toona sinensis clones exhibiting a range of leaf colors, including red, light red, pink, dark, and green, to clarify regulatory networks for biosynthesis-related genes and key metabolomes. Overall, our findings unveil the regulatory mechanisms driving the coloration of Toona sinensis leaves.
Morphological Observation and Anthocyanin Content of Leaves in Toona sinensis
Five clones of Toona sinensis with distinct leaf colors were selected to analyze the regulatory mechanism of anthocyanin biosynthesis. Among the clones, JFC Toona is characterized by brilliant-red leaves and is commonly cultivated in Zhejiang province. The leaf color of other clones was pink buds (FHXN), light-red with a little green buds (BSH), dark-purple buds (HBG), and green buds (LFC) (Figure 1A). Then, the total anthocyanin was extracted and the resulting color were displayed in Figure 1B, which reflect the anthocyanin content of different clones. Total anthocyanin present in the leaves were quantified, with JFC Toona sinensis found to have the highest concentration of anthocyanin, followed by BSH, FHXN and HBG, and LFC (Figure 1C). Obviously, LFC exhibited green leaves due to a lack of anthocyanin accumulation.
The leaf color of other clones was pink buds (FHXN), light-red with a little green buds (BSH), dark-purple buds (HBG), and green buds (LFC) (Figure 1A). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. In terms of high-temperature stress, different results have been observed, with an increase in the plasmatic activity of AST and ALT in pufferfish (Takifugu obscurus) [44], which is consistent with our results for red cusk-eel, while no effect for AST and ALT was observed in Turbot (Scophthalmus maximus) [45] under high-temperature stress, evidencing that the hepatic response to thermal stress could vary according to fish species. The increased levels on ALT, AST an AP enzymes could be indicative of liver dysfunction, reflecting hepatocyte damage due to thermal stress, which is concordant with the results observed in mammals [46].
Oxidative Stress under High-Temperature Stress
Oxidative stress corresponds to a disturbance between the production of ROS, which can accumulate in cells, and the antioxidant defenses generated by the cellular systems to detoxify these ROS [47]. OS participate in different normal cellular functions, acting as a second messenger in signal transduction [48], but they can also generate cellular damage, including oxidative damage generating lipid peroxidation, as well as damage to proteins and DNA [49]. Environmental stress, including thermal stress, can lead to oxidative stress status in marine animals [3]. It was previously observed that thermal stress could modulate the oxidative stress status in several tissues of teleost fish [50,51]. One of the relevant organs in which oxidative status could be affected by thermal stress is the liver, as it was previously observed that thermal stress by low temperature could increase antioxidant enzymes in milkfish (Chanos Chanos) [52], as well as generate oxidative damage, leading to lipid peroxidation in Pacu (Piaractus mesopotamicus) under low-temperature stress [53,54]. High-temperature stress could also generate a relevant impact on the oxidative status of fish, with increased antioxidant enzyme activities, as observed for Senegalese sole (Solea senegalensis) [55]. Additionally, oxidative damage was observed in the liver of rohu (Labeo rohita), with lipid peroxidation and DNA fragmentation [56]. This is consistent with the findings of our study, evidencing that thermal stress generates an important oxidative effect on the liver of red cusk-eel. This response was also observed at the transcriptional level, with the up-regulation of GPx genes in several tissues of teleost fish under thermal stress, including black porgy (Acanthopagrus schlegeli) [57] and pufferfish [58], as well as in red cusk-eel eggs under thermal stress [23], where gpx1 was increased, an effect not observed for this species in skeletal muscle [24]. Additionally, the effect of high-temperature stress on the liver related to DNA damage was also observed at the transcriptional level, with the up-regulation of genes involved in DNA mismatch repair (msh2 and msh3), concordant with the previously reported effect of thermal stress on zebrafish (Danio rerio) [59] and American lobster [60]. However, the thermal stress response associated with oxidative stress in teleost fish could vary according to species and tissues, as observed in sheepshead minnow (Cyprinodon variegatus), where a limited effect on antioxidant enzymes and no lipid peroxidation were present [61], in contrast to the variable lipid peroxidation observed in Senegalese sole [55]. We previously observed in red cusk-eel that the impact of high-temperature stress on oxidative damage could vary according to tissue, with lipid peroxidation and DNA damage observed for skeletal muscle [24], but no oxidative damage was observed in the ovaries under high-temperature stress [23], showing that the liver is a sensitive organ under thermal stress in red cusk-eel under an ENSO temperature increase scenario.
High-Temperature Stress in Hepatic Protein Processing and Folding
Temperature can modulate several cellular processes through gene expression regulation [2]. Here, we used RNA-seq analysis to evaluate, for the first time, the effect of thermal stress on the liver of red cusk-eel, considering that no studies have previously evaluated the temperature effect on the liver in any species of the Genypterus genus. We observed a higher hepatic transcriptional response to high-temperature stress compared to other types of stressors, such as handling stress, previously observed for red cusk-eel (4.6 times the differentially expressed transcripts) [25], evidencing that a high temperature could have a higher transcriptional impact on the liver than others stressors in this species. At the cellular level, the function of endoplasmic reticulum is key to protein synthesis, folding, and exporting [62]. However, external processes such as thermal stress could generate alterations in homeostasis, affecting normal protein folding, which leads to endoplasmic reticulum (ER) stress. To alleviate this stress, the unfolded protein response molecular mechanism is activated in cells [63]. This ER stress and subsequent unfolded protein response is concordant with our results in the liver, where we observed enriched processes associated with this type of response in the up-regulated genes in the stress group, including protein folding, protein transport, protein localization, protein retention in ER lumen, and unfolded protein binding. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Purple circles represent transcription factors, light-purple triangle represent plant hormones related genes, green round rectangle represent the metabolites. The size of the cells represented the log2FC (gene or compounds).(B) Regulatory network between transcription factors and structural genes responsible for anthocyanin biosynthesis. Purple circles represent transcription factors, light-purple circles represent structural genes involved in the biosynthesis of anthocyanin pathway.
Figure 7 .
Figure 7. Quantitative real-time RT-PCR (qRT-PCR) and RNA-seq analysis of genes involved in anthocyanin biosynthesis pathway and putative transcription regulators.(A) Relative expression level of transcription factors.(B) Relative expression level of structural genes. Data are shown as mean ± standard deviation of three biological replicates.
Figure 7 .
Figure 7. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. Paired-end sequencing (2 × 100 bp) was performed on a Hiseq 2500 (Illumina ® ) platform in Macrogen Inc. (Seul, South Korea).
Reads Filtering, Differential Expression, and GO Enrichment Analysis
The raw reads obtained from Illumina sequencing were trimmed to remove the remaining Illumina adapter, low-quality sequences, and short sequences (<50 bp), using the CLC Genomics Workbench v.7.0.3 software. The filtered reads were mapped to a G. chilensis reference transcriptome previously annotated by our group ( [28], NCBI accession number SRS614525) using the CLC Genomics Workbench v.7.0.3 software with the following parameters: mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.8, and a similarity fraction = 0.8. The expression values were used for clustering and heatmap chart generation with R. Differential expression analysis was performed with the R package DESeq2 (version 1.2.10) [29] to determine differentially expressed transcripts in the liver between the control and stress groups. Transcripts presenting an adjusted p-value of <0.05 and an absolute log2 fold change of >1 were considered as differentially expressed between the groups. Enrichment analysis was performed on the list of differentially expressed transcripts and GO terms to determine the overrepresented processes in response to high-temperature stress in the liver, considering up and down-expressed transcripts. This analysis was performed using the enrichment analysis tool implemented in the Blast2GO software [30]. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database was used to build the represented pathways through the KEGG Automatic Annotation Server (KAAS) [31] using the KEGG Orthology (KO) identifiers of the differentially expressed list.
RNA-seq Validation by qPCR
Total RNA previously extracted was DNAse-treated to remove residual gDNA, and 1 µg of RNA was reverse transcribed into cDNA using the QuantiTect ® Reverse Transcription kit (Qiagen, Germantown, MD, USA), following the manufacturer's protocol. A total of thirteen differentially expressed transcripts were selected for qPCR validation, corresponding to: hsp60, hsp70, gpx7, ddit4, leptin, msh2, msh3, c1ql1, ccl20, atg12, atg4b, casp3 and c3. Primers were designed with Primer 3 software v0.4.0 ( [URL]3/, accessed on 6 May 2021) using the reference transcriptome previously described. The primer sequences, amplicon size, Tm, and efficiency are presented in Table 1. The qPCR was performed in a Stratagene MX3000P qPCR system (Stratagene, La Jolla, CA, USA). All qPCR assays were performed in triplicates, using no-template and no-RT controls, in compliance with the MIQE guidelines [32]. The qPCR reaction mixture contained 7.5 µL of 2× Brilliant ® II SYBR ® Green master mix (Agilent Technologies, CA, USA), 100 ng of cDNA per reaction and 200 nM of each primer in a 15 µL final volume. Thermal cycling conditions were an initial activation of 2 min at 95 • C, followed by 40 cycles of 30 s at 95 • C, 30 s at 62 • C, and 30 s at 72 • C. A melt curve analysis was performed to confirm a single qPCR product and a standard curve using two-fold series dilutions was used to estimate the efficiency of each primer set. The expression of target genes was normalized using the geometric means of two reference genes (actb and taf12) previously validated for red cusk-eel in the liver [26] and following the methodology described by [33].
Statistical Analysis
Significant differences between means of the control and stress groups for DNA damage (AP sites), enzymatic activity (AST, ALT, and AP) and differential expression of the qPCR validated genes were determined using a t-test with a significance threshold of p < 0.05. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science<|endoftext|>High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. SerpinH1 (also known as Hsp47) is a chaperone involved in the biosynthesis of collagen at the ER [76], with a key role in the restoration of homeostasis during high-temperature stress and oxidative stress [22]. It was shown to be a good biomarker for thermal stress in several salmonid species, including rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), and Chinook salmon (Oncorhynchus tshawytscha) [3,77], as well as zebrafish [78], evidencing that sherpinh1 is one of the most suitable transcriptional biomarkers of high-temperature stress in fish. This was also the case for red cusk-eel in this study, representing a useful tool to evaluate thermal stress status in this species under a climate change scenario.
Conclusions
The present study evaluated for the first time the effects of high-temperature stress in the liver of G. chilensis, using a multiple approach of plasmatic hepatic enzymes, oxidative damage evaluation and RNA-seq analysis. We showed that high-temperature stress under heatwaves ENSO-associated scenario generated a major effect in the liver, affecting hepatic enzymes, generating oxidative damage in this tissue, as well as generating an unfolded protein response at the molecular level in several associated pathways, including a heat shock response, evidencing the affection of red cusk-eel under this type of stressor. This study contributes to knowledge about thermal stress under a climate change scenario, generating candidate biomarkers for thermal stress evaluation in this species, information that should be relevant for the aquaculture and fisheries industry of red cusk-eel.
Supplementary Materials: The following supporting information can be downloaded at: [URL]:// www.mdpi.com/article/10.3390/biology11070990/s1, Figure S1: Differentially expressed transcripts in response to high-temperature stress in G. chilensis expressed as average transcription per group; Figure S2: Differentially expressed transcripts and clustering groups in response to high-temperature stress in G. chilensis; Figure S3: Validation of selected differentially expressed transcripts by qPCR on liver of G. chilensis on response to high-temperature stress; Table S1: Down-regulated differentially expressed transcripts between control and stress groups of G. chilensis; Table S2: Up-regulated differentially expressed transcripts between control and stress groups of G. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Data acquisition was conducted through Analyst 1.6.3software (Sciex). Quantification of all metabolites was performed using Multiquant 3.0.3software (Sciex). Further optimization of declustering potentials (DP) and collision energies (CE) for individual MRM transitions was carried out. A specific set of MRM transitions was monitored for each period, corresponding to the elution of metabolites during that timeframe.
Identification of Differentially Accumulated Metabolites
Significantly regulated metabolites between groups using the identification criterion of the absolute |log 2 (fold-change)| ≥ 1 and p value < 0.05, based on the Student's t-test. Identified metabolites using the Kyoto Encyclopedia of Gene and Genomes (KEGG) compound database available at [URL] on 20 June 2022. Next, we mapped the annotated metabolites to KEGG Pathway database found at [URL] on 20 June 2022. The pathways that significantly regulated metabolites mapped to were integrated into MSEA (metabolite sets enrichment analysis) and evaluated for significance by checking the hypergeometric test's p-values.
RNA Sequencing
Total RNA was extracted from frozen leaves utilizing the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). The Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to evalute the quality of the gathered RNAs by examining their integrity. Subsequently, Poly (A) mRNA was enriched from total RNA using Oligo (dT) magnetic beads. To facilitate sequencing, Poly (A) mRNA was fragmented by an RNA fragmentation kit (Ambion, Austin, TX, USA). First-strand cDNA was produced via transcriptase reaction with random hexamer primers. Next, DNA polymerase I and RNase H enzymes were employed to create the second-strand cDNA (Invitrogen, Carlsbad, CA, USA). Following this, DNA fragments of suitable lengths were obtained, end-repaired, poly(A)-tailed, and connected with sequencing adaptors. Eventually, these fragments underwent Illumina HiSeq™ 2500 platform sequencing.
Transcript Profiles and Annotation
High-quality reads were obtained by processing the raw reads in fastq format using Perl scripts developed in-house. Clean reads were obtained from the raw data by eliminating adaptor sequences, low-quality reads, and reads containing polyN. All downstream analyses were based on clean, highquality data. Gene function was annotated employing several databases: KEGG pathway database, the NCBI non-redundant (Nr) database, the Swiss-Prot protein database, the euKaryotic Clusters of Orthologous Groups (KOG) database, the Gene Ontology (GO) database, and the Pfam database. We analyzed the differentially expressed genes of both groups by utilizing the DESeq R package (version 1.10.1). The DESeq R package utilizes a statistical model based on the negative binomial distribution to identify differentially expressed genes. We corrected the outcomes of all statistical tests by employing the false discovery rate of Benjamini and Hochberg to account for multiple testing. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>This below document has 2 sentences that end with 'differentially expressed genes'. It has approximately 494 words, 28 sentences, and 11 paragraph(s).
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. First-strand cDNA was produced via transcriptase reaction with random hexamer primers. Next, DNA polymerase I and RNase H enzymes were employed to create the second-strand cDNA (Invitrogen, Carlsbad, CA, USA). Following this, DNA fragments of suitable lengths were obtained, end-repaired, poly(A)-tailed, and connected with sequencing adaptors. Eventually, these fragments underwent Illumina HiSeq™ 2500 platform sequencing.
Transcript Profiles and Annotation
High-quality reads were obtained by processing the raw reads in fastq format using Perl scripts developed in-house. Clean reads were obtained from the raw data by eliminating adaptor sequences, low-quality reads, and reads containing polyN. All downstream analyses were based on clean, highquality data. Gene function was annotated employing several databases: KEGG pathway database, the NCBI non-redundant (Nr) database, the Swiss-Prot protein database, the euKaryotic Clusters of Orthologous Groups (KOG) database, the Gene Ontology (GO) database, and the Pfam database. We analyzed the differentially expressed genes of both groups by utilizing the DESeq R package (version 1.10.1). The DESeq R package utilizes a statistical model based on the negative binomial distribution to identify differentially expressed genes. We corrected the outcomes of all statistical tests by employing the false discovery rate of Benjamini and Hochberg to account for multiple testing. According to DESeq, genes were considered substantially differentially expressed if their adjusted p-value was less than 0.05. We used the top GO R package v.1, which is based on the Kolmogorov-Smirnov test, to carry out GO enrichment analysis of the differentially expressed genes. We performed pathway analysis utilizing the KEGG database ( [URL] on 10 June 2022) to investigate relevant pathways of substantially differentially expressed genes [56][57][58].
Correlation Analysis of Transcriptome and Metabolome
To integrate transcriptome and metabolome datasets, Pearson correlation coefficients were utilized. Gene-metabolite coefficients were obtained by calculating the average expression levels of transcripts and metabolite contents. The fold changes for both transcriptome and metabolome data in each group were also computed. A correlation was considered significant if the Pearson correlation coefficients exceed 0.8 and p-values were less than 0.05 (Table 1). Significant positive correlations between transcription factors (TFs) and anthocyanin derivatives in groups JFC and LFC were detected and visually presented utilizing Cytoscape 3.10.0.
Verification of RNA-Seq Data by qRT-PCR
Total RNA was extracted from Toona sinesis leaves and reverse-transcribed using the Quantscript Reverse Transcriptase Kit. Each clone had three biological replicates, and each sample had three technical replicates. The obtained cDNA served as a template for determining gene expression levels, employing specific primers for genes linked to anthocyanin biosynthesis as well as ACTIN gene (used as an internal control). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. The log 2 FC represents the expression change in the stress group compared with the control group. Results are expressed as the mean ± standard error. Significant differences in the validated qPCR data between control and stress groups are indicated by asterisks in the log2 Fold qPCR bars; (* p-value < 0.05).
Discussion
Stress in fish is a relevant issue for aquaculture species and native populations of fish [15]. The environmental stress factor associated with water parameters represents a key issue in fish, especially considering the variations in the environment in the short-and long-term associated with global warming, climate change, ENSO, and pollution of the oceans, which could lead to modification of sea temperature, pH, and DO level, as well as increased levels of pollutants, including microplastics and toxic compounds, which could generate stress in teleost fish [7,8,[34][35][36]. These environmental stressors could also affect the red cusk-eel, a relevant species for Chilean fisheries and aquaculture diversification. However, studies aimed at understanding the effect of stressors in Genypterus species are limited [25,26,37,38], with no information about the impact of thermal stress on the liver for Genypterus species. In this sense, thermal stress has been previously studied in other tissues of Genypterus species, including the skeletal muscle, ovary, and post-ovulatory eggs [23,24]. Therefore, to understand the effect of high-temperature stress at the hepatic level, we evaluated the hepatic enzymes, oxidative stress response, and transcriptional regulation in red cusk-eel.
High-Temperature Effect on Hepatic Enzymes
In a previous study, we determined that high temperature (19 • C) could generate a stress response in red cusk-eel with an increase in the plasmatic level of cortisol and glucose [24]. In the present study, we determined that a high temperature increased the plasmatic activity of ALT, AST, and AP enzymes, evidencing the effect at the hepatic level. This effect was previously observed in other teleost fish under stress conditions, including the plasmatic activity of ALT and AST under high densities and metal pollution in rohu (Labeo rohita) [39,40], metal toxicity and pesticides in Nile tilapia (Oreochromis niloticus) [41,42] and spotted snakehead (Channa punctatus) [43], and elevated ALT, AST and AP under handling stress in red cusk-eel [25]. In terms of high-temperature stress, different results have been observed, with an increase in the plasmatic activity of AST and ALT in pufferfish (Takifugu obscurus) [44], which is consistent with our results for red cusk-eel, while no effect for AST and ALT was observed in Turbot (Scophthalmus maximus) [45] under high-temperature stress, evidencing that the hepatic response to thermal stress could vary according to fish species. The increased levels on ALT, AST an AP enzymes could be indicative of liver dysfunction, reflecting hepatocyte damage due to thermal stress, which is concordant with the results observed in mammals [46].
Oxidative Stress under High-Temperature Stress
Oxidative stress corresponds to a disturbance between the production of ROS, which can accumulate in cells, and the antioxidant defenses generated by the cellular systems to detoxify these ROS [47]. OS participate in different normal cellular functions, acting as a second messenger in signal transduction [48], but they can also generate cellular damage, including oxidative damage generating lipid peroxidation, as well as damage to proteins and DNA [49]. Environmental stress, including thermal stress, can lead to oxidative stress status in marine animals [3]. It was previously observed that thermal stress could modulate the oxidative stress status in several tissues of teleost fish [50,51]. One of the relevant organs in which oxidative status could be affected by thermal stress is the liver, as it was previously observed that thermal stress by low temperature could increase antioxidant enzymes in milkfish (Chanos Chanos) [52], as well as generate oxidative damage, leading to lipid peroxidation in Pacu (Piaractus mesopotamicus) under low-temperature stress [53,54]. High-temperature stress could also generate a relevant impact on the oxidative status of fish, with increased antioxidant enzyme activities, as observed for Senegalese sole (Solea senegalensis) [55]. Additionally, oxidative damage was observed in the liver of rohu (Labeo rohita), with lipid peroxidation and DNA fragmentation [56]. This is consistent with the findings of our study, evidencing that thermal stress generates an important oxidative effect on the liver of red cusk-eel. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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This below document has 2 sentences that end with 'USA) following the manufacturer's protocol',
3 sentences that end with 'USA), following the manufacturer's protocol'. It has approximately 684 words, 25 sentences, and 7 paragraph(s).
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. Then, the apurinic/apyrimidinic sites (AP sites) were determined in the gDNA using the OxiSelect Oxidative DNA Damage Quantification Kit (Cell Biolabs, CA, USA) following the manufacturer's protocol. The protein carbonylation assay was performed using total protein extracted from 100 mg of the liver in 1 mL of lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% NP-40, solubilized at 4 • C after 12,000× g centrifugation. Proteins were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, Batavia, IL, USA). Then, carbonylated protein content was quantified using the OxiSelect Protein Carbonyl Spectrophotometric Assay (Cell Biolabs, San Diego, CA, USA) following the manufacturer's protocol. The lipid peroxidation determination in the liver was performed using the OxiSelect HNE Adduct Competitive ELISA Kit (Cell Biolabs, San Diego, CA, USA), following the manufacturer's protocol. This kit determines lipid peroxidation through the quantification of hydroxynonenal (HNE) protein adducts in the extracted proteins of the liver.
Liver RNA Extraction, Library Preparation and Illumina Sequencing
Total RNA was extracted from the liver stored in RNAsave solution (Biological Industries, Cromwell, CT, USA) using the TRIzol ® reagent (Invitrogen, Carlsbad, CA, USA) protocol. Total RNA was quantified by fluorometry with the Qubit ® RNA quantitation assay (Invitrogen, Carlsbad, CA, USA) and purity was determined according to 260/280 ratio using the Epoch Spectrophotometer System (BioTek, Winooski, VT, USA). The RNA integrity was measured according to RNA Quality Measurement Number (RQN) through a Fragment Analyzer with the Standard Sensitivity RNA Analysis kit (Advanced Analytical Technologies, Fiorenzuola, Italy), selecting samples with RQN ≥ 8. The cDNA libraries construction were performed with 1 µg of total RNA per sample using the Illumina ® TruSeq RNA Sample Prep Kit v2 (Illumina ® , San Diego, CA, USA), following the manufacturer's protocol. The sizes of the mRNA libraries were determined through a Fragment Analyzer using the NGS Fragment Analysis kit (Advanced Analytical Technologies) and quantified by qPCR using the Kapa Library Quantification kit (Roche, Little Falls, NJ, USA). Paired-end sequencing (2 × 100 bp) was performed on a Hiseq 2500 (Illumina ® ) platform in Macrogen Inc. (Seul, South Korea).
Reads Filtering, Differential Expression, and GO Enrichment Analysis
The raw reads obtained from Illumina sequencing were trimmed to remove the remaining Illumina adapter, low-quality sequences, and short sequences (<50 bp), using the CLC Genomics Workbench v.7.0.3 software. The filtered reads were mapped to a G. chilensis reference transcriptome previously annotated by our group ( [28], NCBI accession number SRS614525) using the CLC Genomics Workbench v.7.0.3 software with the following parameters: mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.8, and a similarity fraction = 0.8. The expression values were used for clustering and heatmap chart generation with R. Differential expression analysis was performed with the R package DESeq2 (version 1.2.10) [29] to determine differentially expressed transcripts in the liver between the control and stress groups. Transcripts presenting an adjusted p-value of <0.05 and an absolute log2 fold change of >1 were considered as differentially expressed between the groups. Enrichment analysis was performed on the list of differentially expressed transcripts and GO terms to determine the overrepresented processes in response to high-temperature stress in the liver, considering up and down-expressed transcripts. This analysis was performed using the enrichment analysis tool implemented in the Blast2GO software [30]. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database was used to build the represented pathways through the KEGG Automatic Annotation Server (KAAS) [31] using the KEGG Orthology (KO) identifiers of the differentially expressed list.
RNA-seq Validation by qPCR
Total RNA previously extracted was DNAse-treated to remove residual gDNA, and 1 µg of RNA was reverse transcribed into cDNA using the QuantiTect ® Reverse Transcription kit (Qiagen, Germantown, MD, USA), following the manufacturer's protocol. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. These results revealed that the expression of genes in anthocyanin biosynthesis was consistent with accumulation of anthocyanins.
Correlation Analysis of Differentially Expressed Transcripts and Anthocyanin Compounds to Identify Key TFs and Plant Hormones Genes
To achieve the regulatory network between regulatory genes, including transcription factors and plant hormone-related genes, and anthocyanin metabolites, an association analysis was conducted between transcripts and metabolome (Supplementary Figure S2). The major transcription factors and plant hormone-related genes were then identified using differentially expressed genes and metabolites that showed significant differences between JFC (red) and LFC (green). A conjoint analysis was conducted to establish relationships between 510 genes and 23 metabolites with a positive correlation coefficient greater than 0.8 within JFC and LFC. Among these genes and metabolites, 23 TFs and
Correlation Analysis of Differentially Expressed Transcripts and Anthocyanin Compounds to Identify Key TFs and Plant Hormones Genes
To achieve the regulatory network between regulatory genes, including transcription factors and plant hormone-related genes, and anthocyanin metabolites, an association analysis was conducted between transcripts and metabolome (Supplementary Figure S2). The major transcription factors and plant hormone-related genes were then identified using differentially expressed genes and metabolites that showed significant differences between JFC (red) and LFC (green). A conjoint analysis was conducted to establish relationships between 510 genes and 23 metabolites with a positive correlation coefficient greater than 0.8 within JFC and LFC. Among these genes and metabolites, 23 TFs and phytohormonerelated genes were identified and showed strong correlations with 13 anthocyanin derivates (Table 1). Using Cytoscape 3.10.0,a regulatory network was constructed between these 23 genes and 13 anthocyanin derivatives (Figure 5A). The top 3 TFs associated with the biosynthesis of anthocyanins belonged to AGL9 (MADS-box), SOC1 (MADS-box), MYB (CPC) (Table 1). Furthermore, among the top five compounds, Cyanidin-3-O-arabinoside and Cyanidin-3-O-galactoside emerged as the most significantly different anthocyanin derivatives in Toona sinensis (Table 1). Consequently, a regulatory network was constructed between these top transcription factors and structural regulatory genes involved in anthocyanin biosynthesis (Figure 5B). These findings suggest that MADS-box, MYB, and bHLH transcription factors may regulate DFR, ANS, UFGT, and other structural genes involved in the biosynthesis of anthocyanin in Toona sinensis by either affecting or participating in the regulation of structural gene.
Int. J. Mol. Sci.2023, 24, x FOR PEER REVIEW 9 of 22 phytohormone-related genes were identified and showed strong correlations with 13 anthocyanin derivates (Table 1). Using Cytoscape 3.10.0,a regulatory network was constructed between these 23 genes and 13 anthocyanin derivatives (Figure 5A). The top 3 TFs associated with the biosynthesis of anthocyanins belonged to AGL9 (MADS-box), SOC1 (MADS-box), MYB (CPC) (Table 1). Furthermore, among the top five compounds, Cyanidin-3-O-arabinoside and Cyanidin-3-O-galactoside emerged as the most significantly different anthocyanin derivatives in Toona sinensis (Table 1). Consequently, a regulatory network was constructed between these top transcription factors and structural regulatory genes involved in anthocyanin biosynthesis (Figure 5B). These findings suggest that MADS-box, MYB, and bHLH transcription factors may regulate DFR, ANS, UFGT, and other structural genes involved in the biosynthesis of anthocyanin in Toona sinensis by either affecting or participating in the regulation of structural gene.
Expression Profiles of TFs and Plant Hormone Genes Related to Anthocyanin Biosynthesis
Previous studies have demonstrated that the biosynthesis of anthocyanins requires the involvement of transcription factors, such as MADS-box, MYB, bHLH, and bZIP. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. Additionally, high-temperature stress generated oxidative stress in the liver, with oxidative damage and transcriptional modulation of the antioxidant response. Furthermore, an unfolded protein response was observed, with several pathways enriched, as well as a heat shock response, with several heat shock proteins up regulated, suggesting candidate biomarkers (i.e., serpinh1) for thermal stress evaluation in this species. The present study shows that high-temperature stress generated a major effect on the liver of red cusk-eel, knowledge to consider for the aquaculture and fisheries of this species.
Introduction
Environmental factors are important for the physiology of fish, particularly those associated with water conditions. Among these, temperature, pH, and dissolved oxygen (DO) are crucial for the homeostasis of the fish in marine environments, and changes in these factors could lead to generating a stressful status for the animal [1]. It has been reported that an increase in water temperature could lead to stress and negative effects on marine fish, including salmonids [2,3], catfish [4], Atlantic cod [5], and gilthead seabream [6]. Understanding the effects of water temperature on fish is crucial in the actual scenario of climate change, considering the average rising sea temperature per decade [7]. Additionally, the effect of climate change on sea temperatures is expected to influence relevant phenomena of the Pacific coast, such as El Niño-Southern Oscillation (ENSO), increasing their intensity and frequency [8], which is relevant for marine species of the South Pacific coast, considering the increase in water temperatures associated with this phenomena. Therefore, it is important to understand the effect of an increase in temperature and how it affects the stress response in marine fish with aquaculture potential.
Chile is a relevant country in seafood production, with an important fishery and aquaculture industry. Additionally, the Chilean aquaculture sector is recognized for its important salmon and mussel industries [9]. However, in the last decade, an important effort has been made by the public private association to diversify Chilean aquaculture with native fish of commercial value. One of these species is the red cusk-eel (Genypterus chilensis), part of the Genypterus genus, endemic to the South Pacific coast, an economically relevant fish for fisheries and, recently, part of the Chilean aquaculture diversification program [10,11], with recent elucidation of the complete production cycle [12]. The red cusk-eel is a demersal fish with a carnivorous diet; it lives on rocky bottoms [13] and is characterized reproductively as a multiple spawner [11]. The high value of its flesh makes this species an attractive product; however, tons of fisheries have presented variable levels, with a decreasing tendency in the last decade [14]. In this sense, it is important to understand how environmental factors could affect this species and the mechanisms involved.
Stress in fish can be characterized as an adaptative response to danger, which generates physiological changes to prepare the fish to respond and survive threats. This stress response in fish is mediated by the hypothalamic-pituitary-interrenal (HPI) axis though several key chemical mediators, including corticotropin releasing factor, adrenocorticotropic hormone, α-melanocyte-stimulating hormone, adrenaline, and cortisol, a key hormone that increases in plasma under stress [15]. If the stressor is maintained for prolongated periods, a chronic stress status is generated in the fish, leading to several negative physiological effects, including decreased growth, reproductive problems, behavior modifications, and immune response [16,17]. Moreover, at a cellular level, the stress can lead to an increase in reactive oxygen species (ROS), which could lead to an oxidative stress status, as previously observed for several teleost fish species [18][19][20][21][22], an effect also observed for red cusk-eel in response to several stressors [23][24][25]. Red cusk-eel has shown low tolerance to intensive farming stressors, with limited information related to the stress response capacity in this species [13]. However, the specific stress response varies according to species, as well as the effect on each tissue. Our previous studies on G. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. In LFC vs. BSH, there were 24 upregulated metabolites and 6 downregulated metabolites (Figure 2B). In BSH vs. JFC, there were 10 upregulated metabolites and 11 downregulated metabolites (Figure 2B). Counting the differential metabolites compared with both LFC and JFC for each individual plant, a Venn diagram was used to show that there were 6 differential metabolites in the comparison group compared with JFC and 24 common differential metabolites compared with LFC (Figure 2C,D). Thirty-one common detected metabolites were used to draw clustering heatmap by TBtools ( [URL] on 13 September 2023). The contents of cyanidin and delphinidin could be mainly responsible for the leaves color of Toona sinensis (Figure 2E).
Metabolome Analysis of Anthocyanin in Five Toona Clones
To compare the flavonoids and anthocyanin compound composition among five Toona clones, three biological replicates of each clone were sampled to detect anthocyanin-related metabolites employing the UPLC-MS/MS platform. The resulting metabolite composition data sets underwent PCA analysis (Figure 2A). The PCA plot for the anthocyanin-related metabolites showed a clear separation between the red, pink, light red, green, dark-purple samples. A total of 43, 40, 44, 40, and 32 anthocyanin-related metabolites were identified in leaves of JFC, BSH, FHXN, HBG, and LFC, respectively (Table S1). A total of 31 anthocyanins were identified in all of the samples. Then, the |log2(fold-change)| ≥ 1 and p value < 0.05 was used to determine significantly metabolites between groups (Figure 2B). In group LFC vs. JFC, 23 upregulated metabolites and 8 downregulated metabolites were found to be significantly different (Figure 2B). In LFC vs. BSH, there were 24 upregulated metabolites and 6 downregulated metabolites (Figure 2B). In BSH vs. JFC, there were 10 upregulated metabolites and 11 downregulated metabolites (Figure 2B). Counting the differential metabolites compared with both LFC and JFC for each individual plant, a Venn diagram was used to show that there were 6 differential metabolites in the comparison group compared with JFC and 24 common differential metabolites compared with LFC (Figure 2C,D). Thirty-one common detected metabolites were used to draw clustering heatmap by TBtools ( [URL] on 13 September 2023). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Morphological observation and anthocyanin content of leaves from different Toona clones (JFC, FHXN, BSH, HBG, and LYC).(A) The morphological characteristics of leaves from the five Toona clones.(B) The color of anthocyanin present in the leaves of these clones.(C) The quantification of anthocyanin content in each of the Toona clones. Data are shown as mean ± standard deviation, n = 3. Bars with different letters are significantly different at p < 0.05.
Figure 2 .
Figure 2. Targeted metabolome profiles of the anthocyanin biosynthesis pathway.(A) A PCA score plot demonstrates variations among different colored Toona clones.(B) The number of up-and down-regulated metabolites varies across different comparison groups.(C,D) Venn diagram illustrate the number of differential metabolites when compared with JFC and LFC.(E) Heatmaps reveals the differential metabolites present in diverse Toona clones. Three independent replicates of each clone are displayed in the heatmap.
Figure 2 .
Figure 2. Targeted metabolome profiles of the anthocyanin biosynthesis pathway.(A) A PCA score plot demonstrates variations among different colored Toona clones.(B) The number of up-and downregulated metabolites varies across different comparison groups.(C,D) Venn diagram illustrate the number of differential metabolites when compared with JFC and LFC.(E) Heatmaps reveals the differential metabolites present in diverse Toona clones. Three independent replicates of each clone are displayed in the heatmap.
2 Figure 3 .
Figure 3. Transcriptome analysis of leaves among Toona clones.(A) A PCA score plot of the RNA seq results of various colored Toona.(B) The number of up-and down-regulated genes differs acros different comparison groups.(C) A shared number of DEGs across varying comparison groups.(D A KEGG pathway enrichment bubble plot for DEGs between different comparison groups.
Figure 3 .
Figure 3. Transcriptome analysis of leaves among Toona clones.(A) A PCA score plot of the RNA-seq results of various colored Toona.(B) The number of up-and down-regulated genes differs across different comparison groups.(C) A shared number of DEGs across varying comparison groups.(D) A KEGG pathway enrichment bubble plot for DEGs between different comparison groups.
Figure 4 .
Figure 4. Biosynthetic pathway of anthocyanins. The construction of this pathway is based on the KEGG pathway and pertinent literature references. The red box indicated the expression of genes, and the blue box indicated the content of compounds in this pathway.
Figure 4 .
Figure 4. Biosynthetic pathway of anthocyanins. The construction of this pathway is based on the KEGG pathway and pertinent literature references. The red box indicated the expression of genes, and the blue box indicated the content of compounds in this pathway.
Figure 5 .
Figure 5. Regulatory network related to the anthocyanin biosynthesis pathway.(A) Connection network between regulatory genes and metabolites involved in anthocyanin biosynthesis. Purple circles represent transcription factors, light-purple triangle represent plant hormones related genes, green round rectangle represent the metabolites. The size of the cells represented the log2FC (gene or compounds).(B) Regulatory network between transcription factors and structural genes responsible for anthocyanin biosynthesis. Purple circles represent transcription factors, light-purple circles represent structural genes involved in the biosynthesis of anthocyanin pathway.
Figure 5 .
Figure 5. Regulatory network related to the anthocyanin biosynthesis pathway.(A) Connection network between regulatory genes and metabolites involved in anthocyanin biosynthesis. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
==
Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The construction of this pathway is based on the KEGG pathway and pertinent literature references. The red box indicated the expression of genes, and the blue box indicated the content of compounds in this pathway.
Figure 4 .
Figure 4. Biosynthetic pathway of anthocyanins. The construction of this pathway is based on the KEGG pathway and pertinent literature references. The red box indicated the expression of genes, and the blue box indicated the content of compounds in this pathway.
Figure 5 .
Figure 5. Regulatory network related to the anthocyanin biosynthesis pathway.(A) Connection network between regulatory genes and metabolites involved in anthocyanin biosynthesis. Purple circles represent transcription factors, light-purple triangle represent plant hormones related genes, green round rectangle represent the metabolites. The size of the cells represented the log2FC (gene or compounds).(B) Regulatory network between transcription factors and structural genes responsible for anthocyanin biosynthesis. Purple circles represent transcription factors, light-purple circles represent structural genes involved in the biosynthesis of anthocyanin pathway.
Figure 5 .
Figure 5. Regulatory network related to the anthocyanin biosynthesis pathway.(A) Connection network between regulatory genes and metabolites involved in anthocyanin biosynthesis. Purple circles represent transcription factors, light-purple triangle represent plant hormones related genes, green round rectangle represent the metabolites. The size of the cells represented the log2FC (gene or compounds).(B) Regulatory network between transcription factors and structural genes responsible for anthocyanin biosynthesis. Purple circles represent transcription factors, light-purple circles represent structural genes involved in the biosynthesis of anthocyanin pathway.
Figure 7 .
Figure 7. Quantitative real-time RT-PCR (qRT-PCR) and RNA-seq analysis of genes involved in anthocyanin biosynthesis pathway and putative transcription regulators.(A) Relative expression level of transcription factors.(B) Relative expression level of structural genes. Data are shown as mean ± standard deviation of three biological replicates.
Figure 7 .
Figure 7. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
==
Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The qRT-PCR outcomes of structural genes were also significantly correlated with RNA-seq (Figure 7B). Moreover, our data suggested that the expression levels of bHLH162 (Maker00021798, 2.99 times higher in JFC than in LFC), CPC (Maker00014746, 10.48 times), and SOC1 (Maker00029632, 6.67 times) were synchronized with DFR (Marker00011618, 1.84 times), ANS (Marker00000819, 2.95 times), and UFGT-1 (Marker00029333, 3.07 times), indicating that they may be involved in regulating anthocyanin production. These findings demonstrated that transcription data accurately reflect gene expression in the anthocyanin biosynthesis of Toona sinesis.
Discussion
Anthocyanins present in Toona sinensis leaves possess various health benefits, including antioxidant and anti-inflammatory properties, and potential cancer-preventing properties. Understanding the regulatory mechanisms involved in anthocyanin biosynthesis in Toona sinensis leaves has significant implications for human health and agriculture. In this study, we utilized metabolomic and transcriptomic approaches to elucidate the molecular mechanisms and regulatory network involved in anthocyanin biosynthesis in Toona. A total of 32-44 metabolites related to anthocyanins were identified in five different clones of Toona leaves using UPLC-MS/MS. We found that JFC had the highest total anthocyanin content, with Delphinidin, Cyanidin, Peonidin being the most abundant pigments in JFC. Specifically, Cyanidin-3-O-arabinoside was found to be 520.93 times more prevalent in JFC than in LFC, indicating that cyanidin compounds are the dominant anthocyanin in JFC. These findings are consistent with those of previous studies on strawberry petals, which have shown that cyanidins are the primary anthocyanin compound present [37].
Transcriptome sequencing of Toona leaves with different colors can provide valuable insights into the regulating genes involved in anthocyanin biosynthesis. The KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in colored leaves of different clones compared to green leaves revealed significant enrichment of genes involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and the anthocyanin biosynthesis pathway. This finding supports previous studies demonstrating the involvement of multiple enzymes encoded by early biosynthesis genes and anthocyanin biosynthesis genes in anthocyanin biosynthesis. The study indicates that the biosynthesis of naringenin is a crucial step in the flavonoids metabolic pathway and plays a decisive role in the synthesis of anthocyanin compounds. The PAL, C4H, 4CL, CHS, and CHI are key genes in the flavonoids metabolic pathway and play a decisive role in determining naringenin production. The study found that JFC had a higher naringenin content than BSH and LFC, but only two 4CL genes (Maker00007314, novel.5717)were expressed at high levels in JFC and low levels in LFC. The expression of 4CL genes was highly positively correlated with naringenin content, indicating that the number and function of genes in the naringenin pathway may affect naringenin content. During the biosynthesis of anthocyanins, DFR catalyzes the reduction reaction of flavonoid-3 ,5 -hydroxylase to form anthocyanins, which was confirmed as a key step for regulating anthocyanin types. ANS converts anthocyanin precursors to anthocyanin glycosides. UFGT can catalyze the conversion of unstable anthocyanidins into stable anthocyanins. The absence of these genes directly affects anthocyanins biosynthesis, leading to pigment loss [38,39]. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
==
Domain: Biology Environmental Science<|endoftext|>This below document has 2 paragraphs that end with 'Response to High-Temperature Stress'. It has approximately 595 words, 24 sentences, and 7 paragraph(s).
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. A total of thirteen differentially expressed transcripts were selected for qPCR validation, corresponding to: hsp60, hsp70, gpx7, ddit4, leptin, msh2, msh3, c1ql1, ccl20, atg12, atg4b, casp3 and c3. Primers were designed with Primer 3 software v0.4.0 ( [URL]3/, accessed on 6 May 2021) using the reference transcriptome previously described. The primer sequences, amplicon size, Tm, and efficiency are presented in Table 1. The qPCR was performed in a Stratagene MX3000P qPCR system (Stratagene, La Jolla, CA, USA). All qPCR assays were performed in triplicates, using no-template and no-RT controls, in compliance with the MIQE guidelines [32]. The qPCR reaction mixture contained 7.5 µL of 2× Brilliant ® II SYBR ® Green master mix (Agilent Technologies, CA, USA), 100 ng of cDNA per reaction and 200 nM of each primer in a 15 µL final volume. Thermal cycling conditions were an initial activation of 2 min at 95 • C, followed by 40 cycles of 30 s at 95 • C, 30 s at 62 • C, and 30 s at 72 • C. A melt curve analysis was performed to confirm a single qPCR product and a standard curve using two-fold series dilutions was used to estimate the efficiency of each primer set. The expression of target genes was normalized using the geometric means of two reference genes (actb and taf12) previously validated for red cusk-eel in the liver [26] and following the methodology described by [33].
Statistical Analysis
Significant differences between means of the control and stress groups for DNA damage (AP sites), enzymatic activity (AST, ALT, and AP) and differential expression of the qPCR validated genes were determined using a t-test with a significance threshold of p < 0.05. All statistical analyses were performed using GraphPad Prism, v.5.00 (GraphPad Software, San Diego, CA, USA).
Hepatic Enzyme Activity and Oxidative Stress Response to High-Temperature Stress
We previously reported that thermal stress for 5 days significantly increased plasmatic levels of cortisol and glucose in G. chilensis [24]. To understand how thermal stress affects the metabolism in this species, we studied the effect of high temperature on the liver. To evaluate the effect on the hepatic function of this type of stressor, we measured plasmatic markers of liver damage, i.e., ALT, AST, and AP enzymatic activity. High-temperature stress significantly increased the plasmatic activity of ALT ( Figure 1A), AST ( Figure 1B) and AP ( Figure 1C) in the stress group, evidencing altered hepatic function in the liver of stressed fish. To determine the oxidative damage in the liver generated by high-temperature stress, the DNA oxidative damage, protein carbonylation and lipid peroxidation were determined in the liver. High temperatures generated DNA damage, evidenced by the significant increase in apurinic/apyrimidinic sites in the stressed group (Figure 2A). Oxidative damage was also observed in lipid and proteins, determined by a significant increase in protein carbonylation ( Figure 2B) and lipid peroxidation ( Figure 2C) in response to high temperature in the stress group.
Differentially Expressed Transcripts in Hepatic Response to High-Temperature Stress
To understand the complexity of the stress response in the liver of G. chilensis, we performed RNA-seq analysis on the liver of each experimental group. The sequencing generated a total of 754,678,455 paired-end reads, with an average of 58,052,189 raw paired-end reads per library. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. Significant differences in the validated qPCR data between control and stress groups are indicated by asterisks in the log2 Fold qPCR bars; (* pvalue < 0.05).
Discussion
Stress in fish is a relevant issue for aquaculture species and native populations of fish [15]. The environmental stress factor associated with water parameters represents a key issue in fish, especially considering the variations in the environment in the shortand long-term associated with global warming, climate change, ENSO, and pollution of the oceans, which could lead to modification of sea temperature, pH, and DO level, as well as increased levels of pollutants, including microplastics and toxic compounds, which could generate stress in teleost fish [7,8,[34][35][36]. These environmental stressors could also affect the red cusk-eel, a relevant species for Chilean fisheries and aquaculture diversification. However, studies aimed at understanding the effect of stressors in Genypterus species are limited [25,26,37,38], with no information about the impact of thermal stress on the liver for Genypterus species. In this sense, thermal stress has been previously studied in other tissues of Genypterus species, including the skeletal muscle, ovary, and post-ovulatory eggs [23,24]. Therefore, to understand the effect of high- Figure 5. qPCR validation of selected differentially expressed transcripts in the liver of G. chilensis in response to high-temperature stress. The transcript expression levels were normalized with the geometric means of actb and taf12. The differential expression levels according to qPCR (black bars) and RNA-seq (gray bars) for these selected genes are expressed as log 2 fold changes. The log 2 FC represents the expression change in the stress group compared with the control group. Results are expressed as the mean ± standard error. Significant differences in the validated qPCR data between control and stress groups are indicated by asterisks in the log2 Fold qPCR bars; (* p-value < 0.05).
Discussion
Stress in fish is a relevant issue for aquaculture species and native populations of fish [15]. The environmental stress factor associated with water parameters represents a key issue in fish, especially considering the variations in the environment in the short-and long-term associated with global warming, climate change, ENSO, and pollution of the oceans, which could lead to modification of sea temperature, pH, and DO level, as well as increased levels of pollutants, including microplastics and toxic compounds, which could generate stress in teleost fish [7,8,[34][35][36]. These environmental stressors could also affect the red cusk-eel, a relevant species for Chilean fisheries and aquaculture diversification. However, studies aimed at understanding the effect of stressors in Genypterus species are limited [25,26,37,38], with no information about the impact of thermal stress on the liver for Genypterus species. In this sense, thermal stress has been previously studied in other tissues of Genypterus species, including the skeletal muscle, ovary, and post-ovulatory eggs [23,24]. Therefore, to understand the effect of high-temperature stress at the hepatic level, we evaluated the hepatic enzymes, oxidative stress response, and transcriptional regulation in red cusk-eel.
High-Temperature Effect on Hepatic Enzymes
In a previous study, we determined that high temperature (19 • C) could generate a stress response in red cusk-eel with an increase in the plasmatic level of cortisol and glucose [24]. In the present study, we determined that a high temperature increased the plasmatic activity of ALT, AST, and AP enzymes, evidencing the effect at the hepatic level. This effect was previously observed in other teleost fish under stress conditions, including the plasmatic activity of ALT and AST under high densities and metal pollution in rohu (Labeo rohita) [39,40], metal toxicity and pesticides in Nile tilapia (Oreochromis niloticus) [41,42] and spotted snakehead (Channa punctatus) [43], and elevated ALT, AST and AP under handling stress in red cusk-eel [25]. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. The primers used in the qRT-PCR analysis were shown in Table 2. The melt curve of ACTIN in the qPCR products was presented in Supplementary Figure S3. The reaction system contained 5 µL 2 × Q3 SYBR qPCR Master Mix-Universal (TOLOBIO), 1 µL cDNA template, 0.5 µL of each forward and reverse primer, and 3 µL of RNase-free water.qRT-PCR was performed using Applied Biosystems 7500 Fast Real-Time PCR System.
Conclusions
In this study, an integrative analysis of the transcriptome and metabolome of five distinct Toona sinensis clones were performed to explore the related genes and metabolites associated with the anthocyanin biosynthesis. Furthermore, our analyses found that the red leaves 'JFC' contained the highest content of stable cyanidin, delphinidin, and peonidin, especially Cyanidin-3-O-arabinoside. The integrated analysis also identified the major transcription factor SOC1, CPC, and bHLH162. Moreover, the regulatory network construction of TFs, metabolic, structural genes reveals the underlying mechanisms of the anthocyanin biosynthesis pathway in Toona sinensis. Overall, this study provides a foundation for future research aimed at manipulating anthocyanin biosynthesis to improve plant coloration or to derive human health benefits.
Figure 1 .
Figure 1. Morphological observation and anthocyanin content of leaves from different Toona clones (JFC, FHXN, BSH, HBG, and LYC).(A) The morphological characteristics of leaves from the five Toona clones.(B) The color of anthocyanin present in the leaves of these clones.(C) The quantification of anthocyanin content in each of the Toona clones. Data are shown as mean ± standard deviation, n = 3. Bars with different letters are significantly different at p < 0.05.
Figure 1 .
Figure 1. Morphological observation and anthocyanin content of leaves from different Toona clones (JFC, FHXN, BSH, HBG, and LYC).(A) The morphological characteristics of leaves from the five Toona clones.(B) The color of anthocyanin present in the leaves of these clones.(C) The quantification of anthocyanin content in each of the Toona clones. Data are shown as mean ± standard deviation, n = 3. Bars with different letters are significantly different at p < 0.05.
Figure 2 .
Figure 2. Targeted metabolome profiles of the anthocyanin biosynthesis pathway.(A) A PCA score plot demonstrates variations among different colored Toona clones.(B) The number of up-and down-regulated metabolites varies across different comparison groups.(C,D) Venn diagram illustrate the number of differential metabolites when compared with JFC and LFC.(E) Heatmaps reveals the differential metabolites present in diverse Toona clones. Three independent replicates of each clone are displayed in the heatmap.
Figure 2 .
Figure 2. Targeted metabolome profiles of the anthocyanin biosynthesis pathway.(A) A PCA score plot demonstrates variations among different colored Toona clones.(B) The number of up-and downregulated metabolites varies across different comparison groups.(C,D) Venn diagram illustrate the number of differential metabolites when compared with JFC and LFC.(E) Heatmaps reveals the differential metabolites present in diverse Toona clones. Three independent replicates of each clone are displayed in the heatmap.
2 Figure 3 .
Figure 3. Transcriptome analysis of leaves among Toona clones.(A) A PCA score plot of the RNA seq results of various colored Toona.(B) The number of up-and down-regulated genes differs acros different comparison groups.(C) A shared number of DEGs across varying comparison groups.(D A KEGG pathway enrichment bubble plot for DEGs between different comparison groups.
Figure 3 .
Figure 3. Transcriptome analysis of leaves among Toona clones.(A) A PCA score plot of the RNA-seq results of various colored Toona.(B) The number of up-and down-regulated genes differs across different comparison groups.(C) A shared number of DEGs across varying comparison groups.(D) A KEGG pathway enrichment bubble plot for DEGs between different comparison groups.
Figure 4 .
Figure 4. Biosynthetic pathway of anthocyanins. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
==
Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Then, the total anthocyanin was extracted and the resulting color were displayed in Figure 1B, which reflect the anthocyanin content of different clones. Total anthocyanin present in the leaves were quantified, with JFC Toona sinensis found to have the highest concentration of anthocyanin, followed by BSH, FHXN and HBG, and LFC (Figure 1C). Obviously, LFC exhibited green leaves due to a lack of anthocyanin accumulation.
Metabolome Analysis of Anthocyanin in Five Toona Clones
To compare the flavonoids and anthocyanin compound composition among five Toona clones, three biological replicates of each clone were sampled to detect anthocyaninrelated metabolites employing the UPLC-MS/MS platform. The resulting metabolite composition data sets underwent PCA analysis (Figure 2A). The PCA plot for the anthocyaninrelated metabolites showed a clear separation between the red, pink, light red, green, dark-purple samples. A total of 43, 40, 44, 40, and 32 anthocyanin-related metabolites were identified in leaves of JFC, BSH, FHXN, HBG, and LFC, respectively (Table S1). A total of 31 anthocyanins were identified in all of the samples. Then, the |log2(fold-change)| ≥ 1 and p value < 0.05 was used to determine significantly metabolites between groups (Figure 2B). In group LFC vs. JFC, 23 upregulated metabolites and 8 downregulated metabolites were found to be significantly different (Figure 2B). In LFC vs. BSH, there were 24 upregulated metabolites and 6 downregulated metabolites (Figure 2B). In BSH vs. JFC, there were 10 upregulated metabolites and 11 downregulated metabolites (Figure 2B). Counting the differential metabolites compared with both LFC and JFC for each individual plant, a Venn diagram was used to show that there were 6 differential metabolites in the comparison group compared with JFC and 24 common differential metabolites compared with LFC (Figure 2C,D). Thirty-one common detected metabolites were used to draw clustering heatmap by TBtools ( [URL] on 13 September 2023). The contents of cyanidin and delphinidin could be mainly responsible for the leaves color of Toona sinensis (Figure 2E).
Metabolome Analysis of Anthocyanin in Five Toona Clones
To compare the flavonoids and anthocyanin compound composition among five Toona clones, three biological replicates of each clone were sampled to detect anthocyanin-related metabolites employing the UPLC-MS/MS platform. The resulting metabolite composition data sets underwent PCA analysis (Figure 2A). The PCA plot for the anthocyanin-related metabolites showed a clear separation between the red, pink, light red, green, dark-purple samples. A total of 43, 40, 44, 40, and 32 anthocyanin-related metabolites were identified in leaves of JFC, BSH, FHXN, HBG, and LFC, respectively (Table S1). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
==
Domain: Biology Environmental Science<|endoftext|>High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. All statistical analyses were performed using GraphPad Prism, v.5.00 (GraphPad Software, San Diego, CA, USA).
Hepatic Enzyme Activity and Oxidative Stress Response to High-Temperature Stress
We previously reported that thermal stress for 5 days significantly increased plasmatic levels of cortisol and glucose in G. chilensis [24]. To understand how thermal stress affects the metabolism in this species, we studied the effect of high temperature on the liver. To evaluate the effect on the hepatic function of this type of stressor, we measured plasmatic markers of liver damage, i.e., ALT, AST, and AP enzymatic activity. High-temperature stress significantly increased the plasmatic activity of ALT ( Figure 1A), AST ( Figure 1B) and AP ( Figure 1C) in the stress group, evidencing altered hepatic function in the liver of stressed fish. To determine the oxidative damage in the liver generated by high-temperature stress, the DNA oxidative damage, protein carbonylation and lipid peroxidation were determined in the liver. High temperatures generated DNA damage, evidenced by the significant increase in apurinic/apyrimidinic sites in the stressed group (Figure 2A). Oxidative damage was also observed in lipid and proteins, determined by a significant increase in protein carbonylation ( Figure 2B) and lipid peroxidation ( Figure 2C) in response to high temperature in the stress group.
Differentially Expressed Transcripts in Hepatic Response to High-Temperature Stress
To understand the complexity of the stress response in the liver of G. chilensis, we performed RNA-seq analysis on the liver of each experimental group. The sequencing generated a total of 754,678,455 paired-end reads, with an average of 58,052,189 raw paired-end reads per library. The raw data are available from NCBI under BioProject , and lipid peroxidation in terms of HNE adducts (C) in control and stress groups. Bars represent the mean ± SEM. Significant differences between the control and stress groups are indicated by asterisks; * (p < 0.05), ** (p < 0.01) and **** (p < 0.0001).
Differentially Expressed Transcripts in Hepatic Response to High-Temperature Stress
To understand the complexity of the stress response in the liver of G. chilensis, we performed RNA-seq analysis on the liver of each experimental group. The sequencing The raw data are available from NCBI under BioProject PRJNA835467 with BioSamples accession number SAMN28102858, SAMN28102859, SAMN28102860, SAMN28102861, SAMN28102862, SAMN28102863, SAMN28102864, SAMN28102865, SAMN28102866, and SAMN28102867. After trimming by quality, adapters, and size, we obtained an average of 58,018,122 high-quality filtered paired-end reads per library (Table 2). These reads were mapped to the G. chilensis reference transcriptome (NCBI accession number SRS614525), obtaining an average of 85.2% mapped reads (Table 2). Expression values were used for normalization and differential expression analysis with the R package DESeq2 (version 1.2.10) [29], obtaining a total of 2578 differentially expressed transcripts between control and stressed groups. Of these transcripts, 1239 were down-regulated (Table S1) and 1339 were up-regulated (Table S2) in the stressed group (Figure 3 and Figure S1), evidencing five clusters of transcripts with different patterns of expression for the control and thermal stress groups ( Figure S2). The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. One of these species is the red cusk-eel (Genypterus chilensis), part of the Genypterus genus, endemic to the South Pacific coast, an economically relevant fish for fisheries and, recently, part of the Chilean aquaculture diversification program [10,11], with recent elucidation of the complete production cycle [12]. The red cusk-eel is a demersal fish with a carnivorous diet; it lives on rocky bottoms [13] and is characterized reproductively as a multiple spawner [11]. The high value of its flesh makes this species an attractive product; however, tons of fisheries have presented variable levels, with a decreasing tendency in the last decade [14]. In this sense, it is important to understand how environmental factors could affect this species and the mechanisms involved.
Stress in fish can be characterized as an adaptative response to danger, which generates physiological changes to prepare the fish to respond and survive threats. This stress response in fish is mediated by the hypothalamic-pituitary-interrenal (HPI) axis though several key chemical mediators, including corticotropin releasing factor, adrenocorticotropic hormone, α-melanocyte-stimulating hormone, adrenaline, and cortisol, a key hormone that increases in plasma under stress [15]. If the stressor is maintained for prolongated periods, a chronic stress status is generated in the fish, leading to several negative physiological effects, including decreased growth, reproductive problems, behavior modifications, and immune response [16,17]. Moreover, at a cellular level, the stress can lead to an increase in reactive oxygen species (ROS), which could lead to an oxidative stress status, as previously observed for several teleost fish species [18][19][20][21][22], an effect also observed for red cusk-eel in response to several stressors [23][24][25]. Red cusk-eel has shown low tolerance to intensive farming stressors, with limited information related to the stress response capacity in this species [13]. However, the specific stress response varies according to species, as well as the effect on each tissue. Our previous studies on G. chilensis showed a variable tissue response under handling stress, with altered metabolic status in the liver, a modulation of the immune response in the head kidney, and an induction of atrophy in skeletal muscle through coding and noncoding regulation [26]. Additionally, it has been observed that thermal stress could induce muscle atrophy in this species [24], as well as oxidative damage in eggs, with a minor effect on the ovary [23]. One of the most important organs for fish metabolism is the liver, which is directly involved in stress response by metabolizing and liberating stored energy to respond to stress [17]. Nevertheless, the liver response to thermal stress in Genypterus species has not been previously studied, nor has the impact of this stressor on the oxidative status of this tissue. It is important to consider that sea temperatures will increase through sea heat waves due to the effect of ENSO under a climate change scenario, which will affect the Chilean coast associated with the geographic range of G. chilensis. Therefore, the objective of this study was to evaluate the effect of high-temperature stress on the liver of G. chilensis in terms of the transcriptomic and oxidative stress status to determine the negative impact of this type of stressor on liver.
Ethics Statement
All procedures with the red cusk-eel individuals and all scientific activities adhered to animal welfare procedures and were approved by the bioethical committees of the Universidad Andres Bello (007/2018) and the National Commission for Scientific and Technological Research (CONICYT) of the Chilean government.
Fish Sampling and Experimental Design
In this study, we used reproductively immature red cusk-eel juveniles (G. chilensis) of 12 months of age (average weight of 665 ± 52.7 g; average length of 60 ± 4.8 cm), collected from the Centro de Investigación Marina de Quintay (CIMARQ), maintained under natural photoperiod conditions (L:D 12:12), and controlled temperature (14 ± 1 • C), and fed daily with commercial pellet food. Fish were separated into control and stress groups, with the stress group subjected to a standardized thermal stress protocol proven to generate stress in red cusk-eel [24]. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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This below document has 2 sentences that end with 'liver of G', 2 sentences that end with 'to high-temperature stress in G', 2 sentences that end with 'and stress groups of G'. It has approximately 835 words, 24 sentences, and 6 paragraph(s).
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High-Temperature Stress Effect on the Red Cusk-Eel (Geypterus chilensis) Liver: Transcriptional Modulation and Oxidative Stress Damage Simple Summary The red cusk-eel (Genypterus chilensis) is a native Chilean mintages important for aquaculture diversification in Chile. In this sense, we found several genes associated with protein processing and folding pathways, including transcripts involved in: protein export, such as signal recognition particle 14 and 19 kDa (srp14 and srp19) and signal peptidase complex subunit 2-like (spcs2) [64]; transcripts associated with protein processing in endoplasmic reticulum (Figure 4), with part of this pathway down-regulated, including TNF receptorassociated factor 2-like (traf2), and mainly an up-regulation of this pathway, including ubiquilin-4 (ubqln4), cytoskeleton-associated protein 4 (ckap4); ribosome-binding protein 1 (rrbp1, also known as p180); and glucosidase 2 subunit beta (prkcsh). Others include transcripts associated with the unfolded protein response, including eukaryotic translation initiation factor 2 alpha kinase 1 (eik2ak1, also known as hri), 3 (eik2ak3, also known as perk) and 5 (eik2ak5), cyclic AMP-dependent transcription factor ATF-4-like (atf4) and DNA damage-inducible transcript 3 -like (ddit4, also known as chop). The unfolded protein response could be initiated by EIK2AK3 kinase activation of eIF2α, leading to ribosome inhibition and attenuating protein synthesis [65]. Additionally, the ATF4 gene was activated, which would lead to DDIT4 gene regulation and an antioxidant response in cells [66], evidencing that thermal stress in the liver of red cusk-eel modulates the protein processing and generates an unfolded protein response that is not able to control the oxidative stress and damage in this tissue. This is concordant with the results observed in previous studies where unfolded protein response genes were activated under thermal stress in mammals [67,68] and fish, e.g., in the liver of Tambaqui (Colossoma macropomum) [69] and gilthead sea bream (Sparus aurata) [70].
Heat Shock Protein as Thermal Stress Biomarkers
The cellular response to thermal stress is a key process to preserve protein integrity; this is known as the heat shock response, which includes heat shock proteins (Hsps) to re-fold the proteins damaged by temperature [71]. This response was observed in the liver of red cusk-eel with the enrichment of the term response to heat (GO:0009408), highlighting several heat shock proteins differentially expressed in response to high-temperature stress, including hsp40, hsp60, hsp70, hsp90, and serpinh1. The up-regulation of these Hsps was previously observed in other fish under thermal stress, with an increase in the liver of three-spined stickleback (Gasterosteus aculeatus) (hsp60, hsp70, and hsp90) [72], Atlantic salmon (Salmo salar) (hsp70) [73,74], Atlantic cod (Gadus morhua) (hsp70 and hsp90) [74], and Wuchang bream (Megalobrama amblycephala) (hsp60, hsp70, and hsp90) [75]. The hsp60 and hsp70 expression levels were also regulated under thermal stress in the postovulatory eggs and skeletal muscle of red cusk-eel [23,24], showing that these genes could be valuable biomarkers of thermal stress in this species. However, the most up-regulated gene in the liver under high-temperature stress was serpinh1 (5.7log 2 Fold Change, Table S2). SerpinH1 (also known as Hsp47) is a chaperone involved in the biosynthesis of collagen at the ER [76], with a key role in the restoration of homeostasis during high-temperature stress and oxidative stress [22]. It was shown to be a good biomarker for thermal stress in several salmonid species, including rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), and Chinook salmon (Oncorhynchus tshawytscha) [3,77], as well as zebrafish [78], evidencing that sherpinh1 is one of the most suitable transcriptional biomarkers of high-temperature stress in fish. This was also the case for red cusk-eel in this study, representing a useful tool to evaluate thermal stress status in this species under a climate change scenario.
Conclusions
The present study evaluated for the first time the effects of high-temperature stress in the liver of G. chilensis, using a multiple approach of plasmatic hepatic enzymes, oxidative damage evaluation and RNA-seq analysis. We showed that high-temperature stress under heatwaves ENSO-associated scenario generated a major effect in the liver, affecting hepatic enzymes, generating oxidative damage in this tissue, as well as generating an unfolded protein response at the molecular level in several associated pathways, including a heat shock response, evidencing the affection of red cusk-eel under this type of stressor. This study contributes to knowledge about thermal stress under a climate change scenario, generating candidate biomarkers for thermal stress evaluation in this species, information that should be relevant for the aquaculture and fisheries industry of red cusk-eel.
Supplementary Materials: The following supporting information can be downloaded at: [URL]:// www.mdpi.com/article/10.3390/biology11070990/s1, Figure S1: Differentially expressed transcripts in response to high-temperature stress in G. chilensis expressed as average transcription per group; Figure S2: Differentially expressed transcripts and clustering groups in response to high-temperature stress in G. chilensis; Figure S3: Validation of selected differentially expressed transcripts by qPCR on liver of G. chilensis on response to high-temperature stress; Table S1: Down-regulated differentially expressed transcripts between control and stress groups of G. chilensis; Table S2: Up-regulated differentially expressed transcripts between control and stress groups of G. The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Down-regulation of the apple DFR gene can inhibit both cyanidin and procyanidin accumulation. In Vitis vinifera, the silencing of the DFR gene leads to the absence of anthocyanins [40]. In Duchesnea indica, a plant belonging to the Rosaceae family, decreased expression of the ANS gene results in white-colored fruits [41]. Conversely, overexpressing the SmANS gene in Salvia miltiorrhiza can increase anthocyanin content but decrease the biosynthesis of salvianolic acid [42]. In our study, we found significantly increased expression of the DFR gene (Marker00011618), ANS gene (Marker00000819), UFGT (Marker 00029333 and novel.8170) in JFC and BSH compared to LFC, which may explain the red color leaves in JFC and the green color leaves in LFC. Difference in gene sequence and promoter regions of these structural genes may be the primary factors contributing to difference in gene function and expression, ultimately impacting anthocyanin biosynthesis in different clones.
The MBW protein complex was proven to activate the expression of structural genes involved in anthocyanin production. In addition to the MBW complex, other TFs such as bZIP [32,43], NAC [44], WRKY [45,46], MADS-box [47,48], and zinc finger [49,50] proteins have also shown to regulate anthocyanin biosynthesis in plants. In Toona, we found that MADS-box, MYB, and C2H2 possessed top five TFs related to anthocyanin derivates according to the joint analysis between transcriptome and metabolome. The gene-gene regulatory network also showed that the SOC1, CPC, and bHLH162 were the major TFs regulating the anthocyanin biosynthesis structural genes. SOC1 encodes a MADS box transcription factor and is involved in the regulation of flowering in response to temperature or light in various plants species, such as Arabidopsis [51], Oryza sativa L. [52], Gossypium hissytum [53]. In Toona sinensis, the young leaf color is highly sensitive to temperature, indicating a critical role of SOC1 in temperature or light-induced anthocyanin biosynthesis. These transcription factors might regulate anthocyanin biosynthesis process by directly active or repress gene expression by binding to the promoter regions, or indirectly though protein-protein interactions in model plants and other fruits.
Plant hormones can affect anthocyanin biosynthesis through various mechanisms. Previous studies have reported that overexpression of MdIAA26 in apple calli and Arabidopsis promotes the accumulation of anthocyanin, while auxin inhibit it by degrading the MdIAA26 protein [54]. Exogenous ethylene treatment increased anthocyanin accumulation in grape skins and induced the expression of structural genes (VvPAL, Vv4CH, VvCHS, VvCHI, VvF3H, and VvUFGT) and regulatory genes (VvMYBA1, VvMYBA2, and VvMYBA3) related to anthocyanin biosynthesis [55]. Our study identified nine hormone-related genes with a high correlation to regulate anthocyanins biosynthesis, which are highly expressed in JFC and play a significant role in the controlling leaf color variation in Toona. Leaf color is an important trait that is susceptible to both endogenous and exogenous influences. Variations in hormone signal transduction gene expression reflect the essential mechanisms by which plant hormones affect the anthocyanin biosynthesis pathway. These findings enhance our understanding of how anthocyanin biosynthesis works in Toona and how it can be controlled, with implications for breeding new Toona cultivars. Identifying and understanding the beneficial metabolic constituents and regulatory networks of Toona can facilitate the development and utilization of anthocyanin-enriched varieties.
Plant Materials
The study selected five Chinese toon clones, JinFuChun (JFC), BaShanHong (BSH), LvFuChun (LFC), FenHongXinNiang (FHXN), and HeBeiGu (HBG), which cultivated in Research Institution of Subtropical Forestry, Chinese Academy of Forestry. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable. Furthermore, we identified several differentially expressed genes that participate in plant hormone signal transduction, including 8 Auxin-related genes, 9 CTK-related genes, 10 GA-related genes, 23 BR-related genes, 7 ABA-related genes, 11 ethlye-responsive genes, 6 JA-related genes, and 3SA-related genes. Among these genes, the following exhibited a positive correlation coefficient greater than 0.8 with anthocyanin compounds: 2 Auxinrelated genes (Maker00008503 and Maker00022256), 1 CTK-related gene (Maker00000350), 1 GA-related gene (Maker000117930), 2 BR-related genes (Maker00026791 and novel.6183), 1 Ethylene-responsive genes (Marker00033233), and 2 SA-related genes (Maker00020104 and Maker00032838) (Figure 6B). This indicates that these genes may positively regulate anthocyanin biosynthesis either directly or indirectly.
Expression Analysis of Genes through qRT-PCR
To validate the RNA-seq results, we utilized qRT-PCR to evaluate the expression of 8 structural genes and 4 TFs involved in the anthocyanin biosynthesis pathway. Our observations revealed that bHLH162, CPC, and SOC1 TFs were significantly upregulated in JFC and exhibited the lowest expression levels in LFC (Figure 7A). Additionally, there was a strong correlation between the RNA-seq and the characteristic leaf color of these samples. The qRT-PCR outcomes of structural genes were also significantly correlated with RNA-seq (Figure 7B). Moreover, our data suggested that the expression levels of bHLH162 (Maker00021798, 2.99 times higher in JFC than in LFC), CPC (Maker00014746, 10.48 times), and SOC1 (Maker00029632, 6.67 times) were synchronized with DFR (Marker00011618, 1.84 times), ANS (Marker00000819, 2.95 times), and UFGT-1 (Marker00029333, 3.07 times), indicating that they may be involved in regulating anthocyanin production. These findings demonstrated that transcription data accurately reflect gene expression in the anthocyanin biosynthesis of Toona sinesis.
Expression Analysis of Genes through qRT-PCR
To validate the RNA-seq results, we utilized qRT-PCR to evaluate the expression of 8 structural genes and 4 TFs involved in the anthocyanin biosynthesis pathway. Our observations revealed that bHLH162, CPC, and SOC1 TFs were significantly upregulated in JFC and exhibited the lowest expression levels in LFC (Figure 7A). Additionally, there was a strong correlation between the RNA-seq and the characteristic leaf color of these samples. The qRT-PCR outcomes of structural genes were also significantly correlated with RNA-seq (Figure 7B). Moreover, our data suggested that the expression levels of bHLH162 (Maker00021798, 2.99 times higher in JFC than in LFC), CPC (Maker00014746, 10.48 times), and SOC1 (Maker00029632, 6.67 times) were synchronized with DFR (Marker00011618, 1.84 times), ANS (Marker00000819, 2.95 times), and UFGT-1 (Marker00029333, 3.07 times), indicating that they may be involved in regulating anthocyanin production. These findings demonstrated that transcription data accurately reflect gene expression in the anthocyanin biosynthesis of Toona sinesis.
Discussion
Anthocyanins present in Toona sinensis leaves possess various health benefits, including antioxidant and anti-inflammatory properties, and potential cancer-preventing properties. Understanding the regulatory mechanisms involved in anthocyanin biosynthesis in Toona sinensis leaves has significant implications for human health and agriculture. In this study, we utilized metabolomic and transcriptomic approaches to elucidate the molecular mechanisms and regulatory network involved in anthocyanin biosynthesis in Toona. A total of 32-44 metabolites related to anthocyanins were identified in five different clones of Toona leaves using UPLC-MS/MS. We found that JFC had the highest total anthocyanin content, with Delphinidin, Cyanidin, Peonidin being the most abundant pigments in JFC. Specifically, Cyanidin-3-O-arabinoside was found to be 520.93 times more prevalent in JFC than in LFC, indicating that cyanidin compounds are the dominant anthocyanin in JFC. These findings are consistent with those of previous studies on strawberry petals, which have shown that cyanidins are the primary anthocyanin compound present [37].
Transcriptome sequencing of Toona leaves with different colors can provide valuable insights into the regulating genes involved in anthocyanin biosynthesis. Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science
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Integrated Transcriptomic and Metabolomic Analysis Reveal the Underlying Mechanism of Anthocyanin Biosynthesis in Toona sinensis Leaves Toona sinensis, commonly known as Chinese Toon, is a plant species that possesses noteworthy value as a tree and vegetable.
The investigation of anthocyanin-regulating genes and associated flavonoid metabolites is critical to elucidate the mechanism that governs the biological synthesis of anthocyanins. To study gene expression and metabolic changes in different cultivars, researchers mainly rely on transcriptomic and metabolic profiling, which complement each other [34,35]. In this study, we conducted a dual-level exploration of genes and metabolites that control the pigmentation of Toona sinensis leaves using transcriptome and metabolomics approaches. Specifically, we analyzed the transcriptome and metabolome data of five Toona sinensis clones exhibiting a range of leaf colors, including red, light red, pink, dark, and green, to clarify regulatory networks for biosynthesis-related genes and key metabolomes. Overall, our findings unveil the regulatory mechanisms driving the coloration of Toona sinensis leaves.
Morphological Observation and Anthocyanin Content of Leaves in Toona sinensis
Five clones of Toona sinensis with distinct leaf colors were selected to analyze the regulatory mechanism of anthocyanin biosynthesis. Among the clones, JFC Toona is characterized by brilliant-red leaves and is commonly cultivated in Zhejiang province. The leaf color of other clones was pink buds (FHXN), light-red with a little green buds (BSH), dark-purple buds (HBG), and green buds (LFC) (Figure 1A). Then, the total anthocyanin was extracted and the resulting color were displayed in Figure 1B, which reflect the anthocyanin content of different clones. Total anthocyanin present in the leaves were quantified, with JFC Toona sinensis found to have the highest concentration of anthocyanin, followed by BSH, FHXN and HBG, and LFC (Figure 1C). Obviously, LFC exhibited green leaves due to a lack of anthocyanin accumulation.
The leaf color of other clones was pink buds (FHXN), light-red with a little green buds (BSH), dark-purple buds (HBG), and green buds (LFC) (Figure 1A). Then, the total anthocyanin was extracted and the resulting color were displayed in Figure 1B, which reflect the anthocyanin content of different clones. Total anthocyanin present in the leaves were quantified, with JFC Toona sinensis found to have the highest concentration of anthocyanin, followed by BSH, FHXN and HBG, and LFC (Figure 1C). Obviously, LFC exhibited green leaves due to a lack of anthocyanin accumulation.
Metabolome Analysis of Anthocyanin in Five Toona Clones
To compare the flavonoids and anthocyanin compound composition among five Toona clones, three biological replicates of each clone were sampled to detect anthocyaninrelated metabolites employing the UPLC-MS/MS platform. The resulting metabolite composition data sets underwent PCA analysis (Figure 2A). The PCA plot for the anthocyaninrelated metabolites showed a clear separation between the red, pink, light red, green, dark-purple samples. A total of 43, 40, 44, 40, and 32 anthocyanin-related metabolites were identified in leaves of JFC, BSH, FHXN, HBG, and LFC, respectively (Table S1). A total of 31 anthocyanins were identified in all of the samples. Then, the |log2(fold-change)| ≥ 1 and p value < 0.05 was used to determine significantly metabolites between groups (Figure 2B). In group LFC vs. JFC, 23 upregulated metabolites and 8 downregulated metabolites were found to be significantly different (Figure 2B). Data are shown as mean ± standard deviation of three biological replicates.
Table 1 .
Correlation analysis of transcription factors and plant hormone-related genes with anthocyanin-related metabolites.
Table 2 .
The primer sequences used in this study.
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Domain: Biology Environmental Science<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Targeting the CD47/SIRPα axis has become a promising strategy to promote tumor elimination through innate immunity. This review focuses on development, safety, and efficacy of agents that target the CD47/SIRPα axis in preclinical and clinical studies.
CD47/SIRPα: the molecules and biology
SIRPα [16,17], also named SHPS-1 or CD172a, is a transmembrane glycoprotein mainly expressed on neurons and myeloid cells that is particular enriched on macrophages. Human SIRPα is coded by the SHPS-1 gene located at human chromosome 20p13. The open reading frame region is composed of eight exons, including a signal peptide, extracellular domain, a transmembrane segment, and three parts of one cytoplasmic domain. The extracellular domain consists of three Ig-like regions, an NH2-terminal immunoglobulin (Ig) variable (V) region (domain 1, D1), and two Ig constant (C) regions (domain 2 and 3). The cytoplasmic region contains two immunoreceptor tyrosine based inhibitory motifs (ITIMs) and a proline-rich region (YYYY), which bind to Src homology (SH2) domain-containing molecules.
CD47 is a 52 kD transmembrane glycoprotein belonging to the immunoglobulin superfamily. Human CD47 is encoded by the CD47 gene located at the q13.12 region of chromosome 3. Human CD47 contains an NH 2 -terminal Ig variable-like extracellular domain (ECD), a 5-transmembrane spanning helical bundle domain, and a short intracellular COOH-terminal domain (CTD) [18]. CD47 is an essential component of the innate immune system, and binding of its extracellular domain with its ligands αVβ3, SIRPα, and thrombospondin-1 (Tsp-1) activates different signaling pathways that control cell proliferation and differentiation, angiogenesis, and immune regulation. The CTD is alternatively spliced and can exist as four isoforms, ranging from 4 to 36 residues. A schematic diagram of the compositions of CD47 and SIRPα proteins are shown in Fig. 1.
Therapeutic strategy
The critical role of the CD47/SIRPα axis in the innate immune response suggests that these two proteins may be attractive therapeutic targets. Antagonists targeting the innate immune checkpoint CD47/SIRPα pathway are currently in clinical development. These antagonists include 1) monoclonal antibodies targeting CD47 or SIRPα, 2) SIRPα-Fc fusion proteins, 3) bispecific antibodies (BsAb), 4) small molecules to down-regulate CD47 on tumor cells, 5) RNAi and, 6) CD47-chimeric antigen receptor-T cell/Macrophages.
Monoclonal antibodies and fc fusion proteins
Three types of agents targeted to the CD47/SIRPα axis were developed: antibodies, SIRPα-Fc fusion proteins targeted to CD47, and antibodies targeted to SIRPα. The mechanisms of CD47-SIRPα blocking agents are summarized in Fig. 3. Agents targeted to CD47 should block the CD47-SIRPα interaction to remove the anti-phagocytic signal and restore the phagocytic activity of macrophages [27]. In addition, engagement of FcRs to limit activity is considered to be necessary for agents targeted to CD47 [28]. In addition, anti-SIRPα antibodies using inert Fc to prevent toxicity resulting from SIRPα expressed on myeloid immune cell perhaps have therapeutic potential.
The second therapeutic mechanism is bridging innate and acquired immunity [29]. Tumor cells are recognized, taken up by antigen presenting cells (APCs, such as dendritic cells and macrophages), and presented to naive T cells, resulting in T cell activation. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. RNA-seq, bioinformatics analysis, and TCGA database analysis were used to explore the target genes and molecular signaling pathways between the proliferation class (G1-G3, group H) and non-proliferation class (G4-G6, group L). We compared two or multiple gene expressions under different conditions using statistical methods, identi ed the speci c genes that correlated with the conditions, and analyzed the biological signi cance (quality control, matching, quantitative analysis process, signi cant difference analysis, and function of enrichment) of these speci c genes.
Statistical analysis
Statistical analysis was conducted by GraphPad Prism version 8 software (GraphPad Software, San Diego, CA, USA) and IBM SPSS Version 25 software. The chi-square test (Fisher exact probability method) was used to analyze the correlation between the G1-G6 molecular classi cation and tumor biological behavior. After checking data for normal distribution and variance homogeneity, comparisons between two groups were evaluated using independent-sample t-tests or Mann-Whitney U test. For three or more groups, differences were statistically analyzed by one-way ANOVA or the non-parametric test (Kruskal-Wallis test). Kaplan-Meier and Cox proportional hazards survival regression analysis were used to evaluate the prognostic signi cance of the G1-G6 molecular classi cations within 36 months. All p values were two-tailed, and P < 0.05 was considered statistically signi cant.
Distribution of G1-G6 subgroups in HCC patients from Europe and China studies Gene expression analysis was performed on surgically resected HCC samples from Chinese patients who were grouped into G1-G6 transcriptomic categories according to the expression of 16 predictor genes. First, we isolated RNA samples from 120 HCC tumor samples for HCC classi cation, and 107 RNA samples with su cient tissue quantity and good quality were analyzed by qPCR. Data from these samples were analyzed according to the standard method proposed by the Europe group [8]. We found that the HCC cases from China (n=107) were distributed as follows: G1: n=19 (17.76%), G2: n=2 (1.87%), G3: n=20 (18.69%), G4: n=10 (9.35%), G5: n=25 (23.36%), and G6: n=31 (28.97%) subgroups.
Several studies have shown that no matter what molecular typing method is used, HCC can be de ned into two major groups, including a proliferation group and a non-proliferation group. Proliferation tumors are associated with aberrant activation of signaling pathways, while non-proliferative tumors display a well-differentiated phenotype [15,16]. Correspondingly, G1-G3 (combined with G1, G2, and G3) of the G1-G6 molecular typing can be classi ed as a proliferation group, while G4-G6 (combined with G4, G5, and G6) was classi ed as a non-proliferative group. Next, we pooled classes into the G1-G3 group and G4-G6 group. We found that the proportions of the G1-G3 group and G4-G6 group in the China cohort were 38.32% and 61.68%, respectively. In the European cohort, 35 I-II 4 2 3 0 6 9 III-IV 15 0 14 10 19 22 TNM stage 107 I-II 8 2 10 6 18 19 III-IV 11 0 To analyze the correlation between proliferation and clinicopathological characteristics of HCC patients based on G1-G6 molecular classi cation, we integrated the proliferative group (G1-G3) and nonproliferative group (G4-G6) by clinicopathological information. We found that the proliferative group (G1-G3) was correlated with high serum AFP level (P=0.002), high copy number of HBV DNA (P=0.047), complex histological subtype (P=0.010), macrovascular invasion (P=0.012), and negative or weak positive Hep-Par1 (P=0.012) ( Table 4). Using follow-up data of patients after hepatectomy, we also analyzed the correlation between HCC prognosis and G1-G6 molecular classi cation. As shown in Fig. 1C, the G1-G3 HCC patients showed a shorter 3-year OS than G4-G6 HCC patients (P=0.010). The recurrence-free survival of the G1-G3 group tended to be shorter than the G4-G6 group (P=0.072) ( Figure 1D). Click to download.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. Next, the sections were incubated overnight with the primary antibodies at 4°C. A section was incubated in antibody diluent alone without the primary antibody as a control. The sections were further incubated with ImmPress horseradish peroxidase anti-rabbit IgG antibody (Maixin Inc., Fujian, China) secondary antibodies. The immunoreactions were visualized using a DAB Kit (Maixin Inc.). Following counterstaining and mounting, digital images from the sections were created using a wholeslide scanner (KF-PRO-005-EX, KFBIO, Ningbo, China) and images were captured and analyzed using K-Viewer version 1.5.3.1 software (KFBIO).
RNA-seq and bioinformatics analysis
Total RNA was extracted from tissues using Trizol reagent (TransGen Biotech) according to the manufacturer's instructions. RNA quality was con rmed using an Agilent 2100 Bioanalyzer. Libraries for sequencing were created with the Illumina NEBNext® Ultra™ RNA Library Prep Kit or NEBNext® Ultra™ Directional RNA Library Prep Kit. Brie y, PCR ampli cation was performed to obtain the nal DNA library.
After the library was constructed, a Qubit2.0 Fluorometer was used for the preliminary quanti cation. RNA-seq was performed on the Illumina sequencing platform by Shanghai Jikai Company (Shanghai, China). Quality control indicated that the sequencing error rates and data ltering reads of the 24 samples were controlled within the acceptable range. RNA-seq, bioinformatics analysis, and TCGA database analysis were used to explore the target genes and molecular signaling pathways between the proliferation class (G1-G3, group H) and non-proliferation class (G4-G6, group L). We compared two or multiple gene expressions under different conditions using statistical methods, identi ed the speci c genes that correlated with the conditions, and analyzed the biological signi cance (quality control, matching, quantitative analysis process, signi cant difference analysis, and function of enrichment) of these speci c genes.
Statistical analysis
Statistical analysis was conducted by GraphPad Prism version 8 software (GraphPad Software, San Diego, CA, USA) and IBM SPSS Version 25 software. The chi-square test (Fisher exact probability method) was used to analyze the correlation between the G1-G6 molecular classi cation and tumor biological behavior. After checking data for normal distribution and variance homogeneity, comparisons between two groups were evaluated using independent-sample t-tests or Mann-Whitney U test. For three or more groups, differences were statistically analyzed by one-way ANOVA or the non-parametric test (Kruskal-Wallis test). Kaplan-Meier and Cox proportional hazards survival regression analysis were used to evaluate the prognostic signi cance of the G1-G6 molecular classi cations within 36 months. All p values were two-tailed, and P < 0.05 was considered statistically signi cant.
Distribution of G1-G6 subgroups in HCC patients from Europe and China studies Gene expression analysis was performed on surgically resected HCC samples from Chinese patients who were grouped into G1-G6 transcriptomic categories according to the expression of 16 predictor genes. First, we isolated RNA samples from 120 HCC tumor samples for HCC classi cation, and 107 RNA samples with su cient tissue quantity and good quality were analyzed by qPCR. Data from these samples were analyzed according to the standard method proposed by the Europe group [8]. We found that the HCC cases from China (n=107) were distributed as follows: G1: n=19 (17.76%), G2: n=2 (1.87%), G3: n=20 (18.69%), G4: n=10 (9.35%), G5: n=25 (23.36%), and G6: n=31 (28.97%) subgroups.
Several studies have shown that no matter what molecular typing method is used, HCC can be de ned into two major groups, including a proliferation group and a non-proliferation group. Click to download.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. The primary antibodies are listed in Table 2. The membranes were washed ve times and then incubated with peroxidase-conjugated secondary antibodies (1:3,000, ab6721, Abcam, Cambridge, MA, USA) in blocking buffer for 1 h at RT.
Band intensities were quantitated by an enhanced chemiluminescence detection system using the SuperSignal™ West Pico Plus Kit (Thermo Fisher Scienti c Inc.). The protein density was quanti ed with Bio-Rad Image Lab software and ImageJ software. Tissue microarray (TMA) construction A total of 58 para n-embedded tissue samples from HCC patients (G1: 14 cases, G2: 1 case, G3: 14 cases, G4: 9 cases, G5: 10 cases, and G6: 10 cases) were randomly selected under a strati ed sampling procedure to generate TMAs. The donor wax blocks were made of the para n sections and stained by hematoxylin-eosin (HE) staining. To exclude tissues with necrotic or bleeding areas, the corresponding positions of cancer tissues and adjacent liver tissues were observed and marked under a microscope.
The recipient block was cast by melting conventional para n wax in molds for making blank blocks. The donor tissue blocks were transferred into the recipient wax wells and prepared with a 1.5 mm perforated needle. Next, we placed the wax blocks in the oven at 65°C for approximately 7-9 min and removed them at the semi-melted state; the blocks were cooled slightly at room temperature and moved to a refrigerator at 4°C. The freeze-thaw process was repeated so that the tissue core in the blocks and the wall of the pore were integrated.
The sections were treated with a 3 % peroxidase solution to block endogenous peroxidase. The sections were then incubated with 5 % BSA blocking solution to reduce the non-speci c background signal and false positives. Next, the sections were incubated overnight with the primary antibodies at 4°C. A section was incubated in antibody diluent alone without the primary antibody as a control. The sections were further incubated with ImmPress horseradish peroxidase anti-rabbit IgG antibody (Maixin Inc., Fujian, China) secondary antibodies. The immunoreactions were visualized using a DAB Kit (Maixin Inc.). Following counterstaining and mounting, digital images from the sections were created using a wholeslide scanner (KF-PRO-005-EX, KFBIO, Ningbo, China) and images were captured and analyzed using K-Viewer version 1.5.3.1 software (KFBIO).
RNA-seq and bioinformatics analysis
Total RNA was extracted from tissues using Trizol reagent (TransGen Biotech) according to the manufacturer's instructions. RNA quality was con rmed using an Agilent 2100 Bioanalyzer. Libraries for sequencing were created with the Illumina NEBNext® Ultra™ RNA Library Prep Kit or NEBNext® Ultra™ Directional RNA Library Prep Kit. Brie y, PCR ampli cation was performed to obtain the nal DNA library.
After the library was constructed, a Qubit2.0 Fluorometer was used for the preliminary quanti cation. RNA-seq was performed on the Illumina sequencing platform by Shanghai Jikai Company (Shanghai, China). Quality control indicated that the sequencing error rates and data ltering reads of the 24 samples were controlled within the acceptable range. Click to download.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. 6A). Compared with group L (G4-G6 subgroup), there were 1,269 up-regulated and 1,445 down-regulated genes in group H (G1-G3 subgroup), and 33,692 genes showed no differential expression between the two groups (Fig. 6B). We also found that 108 genes overlapped between the H group (compared with group L) and CA (compared with group pCA) (Fig. 6C), and 27 genes were commonly down-regulated ( Fig. 6D). Among the 27 genes, four genes (REG1B, REG3G, C19orf18, and ITPR1) were signi cantly upregulated or downregulated in group H compared with group L ( Table 6). Analysis using TCGA database also con rmed that the expression levels of REG1B, REG3G, and ITPR1 were signi cantly increased in HCC liver tissues compared with normal liver tissues (Fig. 6E). Using the follow-up information database of TCGA patients, Kaplan-Meier analysis was conducted to analyze the prognosis of HCC patients. As shown in Fig. 6F, HCC patients with a high expression of ITPR1 showed a signi cantly shorter 3-year OS than HCC patients with low or medium expression of ITPR1. However, there were no statistical differences between HCC patients with high expressions of REG1B or REG3G and the corresponding control groups. These results suggested that ITPR1 may promote the occurrence and development of HCC and affect the prognosis of HCC patients. We next performed KEGG pathway analysis using the KEGG database and Clusterpro le software. Fisher exact analysis was used to test and calculate p-values, and the pathways with a p-value less than 0.05 were retained. The p-value represented the signi cance of enrichment of differential genes; a lower the pvalue indicates a greater correlation between the pathway and the differential genes. A Padj value less than 0.05 was taken as the threshold of signi cant enrichment. The KEGG pathway enrichment analysis revealed that the calcium signaling pathway, cGMP-PKG signaling pathway, renin secretion, neuroactive ligand-receptor interaction, and cell metabolism-related signaling pathways were signi cantly enriched in the G1-G3 subgroups (Fig. 7A-B).
Discussions
Although our results were generally consistent with the studies from Europe and Singapore, there were still many inconsistencies. Click to download.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Six SIRPα-fusion proteins are currently in phase I or phase II clinical trials (Table 2, ordered by clinical trial developmental state).
Evorpacept (ALX148)
Evorpacept is comprised of SIRPα variant 1 domain 1 (V1D1) and inactive human IgG1-Fc [51]. Evorpacept (CV1) was selected from mutant libraries and includes 9 mutations (V6I, A271, I31F, E47V, K53R, E54S, H56P, L66T, V92I), which resulted in a 50,000-fold increase in affinity compared with that of wild type SIRPα. Preclinical data showed that evorpacept augmented macrophage antitumor activity in vitro and in vivo in combination with tumor-opsonizing antibodies (trastuzumab, obinutuzumab and cetuximab). However, no phagocytosis or antitumor activity was observed following treatment with evorpacept alone. A trial [52] in which patients with NHL received evorpacept alone at 10 mg/kg or at 15 mg/kg in combination with rituximab resulted in ORRs of 40.9 and 63.6%, respectively. The CD47 receptor occupancy on RBC and CD4 T cells was approximately 90% at 10-15 mg/ kg. A phase I study [53] of evorpacept in combination with pembrolizumab, trastuzumab, or zanidatamab, and/or chemotherapeutic agents, evaluating treatment of advanced solid malignancy is ongoing. Preliminary results showed anti-cancer activity of evorpacept in combination with pembrolizumab (AP) and/or chemotherapy (5FU + platinum) in patients with second line or greater HNSCC (head and neck squamous cell carcinoma) with prior platinum therapy. The ORR in patients with checkpoint inhibitor-naïve HNSCC (n = 10) treated with AP was 40%, but 0% in patients with HNSCC who had previously received checkpoint inhibitors (n = 10). A phase II study of evorpacept in combination with pembrolizumab for treatment of HNSCC was recently initiated. Treatment with evorpacept in combination
Antibodies targeting SIRPα on myeloid cells
Anti-SIRPα antibodies induce weak or no phagocytic activity alone, but induce significantly increased phagocytic activity when combined with opsonizing antibodies (rituximab, cetuximab) [58][59][60]. Several issues with anti-SIRPα antibodies are important to consider. First, since SIRPα is expressed on myeloid cells, anti-SIRPα antibodies using inactive human IgG-Fc to avoid Fc effector-mediated toxicity on these immune cells may be advantageous [60]. Second, SIRPγ expressed on T and NK cell shares 74.37% amino acid similarity with the extracellular domain with SIRPα [61]. SIRPγ on T cells binds to CD47 on APCs to mediate cell-cell adhesion and enhances antigen presentation, resulting in T cell proliferation and cytokine secretion [62]. Development of a SIRPα targeting antibody with specificity toward SIRPα to avoid interference with the interaction between CD47 and SIRPγ may preserve T cell activity. Third, antibody internalization could lead to rapid clearance of antibody in vivo. Higher doses or multiple dosing is required to ensure that levels remain therapeutically relevant [63,64]. Internalization of SIRPα decreases the inhibitory signal and may enhance the ability of antibodies to restore phagocytic activity [59,65]. Finally, Although SIRPα has ten known variants, V1, V2, and V8 are the most prominent (over 90%) haplotypes in the human population. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. Several studies showed in amed and membrane expression of PD-L1 in ancestral liver cancer HCC, while lymph epithelioma-like HCC is in amed by in ammatory cells that express PD-L1 in large numbers [16,19,20]. The expression pattern of PD-L1 has an important effect on HCC prognosis, which is poor if both tumor cells and macrophages express PD-L1 [21]. Screening of patients who may bene t from PD-1/PD-L1 inhibitor therapy is the most important clinical concern. The correlation between G1-G6 classi cation and tumor cell PD-L1 expression is not clear. Therefore, we examined the expression of the PD-L1 by IHC in a TMA. The TMA comprised 58 resected HCC specimens and 58 specimens of adjacent non-malignant and premalignant para-cancer liver tissue from the same patients. Staining was compared on consecutive tissue sections of the TMA to enable automated (unbiased) image analysis and direct comparison of the samples. The staining was quanti ed according to the percentage of area stained for each protein in comparison with isotype-matched control IgG (Fig. 3A). Representative images for the positive PD-L1 antibody staining are shown in Fig. 3B, along with histograms showing the pooled quanti ed data for the antigen (Fig. 3C). We found that the positive staining of PD-L1 in G1-G3 subgroups was signi cantly higher than in G4-G6 subgroups (P=0.028).
G5 and G6 HCC subgroups are associated with activation of the Wnt/β-catenin pathway signaling Aberrant activation of Wnt/β-catenin signaling plays a key role in HCC progression, with approximately half of HCC cases acquiring mutations in either CTNNB1 or AXIN1 [22]. Glutamine synthetase (GS) is classically overexpressed when the Wnt/β-catenin signaling pathway is activated due to CTNNB1 mutations and could be used as surrogate marker [23]. In our study, we found that the protein levels of βcatenin were substantially higher in G5 and G6 HCC subgroups than in G1-G4 subgroups ( Fig. 4A and B). Additionally, the mRNA expression level of GS in HCC liver tissues was signi cantly increased compared with levels in para-cancer liver tissues, and the G5 and G6 HCC subgroups showed signi cantly higher expression levels of GS than the G1-G4 subgroups (Fig. 4C). In line with these results, western blot analysis showed that protein levels of β-catenin and GS were higher in G5 and G6 subgroups than other subgroups (Fig. 4D). Click to download.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. 3B, along with histograms showing the pooled quanti ed data for the antigen (Fig. 3C). We found that the positive staining of PD-L1 in G1-G3 subgroups was signi cantly higher than in G4-G6 subgroups (P=0.028).
G5 and G6 HCC subgroups are associated with activation of the Wnt/β-catenin pathway signaling Aberrant activation of Wnt/β-catenin signaling plays a key role in HCC progression, with approximately half of HCC cases acquiring mutations in either CTNNB1 or AXIN1 [22]. Glutamine synthetase (GS) is classically overexpressed when the Wnt/β-catenin signaling pathway is activated due to CTNNB1 mutations and could be used as surrogate marker [23]. In our study, we found that the protein levels of βcatenin were substantially higher in G5 and G6 HCC subgroups than in G1-G4 subgroups ( Fig. 4A and B). Additionally, the mRNA expression level of GS in HCC liver tissues was signi cantly increased compared with levels in para-cancer liver tissues, and the G5 and G6 HCC subgroups showed signi cantly higher expression levels of GS than the G1-G4 subgroups (Fig. 4C). In line with these results, western blot analysis showed that protein levels of β-catenin and GS were higher in G5 and G6 subgroups than other subgroups (Fig. 4D). Further statistical analysis found that the ratio of GS expression to β-catenin expression in the G5 and G6 groups was approximately three-fold higher compared with levels in the G1-G4 subgroups, which suggests that the Wnt/β-catenin pathway is highly activated in G5 and G6 HCC. The activation level was highest in G6. These results suggested that G5 and G6 HCC subgroups were closely associated with the robust activation of Wnt/β-catenin signaling.
Transcriptome sequencing analysis for G1-G6 molecular classi cation of Chinese HCC patients The results of our study suggest that G1-G3 group can be used as an independent risk factor for evaluating HCC patients' prognosis and is signi cantly correlated with poor prognosis after hepatectomy.
To clearly understand the poor prognosis group based on G1-G6 molecular classi cation of Chinese HCC patients, 12 samples of HCC tissues (2 cases of each subgroup) and the corresponding adjacent liver tissues were randomly selected. The expression levels of genes related to differentiation, proliferation, and function of tumor cells were examined in these samples and analyzed by RNA-seq and bioinformatics. The error rates of the 24 samples were all within the acceptable range ( Supplementary Fig. 1). The proportions of ltered reads of the 24 samples were all within the acceptable range, and the proportion of clean reads of each sample accounted for more than 90%, which suggested that the data were suitable for subsequent analysis (Supplementary Fig. 2).
The all HCC group (CA group) included 12 samples of HCC tissues, and the para-HCC group (pCA group) included 12 corresponding samples of adjacent liver tissues. High proliferative group (group H) includes 6 samples of G1-G3 cancer tissues, and low proliferative group (group L) includes 6 samples of G4-G6 cancer tissues. Compared with the pCA group, there were 3233 up-regulated genes and 1654 downregulated genes in the CA group, while 33,299 genes showed no changes between the two groups (Fig. Click to download.
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Domain: Biology Medicine
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Golubovskaya et al. [84] showed that CD47-CAR-T cells effectively killed ovarian, pancreatic, and other cancer cells, and induced production of high levels of IL-2, which correlated with expression of CD47 antigens. Treatment with CD47-CAR-T cells may be a novel strategy for treating different types of cancers. Huyen [85] designed a third generation of CD47-CAR-T cell that could effectively kill lung cancer cells (A549) and inhibit lung cancer cell metastasis. A dual CAR-T targeting CD47 and TAG-72 (tumor-associated glycoprotein 72) generated by Shu [86] showed promising results against ovarian cancer in preclinical experiments. An Anti-PD-L1 (A12) CAR-T with the ability to secrete anti-CD47 VHH (variable heavy domain of heavy chain antibodies or nanobodies) (A4), developed by Xie [87], represented a novel strategy for cancer treatment. An A12-A4 CAR-T showed better anti-cancer activity than an A12 CAR-T plus soluble A4 in C57BL/6 PD-L1-KO mice bearing B16F10 cells. Each of these CD47-CAR-T cells are in the preclinical stage, and efficacy and safety should be further investigated. Development of CAR-M1 (M1: classically activated macrophages) is an emerging therapeutic strategy [88], and several studies of engineered CAR-M cells [89][90][91] showed tumor cell elimination activity in vitro and in vivo. These studies showed that CAR-M induced phagocytosis and induced M2 to M1 polarization through secretion of pro-inflammatory factors and chemokines [90]. CD47 is ubiquitously expressed on the surfaces of multiple hematopoietic and solid tumor cells. However, adverse events due to cytokine secretion by macrophages during immune checkpoint activation [92,93] and CAR-T [94,95] treatment was common. Technological improvements for preparation and production of CAR-M are needed to offer scalable and reproducible manufacturing processes.
Small molecules, peptides, and microRNA
RRx-001 [96] is an anticancer agent designed to induce M2 to M1 polarization and to promote recovery of phagocytic activity of macrophages toward tumor cells.
The anti-phagocytic inhibitory signal was removed or reduced through downregulation of both CD47 and SIRPα gene expression on tumor cells and macrophages, respectively. Elimination of tumor cells was shown in in vitro and in vivo. Phase III clinical trials (NCT03699956, NCT02489903) against small cell lung cancer [97,98] are ongoing.
D4-2 [99], a macrocyclic peptide targeted to mouse SIRPα was designed to inhibit the interaction between CD47 and SIRPα and promote macrophage-mediated phagocytosis of tumor cells when combined with rituximab. PKHB1 [100], a TSP-1-derived CD47 agonist peptide, induced cell death (CRT exposure and DAMP release) in chronic lymphocytic leukemia cells.
MicroRNAs (miRNAs), which are 20-22 nucleotides in length, play important roles in cancer pathogenesis and progression since they can repress the target gene at the translational level by directly binding to the 3'untranslated regions (3'UTRs) [101].
Overexpression miR-378a [102] in mice peritoneal macrophages downregulates SIRPα mRNA expression. Phagocytosis of Ishikawa cells by macrophages-miR-378a and macrophages was carried out in vitro. Phagocytic index in macrophages-miR-378a group is 3 times than that in macrophages group.
Zhao [103] reported that miR-200a inhibited the expression of CD47 by directly targeting the 3'UTR of the CD47 mRNA. MicroRNA 200a suppressed nasopharyngeal carcinoma (NPC) cell proliferation, migration, and invasion, and promoted phagocytosis of NPC cells by Fig. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. However, only a portion of patients profit from T cell-based immunotherapy. Macrophages, the most abundant type of innate immune cells in the body, play an important role in eliminating tumor cells and infectious microbes. The phagocytic check point protein CD47 inhibits the phagocytic activity of macrophages through binding to SIRPα expressed on macrophages. Blockade of the interaction between CD47 and SIRPα could restore phagocytic activity and eliminate tumor cells in vitro and in vivo. In this manuscript, we review the mechanism of action and developing status of agents (antibodies targeting CD47 and SIRPα, SIRPα-Fc fusion proteins, and bi-specific antibodies) that block CD47/SIRPα interaction in presymptomatic studies and in the clinical setting. In addition, small molecules, mRNA, and CAR-T/M that target the CD47/SIRPα axis are also reviewed in this article.
Introduction
Tumor cells evade immune destruction by transmitting inhibitory signals to lymphocytes and myeloid cells [1]. Blockade of these inhibitory molecules, which include CTLA4, PD-1, and PD-ligand 1 (PD-L1), could restore T cell function and promote elimination of tumor cells. Immune checkpoint inhibitors (ICIs) have improved outcomes for patients with multiple types of cancers. However, many patients do not respond to this type of immunotherapy [2,3]. In some cases, these therapeutic agents have been associated with disease progression [4,5], the cause of which is currently being investigated. Therefore, drugs that act on a novel class of targets to immobilize a broader immune cell population are needed to improve upon current therapeutic options.
Macrophages [6] are typically the first dedicated innate immune cells to detect the presence of infectious pathogens or tumor cells. Macrophages are derived from monocyte precursors that circulate in the blood and migrate into tissues, after which they differentiate into tissue macrophages such as Kupffer cells in the liver, alveolar macrophages in the lung, and microglia in the brain. Circulating monocytes and resident tissue macrophages can directly kill tumor cells via phagocytosis (innate immune response) and can activate the adaptive immune response. However, these immune responses can be inhibited by ligand binding to inhibitory receptors expressed on the macrophage cell surface [7]. Signal regulatory protein alpha (SIRPα) is a transmembrane protein expressed on all myeloid cells, including monocytes, macrophages, and neutrophils. SIRPα contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can be phosphorylated, resulting in recruitment of inhibitory molecules such as Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2 [8]. Binding of CD47 to SIRPα triggers coupling of SIRPα to these phosphatases, resulting in inhibition of phagocytic activity [9,10]. CD47 is ubiquitously expressed on many types of cells to prevent phagocytosis by phagocytes. However, tumor cells overexpress CD47 to evade the immune system through inhibition of myeloid cell-mediated elimination [11]. Inhibition of CD47-SIRPα interaction restored the phagocytic activity of phagocytes in vitro and in vivo [12][13][14][15]. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. Further analysis showed that G1 patients tended to have a shorter 3-year OS than non-G1 patients (P=0.1867) ( Figure 1E) and G3 patients had the worst prognosis (P=0.0125) ( Figure 1F). Cox regression univariate analysis showed that the TNM stage (P=0.0002), BCLC stage (P=0.001), and G1-G6 molecular classi cation (P=0.012) were important factors that affected the overall prognosis of HCC patients. All factors with a p-value < 0.1 detected in univariate analyses were included in multivariate analyses, and the subsequent results showed that the G1-G6 molecular classi cation (P=0.035) is an independent prognostic risk factor for poor OS of HCC patients (Table 5). Predictor of HCC classi cation using qPCR A total of 19 para-cancer liver tissue samples were randomly selected to investigate the correlation between expression of the 16 genes in the proliferation group (G1-G3) compared with the nonproliferation group (G4-G6). Our results showed that the expression levels of AFP (P=0.0200), CDH2 (P=0.0022), HN1 (P<0.0001), NRAS (P=0.0096), PAK2 (P=0.0024), RAB1A (P=0.0020), and SAE1 (P<0.0001) were signi cantly higher in the proliferation group (G1-G3) than in the non-proliferation group (G4-G6) ( Fig. 2A). Conversely, the expression levels of LAMA3 (P=0.0059) and PAP (P=0.0495) were markedly higher in the non-proliferation group (G4-G6) than in the proliferation group (G1-G3) (Fig. 2B).
There were no signi cant differences in the expressions of the following genes between the proliferation group (G1-G3) and non-proliferation group ( PD-L1 is highly expressed in G1-G3 subgroups Several molecular targeted drugs have entered clinical trials as palliative and complementary treatments for HCC [17]. For example, the clinically approved antibodies targeting PD-1 or its ligand PD-L1 were shown to cause lasting responses in up to 25% of advanced HCC patients in two early trials. In HCC, the proportion of tumor cells expressing PD-L1 is approximately 15% [18]. Several studies showed in amed and membrane expression of PD-L1 in ancestral liver cancer HCC, while lymph epithelioma-like HCC is in amed by in ammatory cells that express PD-L1 in large numbers [16,19,20]. The expression pattern of PD-L1 has an important effect on HCC prognosis, which is poor if both tumor cells and macrophages express PD-L1 [21]. Screening of patients who may bene t from PD-1/PD-L1 inhibitor therapy is the most important clinical concern. The correlation between G1-G6 classi cation and tumor cell PD-L1 expression is not clear. Therefore, we examined the expression of the PD-L1 by IHC in a TMA. The TMA comprised 58 resected HCC specimens and 58 specimens of adjacent non-malignant and premalignant para-cancer liver tissue from the same patients. Staining was compared on consecutive tissue sections of the TMA to enable automated (unbiased) image analysis and direct comparison of the samples. The staining was quanti ed according to the percentage of area stained for each protein in comparison with isotype-matched control IgG (Fig. 3A). Representative images for the positive PD-L1 antibody staining are shown in Fig. Click to download.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. A schematic diagram of the compositions of CD47 and SIRPα proteins are shown in Fig. 1.
Therapeutic strategy
The critical role of the CD47/SIRPα axis in the innate immune response suggests that these two proteins may be attractive therapeutic targets. Antagonists targeting the innate immune checkpoint CD47/SIRPα pathway are currently in clinical development. These antagonists include 1) monoclonal antibodies targeting CD47 or SIRPα, 2) SIRPα-Fc fusion proteins, 3) bispecific antibodies (BsAb), 4) small molecules to down-regulate CD47 on tumor cells, 5) RNAi and, 6) CD47-chimeric antigen receptor-T cell/Macrophages.
Monoclonal antibodies and fc fusion proteins
Three types of agents targeted to the CD47/SIRPα axis were developed: antibodies, SIRPα-Fc fusion proteins targeted to CD47, and antibodies targeted to SIRPα. The mechanisms of CD47-SIRPα blocking agents are summarized in Fig. 3. Agents targeted to CD47 should block the CD47-SIRPα interaction to remove the anti-phagocytic signal and restore the phagocytic activity of macrophages [27]. In addition, engagement of FcRs to limit activity is considered to be necessary for agents targeted to CD47 [28]. In addition, anti-SIRPα antibodies using inert Fc to prevent toxicity resulting from SIRPα expressed on myeloid immune cell perhaps have therapeutic potential.
The second therapeutic mechanism is bridging innate and acquired immunity [29]. Tumor cells are recognized, taken up by antigen presenting cells (APCs, such as dendritic cells and macrophages), and presented to naive T cells, resulting in T cell activation. Antibodies targeted to CD47 could induce direct killing of tumor cells by inhibiting protein kinase A via Giα, resulting in clustering of CD47 in the membrane, and caspase-independent programmed cell death [30].
Anti-CD47 monoclonal antibodies
More than ten anti-CD47 antibodies are in different phases of clinical development ( Table 1). All of these antibodies are based on human IgG4-Fc, except AO-176, which is based on human IgG2-Fc [31]. Clinical study results of these antibodies were as follows (ordered by clinical trial developmental state).
Magrolimab (Hu5F9-G4) Magrolimab, previously known as Hu5F9-G4, was the first anti-CD47 antibody to enter clinical trials, and is currently in Phase III development. The clinical trials for magrolimab are listed in the Table 1. In a preclinical study [32], combined treatment with magrolimab and rituximab showed significant clearance of Raji cells in vitro and elimination of AML tumor cells in vivo. Magrolimab blocks the CD47/ Fig. 2 Biology of CD47/SIRPα interaction. CD47 binds to SIRPα to transmit inhibitory signals to macrophages and to inhibit or lessen phagocytic activity through uncoupling of receptor binding and signal transduction. Therapeutic agents block the interaction between CD47 and SIRPα to remove the inhibitory signal and restore the phagocytic activity SIRPα interaction and the anti-phagocytic signal on macrophages, and binding of rituximab to FcRs initiates pro-phagocytic signaling. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. A phase I trial (NCT03934814) [43] evaluating treatment of R/R advanced solid tumors and lymphoma with TJC4 alone, or in combination with pembrolizumab or rituximab, is ongoing. No DLT or SAE (severe adverse events) were observed, and treatmentassociated anemia occurred in 30% of patients (6 of 20, 2 receiving 3 mg/kg, 2 receiving 10 mg/kg, 1 receiving 20 mg/kg, and 1 receiving 30 mg/kg). Anti-drug antibody (ADA) events occurred in 25% of patients, but there were no concerns regarding safety or pharmacokinetics (PK). Maximal saturation of peripheral T cells was achieved at a dose of 20 mg/kg administered weekly.
AO176 AO176, developed by Arch Oncology, is a humanized IgG2 subclass anti-CD47 antibody [31]. AO176 was shown to bind to integrin-β1 expressed on tumor cells, but not on RBC. Interestingly, AO176 blocked the CD47/SIRPα interaction to stimulate phagocytosis of tumor cells, and also directly killed tumor cells (non-ADCC). A tolerability and hematologic study on Cynomolgus monkeys showed no anemia. In phase I/ II clinical trials [44], Grade 3 TRAE (treatment-related adverse events) were observed in > 10% of patients. In addition, DLT was observed at 20 mg/kg. Studies evaluating AO176 monotherapy and combination therapy with paclitaxel are ongoing.
CC-90002 CC-90002 is a humanized monoclonal IgG4 CD47 antibody. An early clinical trial (NCT02641002) that evaluated treatment of Acute Myeloid Leukemia (AML) and high-risk myelodysplastic syndrome (MDS) was terminated due to poor activity and safety profiles [45,46]. Another phase I trial (NCT02367196) evaluated treatment of advanced solid (alone) and hematologic malignancies (in combination with rituximab). In a combination trial with patients with R/R NHL [47], the ORR (overall response rate) was 13% with 25% achieving stable disease. However, 50% had anemia (of any grade), 33% had thrombocytopenia, and DLTs were observed in 3 subjects (1 subject infusion-related reaction at 15 mg/ kg Q2W and 2 subjects had grade 3 thrombocytopenia requiring platelet transfusion occurring at 30 mg/kg).
SGN-CD47M
SGN-CD47M, developed by Seagen (Seattle Genetics), is a CD47 targeting probody drug conjugate (PDCs). Probody therapeutics [48] are antibody prodrugs designed to remain inactive until proteolytically cleaved and activated in the tumor microenvironment. Probody drug conjugates [49] can be activated by multiple proteases in the tumor microenvironment, but remain inactive in the circulation and in normal tissues.
The efficacy of PDCs depends on multiple factors including binding affinity and specificity for the antigen, efficiency of cleavage in the tumor microenvironment, lack of cleavage in normal tissues, and internalization efficiency. Studies focused on PDCs are in the early clinical stage, and safety and efficacy have yet to be determined. Clinical trial (NCT03957096) of SGN-CD47M for treatment of advanced solid tumors was terminated based on portfolio prioritization.
Other drugs in early clinical development for safety and dosing evaluation are listed in Table 1.
SIRPα-fc Among the 10 allelic variants of SIRPα, SIRPα V1 and V2 are the most prevalent variants [50]. The affinity of wild type SIRPα binding to CD47 is in the micromolar range, which is 1000-fold weaker than that of anti-CD47 antibodies. Soluble SIRPα binding to CD47 on tumor cells could block inhibitory signals and enhance phagocytic activity. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process.
β-catenin is a key molecular marker in the epithelial-mesenchymal transition. In physiological conditions, β-catenin binds cadherin and promotes the release of cadherin when β-catenin translocates to the nucleus [27]. In HCC, E-cadherin is reduced on the cell surface and N-cadherin is expressed once tumor cells undergo epithelial-mesenchymal transition [28]. It implicates that G5 and G6 subgroups may have some features as mentioned above.
We found several additional correlations in this study that had not been reported previously. For example, the G1-G3 group was associated with PD-L1 expression and liver cancer stemness. These ndings provide a new direction for the diagnosis and treatment of HCC. In the future, if G1-G3 patients were treated with targeted therapy for PD-1/PD-L1 and liver cancer stem cells, this may improve the survival and prognosis of a patient with highly malignant HCC with an extremely poor prognosis.
The pathogenesis of HCC is complex and involves multiple signaling pathways [29,30]. Signaling pathways involved in growth factors activated by intracellular calcium ions play a crucial role in various physiological and biochemical processes [31]. HCC encodes a variety of tyrosine kinases, including epidermal growth factor receptor (EGFR), whose functions are mainly in glucose and lipid metabolism, liver brosis, regeneration, and carcinogenesis [32,33]. Modica et al. [34] reported that EGF-dependent HCC proliferation may be affected by calcium ion, and intracellular dissociative calcium is a key regulator for EGFR signal propagation and apoptosis of tumor cells. Calcium not only promotes the proliferation of proliferating HCC cells but also induces the apoptosis of non-proliferating HCC cells, which might contribute to the reason why the proliferation group (G1-G3) has a poor prognosis. Considering the high hepatic activity of calcium and the overexpression of calcium channels in G1-G3 group, manipulation of calcium in ux in HCC cells may inhibit tumor metastasis.
ITPR1 encodes an intracellular receptor for type 1 inositol 1,4,5-triphosphate and mediates the release of calcium from the endoplasmic reticulum. A few recent studies indicated that ITPR1 affects tumor progression by regulating autophagy. For example, ITPR1 can protect renal cancer cells against natural killer cells by inducing autophagy [35,36]. In our study, we found that HCC patients with a high expression level of ITPR1 have a signi cantly lower 3-year OS rate than HCC patients with low or medium expression levels of ITPR1. Therefore, we speculate that ITPR1 may cooperate with calcium ions to promote the proliferation of proliferating HCC cells, thus leading to the increase of postoperative recurrence risk of HCC patients.
As a second messenger of cell information transmission, cGMP promotes cell division and inhibits cell differentiation. Our RNA-seq analysis showed that the cGMP-PKG signaling pathway was enriched in G1-G3 type HCC, which might contribute to the proliferative characteristics of the G1-G3 subgroups. In this study, we found that G1-G3 type HCC patients showed a high serum AFP level. A differential analysis based on the HCC transcriptome data from TCGA showed that the genes expressed in HCC tissues with high AFP expression are involved in neuroactive ligand-receptor interaction pathways [37]. Click to download.
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Domain: Biology Medicine
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Antibodies targeted to CD47 could induce direct killing of tumor cells by inhibiting protein kinase A via Giα, resulting in clustering of CD47 in the membrane, and caspase-independent programmed cell death [30].
Anti-CD47 monoclonal antibodies
More than ten anti-CD47 antibodies are in different phases of clinical development ( Table 1). All of these antibodies are based on human IgG4-Fc, except AO-176, which is based on human IgG2-Fc [31]. Clinical study results of these antibodies were as follows (ordered by clinical trial developmental state).
Magrolimab (Hu5F9-G4) Magrolimab, previously known as Hu5F9-G4, was the first anti-CD47 antibody to enter clinical trials, and is currently in Phase III development. The clinical trials for magrolimab are listed in the Table 1. In a preclinical study [32], combined treatment with magrolimab and rituximab showed significant clearance of Raji cells in vitro and elimination of AML tumor cells in vivo. Magrolimab blocks the CD47/ Fig. 2 Biology of CD47/SIRPα interaction. CD47 binds to SIRPα to transmit inhibitory signals to macrophages and to inhibit or lessen phagocytic activity through uncoupling of receptor binding and signal transduction. Therapeutic agents block the interaction between CD47 and SIRPα to remove the inhibitory signal and restore the phagocytic activity SIRPα interaction and the anti-phagocytic signal on macrophages, and binding of rituximab to FcRs initiates pro-phagocytic signaling. Activation of pro-phagocytic signaling is a beneficial effect of anti-CD47 antibody treatment. Magrolimab caused significant hemagglutination and phagocytosis of RBCs in vitro, which indicated potential toxicity. Non-human primate pharmacokinetic and toxicology studies showed dose-dependent anemia. To overcome treatment-related anemia and thrombocytopenia, a low priming dose was given to stimulate production of new RBCs and to facilitate tolerance of subsequent higher maintenance doses. The same strategy (1 mg/kg priming dose on day 1) effectively controlled anemia during subsequent infusion of magrolimab in a clinical trial. The saturation concentration (receptor occupancy) on circulating white and red cells was 30 mg/ kg [33]. In a phase Ib [34] (NCT02953509) study evaluating relapsed or refractory non-Hodgkin's lymphoma, patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma were treated with magrolimab in combination with rituximab. The results showed that 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. Specifically, the rates of objective response and complete response were 40 and 33%, respectively, among patients with DLBCL, and 71 and 43%, respectively, among those with follicular lymphoma. Calreticulin (CRT) is a member of the endoplasmic reticulum lectin chaperone family of proteins that plays important biological roles in Ca 2+ homeostasis (in the endoplasmic reticulum, ER), integrin-dependent cell adhesion (in the cytoplasm), and immune response activation (on the plasma membrane) [35]. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. The correlation between the G1-G6 molecular classi cation and pathological features, biological behaviors, serum marker levels, 3-year overall survival (OS) rate, and other indicators were analyzed in combination with the clinicopathological data of the HCC patients. We also detected and analyzed molecular markers and molecular signal pathways related to the G1-G6 molecular classi cation. RNA sequencing and bioinformatics analysis were performed to screen related targets and molecular signaling pathways.
HCC samples and clinical data
The use of human clinical samples was approved by the Medical Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University. A total of 120 HCC specimens and paired adjacent noncancerous liver tissues were randomly selected from patients undergoing hepatectomy between January 2014 to December 2017. The patient follow-up information and clinicopathological characteristics were obtained from the biological sample bank and original pathology report. We also collected 12 fresh HCC tissues and paired adjacent noncancerous liver tissues for further analysis by RNA-seq. Data from The Cancer Genome Atlas (TCGA) database were obtained from TCGA portal sites ( [URL] real-time PCR (qPCR)
The qPCR procedures were performed as previously described [13]
Western blot
Tissues were harvested and lysed in cold extraction buffer (RIPA, Beyotime Biotechnology, Shanghai, China) as previously described [14]. Samples were centrifuged at 13,000 × g at 4°C for 15 min, and the protein concentration in the supernatants was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scienti c Inc., Waltham, IL, USA) according to the manufacturer's protocol. Equal amounts of protein samples were subjected to 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently electro-transferred to polyvinylidene di uoride membranes. The membranes were blocked with 5 % BSA diluted in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4) containing 0.05 % Tween-20 for 2 h and then incubated with primary antibodies at 4°C overnight. The primary antibodies are listed in Table 2. The membranes were washed ve times and then incubated with peroxidase-conjugated secondary antibodies (1:3,000, ab6721, Abcam, Cambridge, MA, USA) in blocking buffer for 1 h at RT.
Band intensities were quantitated by an enhanced chemiluminescence detection system using the SuperSignal™ West Pico Plus Kit (Thermo Fisher Scienti c Inc.). The protein density was quanti ed with Bio-Rad Image Lab software and ImageJ software. Tissue microarray (TMA) construction A total of 58 para n-embedded tissue samples from HCC patients (G1: 14 cases, G2: 1 case, G3: 14 cases, G4: 9 cases, G5: 10 cases, and G6: 10 cases) were randomly selected under a strati ed sampling procedure to generate TMAs. The donor wax blocks were made of the para n sections and stained by hematoxylin-eosin (HE) staining. To exclude tissues with necrotic or bleeding areas, the corresponding positions of cancer tissues and adjacent liver tissues were observed and marked under a microscope.
The recipient block was cast by melting conventional para n wax in molds for making blank blocks. The donor tissue blocks were transferred into the recipient wax wells and prepared with a 1.5 mm perforated needle. Next, we placed the wax blocks in the oven at 65°C for approximately 7-9 min and removed them at the semi-melted state; the blocks were cooled slightly at room temperature and moved to a refrigerator at 4°C. The freeze-thaw process was repeated so that the tissue core in the blocks and the wall of the pore were integrated.
The sections were treated with a 3 % peroxidase solution to block endogenous peroxidase. The sections were then incubated with 5 % BSA blocking solution to reduce the non-speci c background signal and false positives. Click to download.
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Domain: Biology Medicine
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Activation of pro-phagocytic signaling is a beneficial effect of anti-CD47 antibody treatment. Magrolimab caused significant hemagglutination and phagocytosis of RBCs in vitro, which indicated potential toxicity. Non-human primate pharmacokinetic and toxicology studies showed dose-dependent anemia. To overcome treatment-related anemia and thrombocytopenia, a low priming dose was given to stimulate production of new RBCs and to facilitate tolerance of subsequent higher maintenance doses. The same strategy (1 mg/kg priming dose on day 1) effectively controlled anemia during subsequent infusion of magrolimab in a clinical trial. The saturation concentration (receptor occupancy) on circulating white and red cells was 30 mg/ kg [33]. In a phase Ib [34] (NCT02953509) study evaluating relapsed or refractory non-Hodgkin's lymphoma, patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma were treated with magrolimab in combination with rituximab. The results showed that 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. Specifically, the rates of objective response and complete response were 40 and 33%, respectively, among patients with DLBCL, and 71 and 43%, respectively, among those with follicular lymphoma. Calreticulin (CRT) is a member of the endoplasmic reticulum lectin chaperone family of proteins that plays important biological roles in Ca 2+ homeostasis (in the endoplasmic reticulum, ER), integrin-dependent cell adhesion (in the cytoplasm), and immune response activation (on the plasma membrane) [35]. CRT translocates from the ER to the cell surface during immunogenic cell death in response to various stress factors such as chemotherapy, irradiation, photodynamic therapy, and cytokines. CRT on the surface of stressed or dying cells acts as an "eat me" signal by binding to LRP1 on macrophages [36].
Azacitidine is a chemotherapeutic drug that could induce ICD through translocation of CRT to the cell surface. Interaction of CRT with LRP1 transmits "eat me" signals to macrophages and promotes phagocytosis of dying cells [37]. Combined treatment with magrolimab and azacitidine in a preclinical study resulted in significantly increased phagocytic activity in vitro, and elimination of HL60 in vivo [38]. Preliminary data from a trial (NCT04778397) of magrolimab combined with azacitidine for treatment of AML resulted in a 65% OR and a 40% CR in 34 patients. Transient on-target anemia was observed, and 56% of patients with AML became red blood cell transfusion independent in response to this Lemzoparlimab (TJC4) Lemzoparlimab (TJC4), from I-MAB, is a human IgG4 antibody targeted to CD47 that was screened using a phage display system. The crystal structure of the TJC4/CD47 complex (straighter headto-head orientation) showed that TJC4 binds to a different epitope than does magrolimab (tilted head-to-head orientation) [42]. In addition, TJC4 showed minimal binding to RBC and no hemagglutination was observed at 100 μg/ml in vitro. No significant erythrocyte toxicity was observed in cynomolgus monkeys dosed with 10-100 mg/kg QW. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. [34] reported that EGF-dependent HCC proliferation may be affected by calcium ion, and intracellular dissociative calcium is a key regulator for EGFR signal propagation and apoptosis of tumor cells. Calcium not only promotes the proliferation of proliferating HCC cells but also induces the apoptosis of non-proliferating HCC cells, which might contribute to the reason why the proliferation group (G1-G3) has a poor prognosis. Considering the high hepatic activity of calcium and the overexpression of calcium channels in G1-G3 group, manipulation of calcium in ux in HCC cells may inhibit tumor metastasis.
ITPR1 encodes an intracellular receptor for type 1 inositol 1,4,5-triphosphate and mediates the release of calcium from the endoplasmic reticulum. A few recent studies indicated that ITPR1 affects tumor progression by regulating autophagy. For example, ITPR1 can protect renal cancer cells against natural killer cells by inducing autophagy [35,36]. In our study, we found that HCC patients with a high expression level of ITPR1 have a signi cantly lower 3-year OS rate than HCC patients with low or medium expression levels of ITPR1. Therefore, we speculate that ITPR1 may cooperate with calcium ions to promote the proliferation of proliferating HCC cells, thus leading to the increase of postoperative recurrence risk of HCC patients.
As a second messenger of cell information transmission, cGMP promotes cell division and inhibits cell differentiation. Our RNA-seq analysis showed that the cGMP-PKG signaling pathway was enriched in G1-G3 type HCC, which might contribute to the proliferative characteristics of the G1-G3 subgroups. In this study, we found that G1-G3 type HCC patients showed a high serum AFP level. A differential analysis based on the HCC transcriptome data from TCGA showed that the genes expressed in HCC tissues with high AFP expression are involved in neuroactive ligand-receptor interaction pathways [37]. Together, these results suggest that the proliferative characteristics of G1-G3 subgroups might be from the enrichment of neuroactive ligand-receptor interaction pathways.
Conclusions
In this study, we found that the G1-G6 transcriptomic molecular classi cation was applicable in the China HCC cohort regardless of the ethnic origin of patients. Through the results of these cohorts, we may be able to assertively establish the clinical and pathological relevance of the 16 gene score and use the classi cation system to develop therapeutic strategies for HCC patients worldwide in the future. Our results help clarify our understanding of the correlation between G1-G6 molecular classi cation and molecular markers and molecular signaling pathways in the Chinese HCC population and initially established a link between the phenotype and molecular characteristics. Click to download.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. The results from these groups were generally in line with the results of Boyault et al, but there were still many inconsistencies. The Singapore study reported that the G1 subgroup had a higher proportion of patients with HBV in Singapore compared with European samples. HBV infection was found in the G2 subgroup of European HCC patients but not found in the G2 subgroup of Singapore HCC patients. In addition, the G6 subgroup was closely associated with satellite nodules in the European HCC population but not in the Singapore HCC population. The Singapore study also found no associations of clinical features with G4-G6 subgroups in the Southeast Asia HCC population. The probability of microvascular invasion (MVI) in the G3 subgroup was 5 times higher than other subgroups in Singapore samples. These differences suggested that the G1-G6 transcriptomic classi cation is not completely applicable to HCC patients in all regions of Asia. Furthermore, the genomic pro les of HCC patients are distinct in populations from China and other countries. For example, the TP53 mutation rate in Chinese populations is signi cantly lower than that in Korean and Singapore populations [11,12]. Therefore, it is valuable to validate the clinical relevance of the 16 gene HCC classi cation method in a Chinese population with HCC.
In this study, to determine whether the HCC molecular typing method established by the European team applies to Chinese HCC patients, a total of 107 HCC cases from China were selected for the G1-G6 transcriptomic molecular classi cation. The correlation between the G1-G6 molecular classi cation and pathological features, biological behaviors, serum marker levels, 3-year overall survival (OS) rate, and other indicators were analyzed in combination with the clinicopathological data of the HCC patients. We also detected and analyzed molecular markers and molecular signal pathways related to the G1-G6 molecular classi cation. RNA sequencing and bioinformatics analysis were performed to screen related targets and molecular signaling pathways.
HCC samples and clinical data
The use of human clinical samples was approved by the Medical Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University. A total of 120 HCC specimens and paired adjacent noncancerous liver tissues were randomly selected from patients undergoing hepatectomy between January 2014 to December 2017. The patient follow-up information and clinicopathological characteristics were obtained from the biological sample bank and original pathology report. We also collected 12 fresh HCC tissues and paired adjacent noncancerous liver tissues for further analysis by RNA-seq. Data from The Cancer Genome Atlas (TCGA) database were obtained from TCGA portal sites ( [URL] real-time PCR (qPCR)
The qPCR procedures were performed as previously described [13]
Western blot
Tissues were harvested and lysed in cold extraction buffer (RIPA, Beyotime Biotechnology, Shanghai, China) as previously described [14]. Samples were centrifuged at 13,000 × g at 4°C for 15 min, and the protein concentration in the supernatants was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scienti c Inc., Waltham, IL, USA) according to the manufacturer's protocol. Equal amounts of protein samples were subjected to 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently electro-transferred to polyvinylidene di uoride membranes. The membranes were blocked with 5 % BSA diluted in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4) containing 0.05 % Tween-20 for 2 h and then incubated with primary antibodies at 4°C overnight. Click to download.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Therefore, drugs that act on a novel class of targets to immobilize a broader immune cell population are needed to improve upon current therapeutic options.
Macrophages [6] are typically the first dedicated innate immune cells to detect the presence of infectious pathogens or tumor cells. Macrophages are derived from monocyte precursors that circulate in the blood and migrate into tissues, after which they differentiate into tissue macrophages such as Kupffer cells in the liver, alveolar macrophages in the lung, and microglia in the brain. Circulating monocytes and resident tissue macrophages can directly kill tumor cells via phagocytosis (innate immune response) and can activate the adaptive immune response. However, these immune responses can be inhibited by ligand binding to inhibitory receptors expressed on the macrophage cell surface [7]. Signal regulatory protein alpha (SIRPα) is a transmembrane protein expressed on all myeloid cells, including monocytes, macrophages, and neutrophils. SIRPα contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can be phosphorylated, resulting in recruitment of inhibitory molecules such as Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2 [8]. Binding of CD47 to SIRPα triggers coupling of SIRPα to these phosphatases, resulting in inhibition of phagocytic activity [9,10]. CD47 is ubiquitously expressed on many types of cells to prevent phagocytosis by phagocytes. However, tumor cells overexpress CD47 to evade the immune system through inhibition of myeloid cell-mediated elimination [11]. Inhibition of CD47-SIRPα interaction restored the phagocytic activity of phagocytes in vitro and in vivo [12][13][14][15]. Targeting the CD47/SIRPα axis has become a promising strategy to promote tumor elimination through innate immunity. This review focuses on development, safety, and efficacy of agents that target the CD47/SIRPα axis in preclinical and clinical studies.
CD47/SIRPα: the molecules and biology
SIRPα [16,17], also named SHPS-1 or CD172a, is a transmembrane glycoprotein mainly expressed on neurons and myeloid cells that is particular enriched on macrophages. Human SIRPα is coded by the SHPS-1 gene located at human chromosome 20p13. The open reading frame region is composed of eight exons, including a signal peptide, extracellular domain, a transmembrane segment, and three parts of one cytoplasmic domain. The extracellular domain consists of three Ig-like regions, an NH2-terminal immunoglobulin (Ig) variable (V) region (domain 1, D1), and two Ig constant (C) regions (domain 2 and 3). The cytoplasmic region contains two immunoreceptor tyrosine based inhibitory motifs (ITIMs) and a proline-rich region (YYYY), which bind to Src homology (SH2) domain-containing molecules.
CD47 is a 52 kD transmembrane glycoprotein belonging to the immunoglobulin superfamily. Human CD47 is encoded by the CD47 gene located at the q13.12 region of chromosome 3. Human CD47 contains an NH 2 -terminal Ig variable-like extracellular domain (ECD), a 5-transmembrane spanning helical bundle domain, and a short intracellular COOH-terminal domain (CTD) [18]. CD47 is an essential component of the innate immune system, and binding of its extracellular domain with its ligands αVβ3, SIRPα, and thrombospondin-1 (Tsp-1) activates different signaling pathways that control cell proliferation and differentiation, angiogenesis, and immune regulation. The CTD is alternatively spliced and can exist as four isoforms, ranging from 4 to 36 residues. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. Further statistical analysis found that the ratio of GS expression to β-catenin expression in the G5 and G6 groups was approximately three-fold higher compared with levels in the G1-G4 subgroups, which suggests that the Wnt/β-catenin pathway is highly activated in G5 and G6 HCC. The activation level was highest in G6. These results suggested that G5 and G6 HCC subgroups were closely associated with the robust activation of Wnt/β-catenin signaling.
Transcriptome sequencing analysis for G1-G6 molecular classi cation of Chinese HCC patients The results of our study suggest that G1-G3 group can be used as an independent risk factor for evaluating HCC patients' prognosis and is signi cantly correlated with poor prognosis after hepatectomy.
To clearly understand the poor prognosis group based on G1-G6 molecular classi cation of Chinese HCC patients, 12 samples of HCC tissues (2 cases of each subgroup) and the corresponding adjacent liver tissues were randomly selected. The expression levels of genes related to differentiation, proliferation, and function of tumor cells were examined in these samples and analyzed by RNA-seq and bioinformatics. The error rates of the 24 samples were all within the acceptable range ( Supplementary Fig. 1). The proportions of ltered reads of the 24 samples were all within the acceptable range, and the proportion of clean reads of each sample accounted for more than 90%, which suggested that the data were suitable for subsequent analysis (Supplementary Fig. 2).
The all HCC group (CA group) included 12 samples of HCC tissues, and the para-HCC group (pCA group) included 12 corresponding samples of adjacent liver tissues. High proliferative group (group H) includes 6 samples of G1-G3 cancer tissues, and low proliferative group (group L) includes 6 samples of G4-G6 cancer tissues. Compared with the pCA group, there were 3233 up-regulated genes and 1654 downregulated genes in the CA group, while 33,299 genes showed no changes between the two groups (Fig. 6A). Compared with group L (G4-G6 subgroup), there were 1,269 up-regulated and 1,445 down-regulated genes in group H (G1-G3 subgroup), and 33,692 genes showed no differential expression between the two groups (Fig. 6B). We also found that 108 genes overlapped between the H group (compared with group L) and CA (compared with group pCA) (Fig. 6C), and 27 genes were commonly down-regulated ( Fig. 6D). Among the 27 genes, four genes (REG1B, REG3G, C19orf18, and ITPR1) were signi cantly upregulated or downregulated in group H compared with group L ( Table 6). Analysis using TCGA database also con rmed that the expression levels of REG1B, REG3G, and ITPR1 were signi cantly increased in HCC liver tissues compared with normal liver tissues (Fig. 6E). Using the follow-up information database of TCGA patients, Kaplan-Meier analysis was conducted to analyze the prognosis of HCC patients. Click to download.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. As shown in Fig. 6F, HCC patients with a high expression of ITPR1 showed a signi cantly shorter 3-year OS than HCC patients with low or medium expression of ITPR1. However, there were no statistical differences between HCC patients with high expressions of REG1B or REG3G and the corresponding control groups. These results suggested that ITPR1 may promote the occurrence and development of HCC and affect the prognosis of HCC patients. We next performed KEGG pathway analysis using the KEGG database and Clusterpro le software. Fisher exact analysis was used to test and calculate p-values, and the pathways with a p-value less than 0.05 were retained. The p-value represented the signi cance of enrichment of differential genes; a lower the pvalue indicates a greater correlation between the pathway and the differential genes. A Padj value less than 0.05 was taken as the threshold of signi cant enrichment. The KEGG pathway enrichment analysis revealed that the calcium signaling pathway, cGMP-PKG signaling pathway, renin secretion, neuroactive ligand-receptor interaction, and cell metabolism-related signaling pathways were signi cantly enriched in the G1-G3 subgroups (Fig. 7A-B).
Discussions
Although our results were generally consistent with the studies from Europe and Singapore, there were still many inconsistencies. First, the distribution of the HCC subgroups in the Chinese cohort was signi cantly different from that in the original European cohort. In the European study, the G4 and G1 subgroups represented the largest and smallest subgroup, respectively. In our study, the G6 and G2 subgroups were the largest and smallest groups, respectively. Second, the Singapore research reported that the incidence of MVI in the G3 subgroup was markedly higher than in other subgroups. However, we did not nd a similar characteristic of the G3 subgroup of China HCC patients. Instead, we found that the incidence of MVI was signi cantly lower in the G5 subgroup than the other subgroups. Third, inconsistent with the European study but in line with the Singapore study, we did not nd any subgroups characterized with satellite nodules. Finally, our data showed that the G1-G3 subgroups had a shorter 3-year OS rate and the G3 subgroup has the worst prognosis, which was different from the studies in Europe and Singapore. These differences suggest that some of the clinical characteristics of HCC patients are distinct in the populations from China and other countries.
In this study, we found a high serum AFP level in the G1 group and a correlation between the G5-G6 group and Wnt/β-catenin pathway activation based on previous studies. Several studies showed that HCC with β-catenin mutations have slow proliferation, well-differentiated characteristics, and trabecular type false adenoid type histology, with cholestasis and intratumoral in ltrating immune de ciency [16,26]. Click to download.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Treatment with evorpacept in combination
Antibodies targeting SIRPα on myeloid cells
Anti-SIRPα antibodies induce weak or no phagocytic activity alone, but induce significantly increased phagocytic activity when combined with opsonizing antibodies (rituximab, cetuximab) [58][59][60]. Several issues with anti-SIRPα antibodies are important to consider. First, since SIRPα is expressed on myeloid cells, anti-SIRPα antibodies using inactive human IgG-Fc to avoid Fc effector-mediated toxicity on these immune cells may be advantageous [60]. Second, SIRPγ expressed on T and NK cell shares 74.37% amino acid similarity with the extracellular domain with SIRPα [61]. SIRPγ on T cells binds to CD47 on APCs to mediate cell-cell adhesion and enhances antigen presentation, resulting in T cell proliferation and cytokine secretion [62]. Development of a SIRPα targeting antibody with specificity toward SIRPα to avoid interference with the interaction between CD47 and SIRPγ may preserve T cell activity. Third, antibody internalization could lead to rapid clearance of antibody in vivo. Higher doses or multiple dosing is required to ensure that levels remain therapeutically relevant [63,64]. Internalization of SIRPα decreases the inhibitory signal and may enhance the ability of antibodies to restore phagocytic activity [59,65]. Finally, Although SIRPα has ten known variants, V1, V2, and V8 are the most prominent (over 90%) haplotypes in the human population. Antibodies targeting all three of these variants may be more potent than those that target a single variant [66,67]. Antibodies targeting SIRPα currently being evaluated are listed in the Table 4. Trials evaluating treatment with SIRPα antibodies in combination with immunotherapies are in the early clinical stage of development, and include OSE-172 [68] from OSE Immunotherapeutics (co-developed with Boehringer Ingelheim), CC-95251 from Celgene, and FSI-189 from Gilead [59].
Bi-specific molecules
Bi-specific molecules bind two targets or two distinct epitopes of one target. The antigen binding sites of bispecific molecules could consist of two antibodies or proteins (ligand or receptor), or could consist of one antibody and one protein. Bi-specific antibodies can bind two target antigens in-cis and in-trans. Rational design of bispecific molecules based on biological activity may result in distinct effects or improved efficacy when compared to combination treatments. Four bispecific antibodies, catumaxomab [69] (withdrawn in 2017), blinatumomab [70], emicizumab [71], and amivantamab-vmjw [72] have been approved by EMA or FDA. Bi-specific molecules can be constructed from CD47 targeting antibodies or SIRPα and other antigen-targeting molecules (Fig. 4). Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. The main causes of HCC include chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, alcoholic or non-alcoholic steatohepatitis, autoimmune hepatitis, and several metabolic diseases. However, the etiologies of HCC between the Euro-American area and Asia-Paci c region vary widely. HBV infection was endemic in China, where the prevalence rate of HBsAg is 7.2% [2], while HCV infection is common in western countries. In China, HCC only has a 5-year survival rate of 14.1% and a recurrence rate of about 70% [3]. Approximately 70-80% of HCC patients are diagnosed at an advanced stage and can receive only palliative care [4]. Although several promising drugs were developed over the last decades, these drugs have failed to meet clinical endpoints in phase III trials [5]. The failure of these trials is, at least in part, because of the lack of effective molecular markers or the minimal validation of known molecular markers in different populations.
However, few studies have investigated the correlation between HCC molecular classi cation and pathological characteristics. A HCC classi cation method based on the expression of 16 genes was proposed by Boyault et al [8]. The authors classi ed tumors into six groups, G1-G6, using a minimal subset of 16 genes. This transcriptomic group classi cation was tested on cohorts from Singapore [9] and South Korea [10] with comparisons to European HCC patients. The results from these groups were generally in line with the results of Boyault et al, but there were still many inconsistencies. The Singapore study reported that the G1 subgroup had a higher proportion of patients with HBV in Singapore compared with European samples. HBV infection was found in the G2 subgroup of European HCC patients but not found in the G2 subgroup of Singapore HCC patients. In addition, the G6 subgroup was closely associated with satellite nodules in the European HCC population but not in the Singapore HCC population. The Singapore study also found no associations of clinical features with G4-G6 subgroups in the Southeast Asia HCC population. The probability of microvascular invasion (MVI) in the G3 subgroup was 5 times higher than other subgroups in Singapore samples. These differences suggested that the G1-G6 transcriptomic classi cation is not completely applicable to HCC patients in all regions of Asia. Furthermore, the genomic pro les of HCC patients are distinct in populations from China and other countries. For example, the TP53 mutation rate in Chinese populations is signi cantly lower than that in Korean and Singapore populations [11,12]. Therefore, it is valuable to validate the clinical relevance of the 16 gene HCC classi cation method in a Chinese population with HCC.
In this study, to determine whether the HCC molecular typing method established by the European team applies to Chinese HCC patients, a total of 107 HCC cases from China were selected for the G1-G6 transcriptomic molecular classi cation. Click to download.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. First, the distribution of the HCC subgroups in the Chinese cohort was signi cantly different from that in the original European cohort. In the European study, the G4 and G1 subgroups represented the largest and smallest subgroup, respectively. In our study, the G6 and G2 subgroups were the largest and smallest groups, respectively. Second, the Singapore research reported that the incidence of MVI in the G3 subgroup was markedly higher than in other subgroups. However, we did not nd a similar characteristic of the G3 subgroup of China HCC patients. Instead, we found that the incidence of MVI was signi cantly lower in the G5 subgroup than the other subgroups. Third, inconsistent with the European study but in line with the Singapore study, we did not nd any subgroups characterized with satellite nodules. Finally, our data showed that the G1-G3 subgroups had a shorter 3-year OS rate and the G3 subgroup has the worst prognosis, which was different from the studies in Europe and Singapore. These differences suggest that some of the clinical characteristics of HCC patients are distinct in the populations from China and other countries.
In this study, we found a high serum AFP level in the G1 group and a correlation between the G5-G6 group and Wnt/β-catenin pathway activation based on previous studies. Several studies showed that HCC with β-catenin mutations have slow proliferation, well-differentiated characteristics, and trabecular type false adenoid type histology, with cholestasis and intratumoral in ltrating immune de ciency [16,26].
β-catenin is a key molecular marker in the epithelial-mesenchymal transition. In physiological conditions, β-catenin binds cadherin and promotes the release of cadherin when β-catenin translocates to the nucleus [27]. In HCC, E-cadherin is reduced on the cell surface and N-cadherin is expressed once tumor cells undergo epithelial-mesenchymal transition [28]. It implicates that G5 and G6 subgroups may have some features as mentioned above.
We found several additional correlations in this study that had not been reported previously. For example, the G1-G3 group was associated with PD-L1 expression and liver cancer stemness. These ndings provide a new direction for the diagnosis and treatment of HCC. In the future, if G1-G3 patients were treated with targeted therapy for PD-1/PD-L1 and liver cancer stem cells, this may improve the survival and prognosis of a patient with highly malignant HCC with an extremely poor prognosis.
The pathogenesis of HCC is complex and involves multiple signaling pathways [29,30]. Signaling pathways involved in growth factors activated by intracellular calcium ions play a crucial role in various physiological and biochemical processes [31]. HCC encodes a variety of tyrosine kinases, including epidermal growth factor receptor (EGFR), whose functions are mainly in glucose and lipid metabolism, liver brosis, regeneration, and carcinogenesis [32,33]. Modica et al. Click to download.
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Domain: Biology Medicine
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Target antigens could include: A) tumor associated cell surface antigens (PD-L1, CD20, CD19, MSLN (Mesothelin), Claudin18.2, and Her2), B) immune checkpoint proteins (PD-1, CD40, 41BB), and C) cytokines or receptors (CSF-2 receptor, VEGF). Reduced affinity for CD47 and increased affinity for the second target may reduce toxicity and enhance efficacy. For type A antibodies, IgG1-Fc was selected to enhance antibodymediated killing of tumor cells (ADCC, ADCP, and CDC). However, inactivated Fc is preferred when used in type B bi-specific molecules. Use of CD47-targeting biologics has shown significant clinical efficacy for treatment of R/R AML, NHL, and MDS. The combination of Hu5F9 and rituximab showed particular promise as a treatment approach for R/R NHL [34]. Several CD47related BsAb are in early-stage clinical trials. NI1701 [73], which targets CD47 and CD19, is an IgG-like BsAb constructed using modified knobs-into-hole technology [74], and contains an IgG1-Fc. Preclinical data showed that NI1701 [75] selectively binds to CD47 and CD19 co-expressing cells, but interacts poorly with normal healthy cells (CD47 + CD19 − ), resulting in avoidance of normal cells acting as sinks for binding of the antibodies, and reduced toxicity. In vitro and in vivo studies showed that NI1701 more potently killed tumor cells than did anti-CD47 and anti-CD19 antibodies alone, or in combination. A BsAb named NI1801 [73] targeted to CD47 and MSLN showed similar preclinical activity as NI1701. IMM0306, a bispecific antibody fusion protein targeted to CD20 (rituximab) and CD47 (SIRPα) with wild type IgG1-Fc [76], is in a phase I trial evaluating treatment of R/R CD20-positive B-cell non-Hodgkin's lymphoma. SL-172154 [77] is a fusion protein targeted to CD47 with SIRPα and CD40 with CD40L, and is in a phase I trial for treatment of solid tumors. A BsAb, HX009, comprised of the extracellular region of SIRPα V2D fused with an anti-PD1 antibody (HX008) was evaluated in a phase I trial. a antibody in clinical trial The study showed that HX009 blocked both CD47/SIRPα and PD-1/PDL-1 interactions, and interacted with CD47 on tumor cells and PD1 on T cells to help present tumor antigens to T cells, resulting in activation of the innate and acquired immune responses. Several BsAbs have been developed to inhibit CD47/SIRPα and PD-1/PD-L1 interactions. One such BsAb, IBI322 [78], was designed as a selective CD47 binding CD47/PD-L1 bispecific antibody. IBI322 showed no negative hematological effects in cynomolgus monkeys, but the binding affinity of IBI322 to cynomolgus CD47 was not disclosed, and IBI322 showed dose-dependent binding to RBC. Other BsAb antibodies listed in Table 2 are in the preclinical proof-ofconcept stage.
Engineered T cells and macrophages
T cells with chimeric antigen receptors (CARs) showed promising therapeutic efficacy against hematologic malignancies, and several CAR-T therapeutics have been approved [79][80][81][82][83]. However, low treatment response rates of solid tumors to CAR-T treatment were observed. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. CRT translocates from the ER to the cell surface during immunogenic cell death in response to various stress factors such as chemotherapy, irradiation, photodynamic therapy, and cytokines. CRT on the surface of stressed or dying cells acts as an "eat me" signal by binding to LRP1 on macrophages [36].
Azacitidine is a chemotherapeutic drug that could induce ICD through translocation of CRT to the cell surface. Interaction of CRT with LRP1 transmits "eat me" signals to macrophages and promotes phagocytosis of dying cells [37]. Combined treatment with magrolimab and azacitidine in a preclinical study resulted in significantly increased phagocytic activity in vitro, and elimination of HL60 in vivo [38]. Preliminary data from a trial (NCT04778397) of magrolimab combined with azacitidine for treatment of AML resulted in a 65% OR and a 40% CR in 34 patients. Transient on-target anemia was observed, and 56% of patients with AML became red blood cell transfusion independent in response to this Lemzoparlimab (TJC4) Lemzoparlimab (TJC4), from I-MAB, is a human IgG4 antibody targeted to CD47 that was screened using a phage display system. The crystal structure of the TJC4/CD47 complex (straighter headto-head orientation) showed that TJC4 binds to a different epitope than does magrolimab (tilted head-to-head orientation) [42]. In addition, TJC4 showed minimal binding to RBC and no hemagglutination was observed at 100 μg/ml in vitro. No significant erythrocyte toxicity was observed in cynomolgus monkeys dosed with 10-100 mg/kg QW. A phase I trial (NCT03934814) [43] evaluating treatment of R/R advanced solid tumors and lymphoma with TJC4 alone, or in combination with pembrolizumab or rituximab, is ongoing. No DLT or SAE (severe adverse events) were observed, and treatmentassociated anemia occurred in 30% of patients (6 of 20, 2 receiving 3 mg/kg, 2 receiving 10 mg/kg, 1 receiving 20 mg/kg, and 1 receiving 30 mg/kg). Anti-drug antibody (ADA) events occurred in 25% of patients, but there were no concerns regarding safety or pharmacokinetics (PK). Maximal saturation of peripheral T cells was achieved at a dose of 20 mg/kg administered weekly.
AO176 AO176, developed by Arch Oncology, is a humanized IgG2 subclass anti-CD47 antibody [31]. AO176 was shown to bind to integrin-β1 expressed on tumor cells, but not on RBC. Interestingly, AO176 blocked the CD47/SIRPα interaction to stimulate phagocytosis of tumor cells, and also directly killed tumor cells (non-ADCC). A tolerability and hematologic study on Cynomolgus monkeys showed no anemia. In phase I/ II clinical trials [44], Grade 3 TRAE (treatment-related adverse events) were observed in > 10% of patients. In addition, DLT was observed at 20 mg/kg. Studies evaluating AO176 monotherapy and combination therapy with paclitaxel are ongoing.
CC-90002 CC-90002 is a humanized monoclonal IgG4 CD47 antibody. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. CD47 is ubiquitously expressed on the surfaces of multiple hematopoietic and solid tumor cells. However, adverse events due to cytokine secretion by macrophages during immune checkpoint activation [92,93] and CAR-T [94,95] treatment was common. Technological improvements for preparation and production of CAR-M are needed to offer scalable and reproducible manufacturing processes.
Small molecules, peptides, and microRNA
RRx-001 [96] is an anticancer agent designed to induce M2 to M1 polarization and to promote recovery of phagocytic activity of macrophages toward tumor cells.
The anti-phagocytic inhibitory signal was removed or reduced through downregulation of both CD47 and SIRPα gene expression on tumor cells and macrophages, respectively. Elimination of tumor cells was shown in in vitro and in vivo. Phase III clinical trials (NCT03699956, NCT02489903) against small cell lung cancer [97,98] are ongoing.
D4-2 [99], a macrocyclic peptide targeted to mouse SIRPα was designed to inhibit the interaction between CD47 and SIRPα and promote macrophage-mediated phagocytosis of tumor cells when combined with rituximab. PKHB1 [100], a TSP-1-derived CD47 agonist peptide, induced cell death (CRT exposure and DAMP release) in chronic lymphocytic leukemia cells.
MicroRNAs (miRNAs), which are 20-22 nucleotides in length, play important roles in cancer pathogenesis and progression since they can repress the target gene at the translational level by directly binding to the 3'untranslated regions (3'UTRs) [101].
Overexpression miR-378a [102] in mice peritoneal macrophages downregulates SIRPα mRNA expression. Phagocytosis of Ishikawa cells by macrophages-miR-378a and macrophages was carried out in vitro. Phagocytic index in macrophages-miR-378a group is 3 times than that in macrophages group.
Zhao [103] reported that miR-200a inhibited the expression of CD47 by directly targeting the 3'UTR of the CD47 mRNA. MicroRNA 200a suppressed nasopharyngeal carcinoma (NPC) cell proliferation, migration, and invasion, and promoted phagocytosis of NPC cells by Fig. 4 Types of bis-specific molecules targeted CD47 and other molecules. Bispecific Ab-type 1: KIH format, targeted to CD47 (yellow) and other tumor associated antigens (CD19, CD20, and MSLN, blue); Bi-specific Ab-type 2: KIH format, targeted to CD47 (yellow) and immune checkpoint molecules (PD-1, CD40, and 41BB, green); Bi-specific Ab-type 3: SIRPα (cyan) fusion to N-terminus of H-chain of IgG (targeting tumor associated antigen, blue); Bi-specific Ab-type 4: ligand/receptor to modulate TME (GM-CSF, and VEGFR2, pink) fusion to the C-terminus of the H-chain of IgG (yellow); Bi-specific Ab-type 4: SIRPα fusion to the N-terminus of IgG-Fc and ligand/receptor to modulate TME (GM-CSF, VEGFR2, pink) fusion to the C-terminus macrophages through down-regulation of CD47 expression on NPC cells.
MicroRNA 708 [104] was directly targeted CD47 and resulted in downregulation of CD47 on T cell acute lymphoblastic leukemia cell line. MicroRNA 708 expression in the T-ALL cell line was sufficient to promote phagocytosis by macrophages in vitro, and inhibited tumor engraftment in vivo.
Conclusions and future perspectives
Following the clinical success of therapeutic antibodies targeting T cell checkpoint molecules, combination therapies using checkpoint inhibitors with other agents have been a major theme of clinical oncology studies. Specifically, inhibitors of the CD47/SIRPα pathway have emerged as promising therapeutic candidates. Overexpression on tumor cells makes CD47 an ideal target for cancer therapy. Antibodies targeting CD47 showed promising results against MDS and AML [105][106][107]. However, side effects such as anemia, hyperbilirubinemia, thrombocytopenia, and lymphopenia induced by CD47-targeting molecules are of specific concern and need to be addressed with development of new therapeutic agents.
In addition, the therapeutic effects of agents targeting the CD47/SIRPα axis on solid tumors are limited. Additional therapeutic strategies, including combination therapy and bi-specific antibodies, may be promising. Combination therapy with opsonizing antibodies, immune checkpoint inhibitors, chemotherapeutic agents to activate FcR on macrophages, and T cell sensitizers that induce immunogenic cell death to stimulate a more potent immunological effect all have potential. Agents targeted to the CD47-SIRPα axis should not only block the CD47/SIRPα interaction, but also activate signaling on macrophages (FcγR, CRT expression). Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. A BsAb named NI1801 [73] targeted to CD47 and MSLN showed similar preclinical activity as NI1701. IMM0306, a bispecific antibody fusion protein targeted to CD20 (rituximab) and CD47 (SIRPα) with wild type IgG1-Fc [76], is in a phase I trial evaluating treatment of R/R CD20-positive B-cell non-Hodgkin's lymphoma. SL-172154 [77] is a fusion protein targeted to CD47 with SIRPα and CD40 with CD40L, and is in a phase I trial for treatment of solid tumors. A BsAb, HX009, comprised of the extracellular region of SIRPα V2D fused with an anti-PD1 antibody (HX008) was evaluated in a phase I trial. a antibody in clinical trial The study showed that HX009 blocked both CD47/SIRPα and PD-1/PDL-1 interactions, and interacted with CD47 on tumor cells and PD1 on T cells to help present tumor antigens to T cells, resulting in activation of the innate and acquired immune responses. Several BsAbs have been developed to inhibit CD47/SIRPα and PD-1/PD-L1 interactions. One such BsAb, IBI322 [78], was designed as a selective CD47 binding CD47/PD-L1 bispecific antibody. IBI322 showed no negative hematological effects in cynomolgus monkeys, but the binding affinity of IBI322 to cynomolgus CD47 was not disclosed, and IBI322 showed dose-dependent binding to RBC. Other BsAb antibodies listed in Table 2 are in the preclinical proof-ofconcept stage.
Engineered T cells and macrophages
T cells with chimeric antigen receptors (CARs) showed promising therapeutic efficacy against hematologic malignancies, and several CAR-T therapeutics have been approved [79][80][81][82][83]. However, low treatment response rates of solid tumors to CAR-T treatment were observed. Golubovskaya et al. [84] showed that CD47-CAR-T cells effectively killed ovarian, pancreatic, and other cancer cells, and induced production of high levels of IL-2, which correlated with expression of CD47 antigens. Treatment with CD47-CAR-T cells may be a novel strategy for treating different types of cancers. Huyen [85] designed a third generation of CD47-CAR-T cell that could effectively kill lung cancer cells (A549) and inhibit lung cancer cell metastasis. A dual CAR-T targeting CD47 and TAG-72 (tumor-associated glycoprotein 72) generated by Shu [86] showed promising results against ovarian cancer in preclinical experiments. An Anti-PD-L1 (A12) CAR-T with the ability to secrete anti-CD47 VHH (variable heavy domain of heavy chain antibodies or nanobodies) (A4), developed by Xie [87], represented a novel strategy for cancer treatment. An A12-A4 CAR-T showed better anti-cancer activity than an A12 CAR-T plus soluble A4 in C57BL/6 PD-L1-KO mice bearing B16F10 cells. Each of these CD47-CAR-T cells are in the preclinical stage, and efficacy and safety should be further investigated. Development of CAR-M1 (M1: classically activated macrophages) is an emerging therapeutic strategy [88], and several studies of engineered CAR-M cells [89][90][91] showed tumor cell elimination activity in vitro and in vivo. These studies showed that CAR-M induced phagocytosis and induced M2 to M1 polarization through secretion of pro-inflammatory factors and chemokines [90]. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. Together, these results suggest that the proliferative characteristics of G1-G3 subgroups might be from the enrichment of neuroactive ligand-receptor interaction pathways.
Conclusions
In this study, we found that the G1-G6 transcriptomic molecular classi cation was applicable in the China HCC cohort regardless of the ethnic origin of patients. Through the results of these cohorts, we may be able to assertively establish the clinical and pathological relevance of the 16 gene score and use the classi cation system to develop therapeutic strategies for HCC patients worldwide in the future. Our results help clarify our understanding of the correlation between G1-G6 molecular classi cation and molecular markers and molecular signaling pathways in the Chinese HCC population and initially established a link between the phenotype and molecular characteristics. Click to download.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. The G1 subgroup was mainly related to liver cancer stemness, and G3 subgroup showed the worst prognosis. The G5 and G6 subgroups were associated with activation of the Wnt/β-catenin pathway. Compared with the G1–G4 group, the G1–G3 group showed signicantly higher expression levels of regenerating family member 1 beta (REG1B), regenerating family member 3 gamma (REG3G), and inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), and enriched calcium signaling pathway. Conclusions: Our results clarify the correlation between G1–G6 molecular classication and molecular markers and molecular signaling pathways in the Chinese HCC population and initially established a link between the phenotype and molecular characteristics. This study enhances our understanding of the heterogenicity of China HCC and indicates that the G1–G6 signatures can be used to identify potential therapeutic biomarkers against HCC patients in China. was used to analyze the correlation between the G1–G6 molecular classication and tumor biological behavior. After checking data for normal distribution and variance homogeneity, comparisons between two groups were evaluated using independent-sample t-tests or Mann–Whitney U test. For three or more groups, differences were statistically analyzed by one-way ANOVA or the non-parametric test (Kruskal– Wallis test). Kaplan–Meier and Cox proportional hazards survival regression analysis were used to evaluate the prognostic signicance of the G1–G6 molecular classications within 36 months. All p values were two-tailed, and P < 0.05 was considered statistically signicant.
Background
Hepatocellular carcinoma (HCC) is the most common malignancy worldwide, with more than half of the new HCC cases and deaths every year occurring in China [1]. The main causes of HCC include chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, alcoholic or non-alcoholic steatohepatitis, autoimmune hepatitis, and several metabolic diseases. However, the etiologies of HCC between the Euro-American area and Asia-Paci c region vary widely. HBV infection was endemic in China, where the prevalence rate of HBsAg is 7.2% [2], while HCV infection is common in western countries. In China, HCC only has a 5-year survival rate of 14.1% and a recurrence rate of about 70% [3]. Approximately 70-80% of HCC patients are diagnosed at an advanced stage and can receive only palliative care [4]. Although several promising drugs were developed over the last decades, these drugs have failed to meet clinical endpoints in phase III trials [5]. The failure of these trials is, at least in part, because of the lack of effective molecular markers or the minimal validation of known molecular markers in different populations.
However, few studies have investigated the correlation between HCC molecular classi cation and pathological characteristics. A HCC classi cation method based on the expression of 16 genes was proposed by Boyault et al [8]. The authors classi ed tumors into six groups, G1-G6, using a minimal subset of 16 genes. This transcriptomic group classi cation was tested on cohorts from Singapore [9] and South Korea [10] with comparisons to European HCC patients. Click to download.
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Domain: Biology Medicine<|endoftext|>>>> Q: Continue the text.
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. 4 Types of bis-specific molecules targeted CD47 and other molecules. Bispecific Ab-type 1: KIH format, targeted to CD47 (yellow) and other tumor associated antigens (CD19, CD20, and MSLN, blue); Bi-specific Ab-type 2: KIH format, targeted to CD47 (yellow) and immune checkpoint molecules (PD-1, CD40, and 41BB, green); Bi-specific Ab-type 3: SIRPα (cyan) fusion to N-terminus of H-chain of IgG (targeting tumor associated antigen, blue); Bi-specific Ab-type 4: ...
>>> Answer:
Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. 4 Types of bis-specific molecules targeted CD47 and other molecules. Bispecific Ab-type 1: KIH format, targeted to CD47 (yellow) and other tumor associated antigens (CD19, CD20, and MSLN, blue); Bi-specific Ab-type 2: KIH format, targeted to CD47 (yellow) and immune checkpoint molecules (PD-1, CD40, and 41BB, green); Bi-specific Ab-type 3: SIRPα (cyan) fusion to N-terminus of H-chain of IgG (targeting tumor associated antigen, blue); Bi-specific Ab-type 4: ligand/receptor to modulate TME (GM-CSF, and VEGFR2, pink) fusion to the C-terminus of the H-chain of IgG (yellow); Bi-specific Ab-type 4: SIRPα fusion to the N-terminus of IgG-Fc and ligand/receptor to modulate TME (GM-CSF, VEGFR2, pink) fusion to the C-terminus macrophages through down-regulation of CD47 expression on NPC cells.
MicroRNA 708 [104] was directly targeted CD47 and resulted in downregulation of CD47 on T cell acute lymphoblastic leukemia cell line. MicroRNA 708 expression in the T-ALL cell line was sufficient to promote phagocytosis by macrophages in vitro, and inhibited tumor engraftment in vivo.
Conclusions and future perspectives
Following the clinical success of therapeutic antibodies targeting T cell checkpoint molecules, combination therapies using checkpoint inhibitors with other agents have been a major theme of clinical oncology studies. Specifically, inhibitors of the CD47/SIRPα pathway have emerged as promising therapeutic candidates. Overexpression on tumor cells makes CD47 an ideal target for cancer therapy. Antibodies targeting CD47 showed promising results against MDS and AML [105][106][107]. However, side effects such as anemia, hyperbilirubinemia, thrombocytopenia, and lymphopenia induced by CD47-targeting molecules are of specific concern and need to be addressed with development of new therapeutic agents.
In addition, the therapeutic effects of agents targeting the CD47/SIRPα axis on solid tumors are limited. Additional therapeutic strategies, including combination therapy and bi-specific antibodies, may be promising. Combination therapy with opsonizing antibodies, immune checkpoint inhibitors, chemotherapeutic agents to activate FcR on macrophages, and T cell sensitizers that induce immunogenic cell death to stimulate a more potent immunological effect all have potential. Agents targeted to the CD47-SIRPα axis should not only block the CD47/SIRPα interaction, but also activate signaling on macrophages (FcγR, CRT expression). Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. Proliferation tumors are associated with aberrant activation of signaling pathways, while non-proliferative tumors display a well-differentiated phenotype [15,16]. Correspondingly, G1-G3 (combined with G1, G2, and G3) of the G1-G6 molecular typing can be classi ed as a proliferation group, while G4-G6 (combined with G4, G5, and G6) was classi ed as a non-proliferative group. Next, we pooled classes into the G1-G3 group and G4-G6 group. We found that the proportions of the G1-G3 group and G4-G6 group in the China cohort were 38.32% and 61.68%, respectively. In the European cohort, 35 I-II 4 2 3 0 6 9 III-IV 15 0 14 10 19 22 TNM stage 107 I-II 8 2 10 6 18 19 III-IV 11 0 To analyze the correlation between proliferation and clinicopathological characteristics of HCC patients based on G1-G6 molecular classi cation, we integrated the proliferative group (G1-G3) and nonproliferative group (G4-G6) by clinicopathological information. We found that the proliferative group (G1-G3) was correlated with high serum AFP level (P=0.002), high copy number of HBV DNA (P=0.047), complex histological subtype (P=0.010), macrovascular invasion (P=0.012), and negative or weak positive Hep-Par1 (P=0.012) ( Table 4). Using follow-up data of patients after hepatectomy, we also analyzed the correlation between HCC prognosis and G1-G6 molecular classi cation. As shown in Fig. 1C, the G1-G3 HCC patients showed a shorter 3-year OS than G4-G6 HCC patients (P=0.010). The recurrence-free survival of the G1-G3 group tended to be shorter than the G4-G6 group (P=0.072) ( Figure 1D). Further analysis showed that G1 patients tended to have a shorter 3-year OS than non-G1 patients (P=0.1867) ( Figure 1E) and G3 patients had the worst prognosis (P=0.0125) ( Figure 1F). Cox regression univariate analysis showed that the TNM stage (P=0.0002), BCLC stage (P=0.001), and G1-G6 molecular classi cation (P=0.012) were important factors that affected the overall prognosis of HCC patients. All factors with a p-value < 0.1 detected in univariate analyses were included in multivariate analyses, and the subsequent results showed that the G1-G6 molecular classi cation (P=0.035) is an independent prognostic risk factor for poor OS of HCC patients (Table 5). Predictor of HCC classi cation using qPCR A total of 19 para-cancer liver tissue samples were randomly selected to investigate the correlation between expression of the 16 genes in the proliferation group (G1-G3) compared with the nonproliferation group (G4-G6). Our results showed that the expression levels of AFP (P=0.0200), CDH2 (P=0.0022), HN1 (P<0.0001), NRAS (P=0.0096), PAK2 (P=0.0024), RAB1A (P=0.0020), and SAE1 (P<0.0001) were signi cantly higher in the proliferation group (G1-G3) than in the non-proliferation group (G4-G6) ( Fig. 2A). Conversely, the expression levels of LAMA3 (P=0.0059) and PAP (P=0.0495) were markedly higher in the non-proliferation group (G4-G6) than in the proliferation group (G1-G3) (Fig. 2B).
There were no signi cant differences in the expressions of the following genes between the proliferation group (G1-G3) and non-proliferation group ( PD-L1 is highly expressed in G1-G3 subgroups Several molecular targeted drugs have entered clinical trials as palliative and complementary treatments for HCC [17]. For example, the clinically approved antibodies targeting PD-1 or its ligand PD-L1 were shown to cause lasting responses in up to 25% of advanced HCC patients in two early trials. In HCC, the proportion of tumor cells expressing PD-L1 is approximately 15% [18]. Click to download.
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Domain: Biology Medicine<|endoftext|>Predictive Value of The G1–G6 Transcriptomic Molecular Classication of Hepatocellular Carcinoma for its Biological Behavior and Clinicopathological Features in China
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and a large number of genetic alterations are involved in the carcinogenetic process. A G1–G6 transcriptomic classication was previously proposed in a French study, and Korean and Singaporean groups indicated its potential application in Asian HCC patients. However, the genomic proles of Chinese patients are distinct from patients of other regions, and therefore the suitability of this method in Chinese HCC patients has remained unknown. Materials/Methods: In this study, we tested the transcriptomic group classication from the French cohort on a cohort of HCC patients from China. a total of 107 HCC cases from China were selected for the G1– G6 transcriptomic molecular classication. The correlation between the G1–G6 molecular classication and clinicopathological features were analyzed. RNA sequencing and bioinformatics analysis were performed to screen related targets and molecular signaling pathways. Results: We investigated the G1–G6 signatures in 107 Chinese HCC patients. HCC cases from China (n=107) were distributed as follows: G1 (17.76%), G2 (1.87%), G3 (18.69%), G4 (9.35%), G5 (23.36%), and G6 (28.97%) groups. We observed concordance between the genetic proles and clinical features of Chinese HCC patients and French HCC patients. We found that the G1–G3 subgroups were associated with high serum alpha-fetoprotein (AFP) level, high copy number of hepatitis B virus (HBV) DNA, complex histopathological structure, macrovascular invasion, negative or weak Hep-Par1 expression, programmed death-ligand 1 expression, and liver cancer stemness. The G1 subgroup was mainly related to liver cancer stemness, and G3 subgroup showed the worst prognosis. The G5 and G6 subgroups were associated with activation of the Wnt/β-catenin pathway. Compared with the G1–G4 group, the G1–G3 group showed signicantly higher expression levels of regenerating family member 1 beta (REG1B), regenerating family member 3 gamma (REG3G), and inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), and enriched calcium signaling pathway. Conclusions: Our results clarify the correlation between G1–G6 molecular classication and molecular markers and molecular signaling pathways in the Chinese HCC population and initially established a link between the phenotype and molecular characteristics. This study enhances our understanding of the heterogenicity of China HCC and indicates that the G1–G6 signatures can be used to identify potential therapeutic biomarkers against HCC patients in China. was used to analyze the correlation between the G1–G6 molecular classication and tumor biological behavior. After checking data for normal distribution and variance homogeneity, comparisons between two groups were evaluated using independent-sample t-tests or Mann–Whitney U test. For three or more groups, differences were statistically analyzed by one-way ANOVA or the non-parametric test (Kruskal– Wallis test). Kaplan–Meier and Cox proportional hazards survival regression analysis were used to evaluate the prognostic signicance of the G1–G6 molecular classications within 36 months. All p values were two-tailed, and P < 0.05 was considered statistically signicant.
Background
Hepatocellular carcinoma (HCC) is the most common malignancy worldwide, with more than half of the new HCC cases and deaths every year occurring in China [1]. Click to download.
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Domain: Biology Medicine
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Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. Antibodies targeting all three of these variants may be more potent than those that target a single variant [66,67]. Antibodies targeting SIRPα currently being evaluated are listed in the Table 4. Trials evaluating treatment with SIRPα antibodies in combination with immunotherapies are in the early clinical stage of development, and include OSE-172 [68] from OSE Immunotherapeutics (co-developed with Boehringer Ingelheim), CC-95251 from Celgene, and FSI-189 from Gilead [59].
Bi-specific molecules
Bi-specific molecules bind two targets or two distinct epitopes of one target. The antigen binding sites of bispecific molecules could consist of two antibodies or proteins (ligand or receptor), or could consist of one antibody and one protein. Bi-specific antibodies can bind two target antigens in-cis and in-trans. Rational design of bispecific molecules based on biological activity may result in distinct effects or improved efficacy when compared to combination treatments. Four bispecific antibodies, catumaxomab [69] (withdrawn in 2017), blinatumomab [70], emicizumab [71], and amivantamab-vmjw [72] have been approved by EMA or FDA. Bi-specific molecules can be constructed from CD47 targeting antibodies or SIRPα and other antigen-targeting molecules (Fig. 4). Target antigens could include: A) tumor associated cell surface antigens (PD-L1, CD20, CD19, MSLN (Mesothelin), Claudin18.2, and Her2), B) immune checkpoint proteins (PD-1, CD40, 41BB), and C) cytokines or receptors (CSF-2 receptor, VEGF). Reduced affinity for CD47 and increased affinity for the second target may reduce toxicity and enhance efficacy. For type A antibodies, IgG1-Fc was selected to enhance antibodymediated killing of tumor cells (ADCC, ADCP, and CDC). However, inactivated Fc is preferred when used in type B bi-specific molecules. Use of CD47-targeting biologics has shown significant clinical efficacy for treatment of R/R AML, NHL, and MDS. The combination of Hu5F9 and rituximab showed particular promise as a treatment approach for R/R NHL [34]. Several CD47related BsAb are in early-stage clinical trials. NI1701 [73], which targets CD47 and CD19, is an IgG-like BsAb constructed using modified knobs-into-hole technology [74], and contains an IgG1-Fc. Preclinical data showed that NI1701 [75] selectively binds to CD47 and CD19 co-expressing cells, but interacts poorly with normal healthy cells (CD47 + CD19 − ), resulting in avoidance of normal cells acting as sinks for binding of the antibodies, and reduced toxicity. In vitro and in vivo studies showed that NI1701 more potently killed tumor cells than did anti-CD47 and anti-CD19 antibodies alone, or in combination. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine<|endoftext|>Targeting CD47/SIRPα as a therapeutic strategy, where we are and where we are headed Immunotherapy using PD-1 and CTLA4 inhibitors to stimulate T cell immunity has achieved substantial clinical success. An early clinical trial (NCT02641002) that evaluated treatment of Acute Myeloid Leukemia (AML) and high-risk myelodysplastic syndrome (MDS) was terminated due to poor activity and safety profiles [45,46]. Another phase I trial (NCT02367196) evaluated treatment of advanced solid (alone) and hematologic malignancies (in combination with rituximab). In a combination trial with patients with R/R NHL [47], the ORR (overall response rate) was 13% with 25% achieving stable disease. However, 50% had anemia (of any grade), 33% had thrombocytopenia, and DLTs were observed in 3 subjects (1 subject infusion-related reaction at 15 mg/ kg Q2W and 2 subjects had grade 3 thrombocytopenia requiring platelet transfusion occurring at 30 mg/kg).
SGN-CD47M
SGN-CD47M, developed by Seagen (Seattle Genetics), is a CD47 targeting probody drug conjugate (PDCs). Probody therapeutics [48] are antibody prodrugs designed to remain inactive until proteolytically cleaved and activated in the tumor microenvironment. Probody drug conjugates [49] can be activated by multiple proteases in the tumor microenvironment, but remain inactive in the circulation and in normal tissues.
The efficacy of PDCs depends on multiple factors including binding affinity and specificity for the antigen, efficiency of cleavage in the tumor microenvironment, lack of cleavage in normal tissues, and internalization efficiency. Studies focused on PDCs are in the early clinical stage, and safety and efficacy have yet to be determined. Clinical trial (NCT03957096) of SGN-CD47M for treatment of advanced solid tumors was terminated based on portfolio prioritization.
Other drugs in early clinical development for safety and dosing evaluation are listed in Table 1.
SIRPα-fc Among the 10 allelic variants of SIRPα, SIRPα V1 and V2 are the most prevalent variants [50]. The affinity of wild type SIRPα binding to CD47 is in the micromolar range, which is 1000-fold weaker than that of anti-CD47 antibodies. Soluble SIRPα binding to CD47 on tumor cells could block inhibitory signals and enhance phagocytic activity. Six SIRPα-fusion proteins are currently in phase I or phase II clinical trials (Table 2, ordered by clinical trial developmental state).
Evorpacept (ALX148)
Evorpacept is comprised of SIRPα variant 1 domain 1 (V1D1) and inactive human IgG1-Fc [51]. Evorpacept (CV1) was selected from mutant libraries and includes 9 mutations (V6I, A271, I31F, E47V, K53R, E54S, H56P, L66T, V92I), which resulted in a 50,000-fold increase in affinity compared with that of wild type SIRPα. Preclinical data showed that evorpacept augmented macrophage antitumor activity in vitro and in vivo in combination with tumor-opsonizing antibodies (trastuzumab, obinutuzumab and cetuximab). However, no phagocytosis or antitumor activity was observed following treatment with evorpacept alone. A trial [52] in which patients with NHL received evorpacept alone at 10 mg/kg or at 15 mg/kg in combination with rituximab resulted in ORRs of 40.9 and 63.6%, respectively. The CD47 receptor occupancy on RBC and CD4 T cells was approximately 90% at 10-15 mg/ kg. A phase I study [53] of evorpacept in combination with pembrolizumab, trastuzumab, or zanidatamab, and/or chemotherapeutic agents, evaluating treatment of advanced solid malignancy is ongoing. Preliminary results showed anti-cancer activity of evorpacept in combination with pembrolizumab (AP) and/or chemotherapy (5FU + platinum) in patients with second line or greater HNSCC (head and neck squamous cell carcinoma) with prior platinum therapy. The ORR in patients with checkpoint inhibitor-naïve HNSCC (n = 10) treated with AP was 40%, but 0% in patients with HNSCC who had previously received checkpoint inhibitors (n = 10). A phase II study of evorpacept in combination with pembrolizumab for treatment of HNSCC was recently initiated. Many promising strategies targeting the CD47-SIRPα axis are in development and offer a great deal of hope to patients with cancer.
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Domain: Biology Medicine
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This below document has 2 sentences that start with 'The synthesized bone mineral',
2 paragraphs that end with 'for bone regeneration'. It has approximately 765 words, 30 sentences, and 18 paragraph(s).
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. The D Release of ICPC + DAMB + fibrin + hPL was significantly higher than that of ICPC + DAMB + hPL ( Figure 3G). This demonstrated that adding fibrin increased the degradation rate of the microbeads. As the microbeads degraded, cells were released, and the D Release of each group increased. There was no difference in D Release between each group at 14 days ( Figure 3G).
Alkaline phosphatase activity of hPDLSCs
Alizarin Red staining of minerals synthesized by hPDLSCs
At 21 days of osteogenic differentiation, the hPDLSCs from the microbeads exhibited a high viability and proliferation. Figures 6A,D,G show that the hPDLSCs released from the microbeads significantly proliferated and adhered to ICPC scaffold. This indicates that the ICPC scaffold was cytocompatible.
Representative ARS staining images of bone mineral secretion by hPDLSCs in the 12-well plate with ICPC are shown in Figures 6B,E,H. The red staining of mineralized nodules formed by the hPDLSCs was denser in the ICPC + DAMB + fibrin + hPL + osteo group than that of the ICPC + DAMB + fibrin + FBS + osteo group Figures 6C,F,I. The synthesized bone mineral in ICPC + DAMB + fibrin + FBS + osteo and ICPC + DAMB + fibrin + hPL + osteo increased with culture time from 1 to 21 days. The synthesized bone mineral amount in the ICPC + DAMB + fibrin + hPL + osteo was 6.9-fold and 13.2-fold that of the control group, at 14 days and 21 days, respectively (Figure 7). These results demonstrate that the hPLbased osteogenic medium significantly enhanced the osteogenic differentiation of the hPDLSCs.
Discussion
This study represents the first report to investigate degradable alginate hydrogel incorporated with hPL inside an
Frontiers in Materials
frontiersin.org 08 injectable calcium phosphate scaffold as a cell delivery system for bone regeneration. The hPDLSCs showed excellent viability while being encapsulated in the DAMB + fibrin + hPL microbeads in ICPC scaffold. When the setting of the ICPC was complete, the hPDLSCs were gradually released from the degradable hydrogel microbeads and grew rapidly in the scaffold. Furthermore, the osteogenic differentiation of hPDLSCs was significantly enhanced by the osteogenic medium supplemented with hPL. This study is schematically shown in Figure 8. These findings demonstrate a promising and novel xeno-free approach to delivering hPDLSCs in ICPC scaffold for bone regeneration.
The ICPC paste can be filled into a bone defect with an intimate adaptation to complex defect cavities and set in situ to form bioresorbable hydroxyapatite Xu et al., 2006). However, ionic activities and pH variations during the setting of ICPC paste could exert a cytotoxic effect on the cells (Matsuya et al., 2000;Simon et al., 2004;Przekora, 2019). TTCP and DCPA dissolved in the chitosan solution as Ca 2+ , PO 4 3-, and OH − ions , which then re-precipitated to form hydroxyapatite: 2Ca 4 (PO 4 ) 2 O+2CaHPO 4 → Ca 10 (PO 4 ) 6 (OH) 2 . It was reported that the pH during the setting of ICPC could be increased to approximately 10 (Simon et al., 2004). Therefore, there was a need to protect the cells from the ICPC setting reaction.
Alginate is among the most common natural polymers for the encapsulation and delivery of cells because of its many outstanding properties such as biocompatibility, gel-forming ability, non-toxicity, and ease of process (Gasperini et al., 2014;Izeia et al., 2020;Xu et al., 2021). However, the absence of alginate degrading enzyme (alginase) in the human body limits the degradability of the alginate. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering<|endoftext|>Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. However, FBS-expanded MSCs could evoke immune responses against xenogenic serum antigens in the human body (Spees et al., 2004;Tonti and Mannello, 2008). In addition, FBS is a potential source of microbial contaminants such as fungi, bacteria, viruses, or prions. Indeed, a 20%-50% contamination rate of the virus was reported for FBS (Even et al., 2006). Furthermore, the ingredients of FBS are not precisely defined, and lot-to-lot variability and unintended interactions with test substances can lead to unexpected or undesired outcomes in the clinical application (Jochems et al., 2002). Therefore, it is critically important and highly desirable to develop a FBS-free medium as a cell nutrition supplement for cell-based therapies.
The use of hPL for MSC expansion was first reported by Doucet et al., in 2005(Doucet et al., 2005. Since then, hPL has been proven as a viable alternative to FBS, enabling efficient propagation of MSC under animal serum-free conditions for clinical application. There are several advantages for hPL for cell growth supplement: 1) hPL can be easily obtained and produced hPDLSCs showed high proliferation in ICPC for 21 days (A,D,G). Representative ARS staining images of mineral synthesis by hPDLSCs with ICPC (B,E,H). Representative images of mineralized nodules formed by the hPDLSCs. More mineralized nodules were present in the ICPC + DAMB + fibrin + hPL + osteo group than in ICPC + DAMB + fibrin + FBS + osteo group and control group (C,F,I).
Frontiers in Materials frontiersin.org using freeze-thaw procedures (Dessels et al., 2016); 2) Because hPL is derived from humans, neither bovine viruses nor immune reactions against bovine proteins are a concern (Saury et al., 2018); 3) hPL can be used in autologous settings to minimize the risk of immunological reactions (Sánchez et al., 2003); 4) hPL is more efficient in terms of costs and proliferation rate than FBS for certain types of MSCs (Burnouf et al., 2016). In the exhibit survey, 2.5% hPL promoted the proliferation of hPDLSCs when compared with the 10% FBS. In a previous study, the use of 10% FBS showed a slightly higher proliferation of hPDLSCs as compared to 5% hPL. However, the difference in colony number was not statistically significant (Abuarqoub et al., 2019). In another study, hPDLSCs were cultured using a medium supplemented with 10% FBS, 5% PL + 5% FBS, or 10% PL. It was revealed that the media containing 5% PL + 5% FBS resulted in more significant stimulation of cell growth, when compared with those containing either 10% FBS or 10% PL. Furthermore, a tendency toward enhanced proliferation was exhibited in media containing 10% PL as compared with media containing 10% FBS (Wu et al., 2017). The differences of hPL concentration or hPDLSCs could be associated with the difference in the preparation method, the variability in the donors, and the storage conditions. Hence, the quality of hPL used by different laboratories reported in the literature may be somewhat different from each other (Christgau et al., 2006;Ogino et al., 2006;Schallmoser et al., 2007).
The implantation of ICPC scaffold with MSCs has achieved ectopic and orthotopic bone formation and critical-sized defect healing Xu et al., 2017). The seeded MSCs could directly deposit bone matrix minerals in the scaffold due to their osteogenic differentiation potential. The therapeutic benefit of the transplanted MSCs was associated with a paracrine mechanism that stimulated the recruitment of host cells. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. The hPDLSCs showed excellent viability while being encapsulated in the DAMB + fibrin + hPL microbeads in ICPC scaffold. When the setting of the ICPC was complete, the hPDLSCs were gradually released from the degradable hydrogel microbeads and grew rapidly in the scaffold. Furthermore, the osteogenic differentiation of hPDLSCs was significantly enhanced by the osteogenic medium supplemented with hPL. This study is schematically shown in Figure 8. These findings demonstrate a promising and novel xeno-free approach to delivering hPDLSCs in ICPC scaffold for bone regeneration.
The ICPC paste can be filled into a bone defect with an intimate adaptation to complex defect cavities and set in situ to form bioresorbable hydroxyapatite Xu et al., 2006). However, ionic activities and pH variations during the setting of ICPC paste could exert a cytotoxic effect on the cells (Matsuya et al., 2000;Simon et al., 2004;Przekora, 2019). TTCP and DCPA dissolved in the chitosan solution as Ca 2+ , PO 4 3-, and OH − ions , which then re-precipitated to form hydroxyapatite: 2Ca 4 (PO 4 ) 2 O+2CaHPO 4 → Ca 10 (PO 4 ) 6 (OH) 2 . It was reported that the pH during the setting of ICPC could be increased to approximately 10 (Simon et al., 2004). Therefore, there was a need to protect the cells from the ICPC setting reaction.
Alginate is among the most common natural polymers for the encapsulation and delivery of cells because of its many outstanding properties such as biocompatibility, gel-forming ability, non-toxicity, and ease of process (Gasperini et al., 2014;Izeia et al., 2020;Xu et al., 2021). However, the absence of alginate degrading enzyme (alginase) in the human body limits the degradability of the alginate. As the setting reaction of ICPC paste is largely complete after 1 day, it would be desirable for the alginate microbeads to quickly degrade, thus releasing the cells from the microbeads for enhanced cell viability and proliferation. A previous study reported that alginate could be chemically modified using oxidizing agents such as sodium periodate to produce oxidized alginate that was hydrolytically degradable Frontiers in Materials frontiersin.org (Zhou and Xu, 2011). In addition, it has been reported that adding fibrin could improve the attachment and proliferation of the cells in the alginate hydrogel, as well as to accelerate the release of cells from the alginate-based microbeads (Liu et al., 2013b). Fibrinogen was converted to fibrin via the mediation of thrombin and self-assembles into fibrin mesh, providing cell binding sites for cell attachment, migration, and proliferation (Li et al., 2015). As the cells migrated out of the microbeads, the porosity of the hydrogel increased, thus accelerating the degradation of the hydrogel. A previous study showed that the cell-encapsulating-alginate-fibrin-microbeads had diameters of about 100-500 μm, and the microbeads started to degrade at day 4 and released the encapsulated hiPSCs (Wang et al., 2016). Another study encapsulated hUCMSCs into the oxidized alginate-fibrin microbeads of about 300 μm in sizes (Chen et al., 2012). The microbeads started to release the cells at 4 days and completely degraded at 21 days (Chen et al., 2012). Therefore, microbeads with sizes of several hundred microns were suitable for injection, and were quickly degraded to release the cells.
FBS is the most widely-used supplement for the cultivation and expansion of eukaryotic cells. FBS contains essential components for cell growth and maintenance such as hormones, vitamins, transport proteins, trace elements, spreading, and growth factors (Brunner et al., 2010), which is effective on most types of human and animal cells. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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@@@ User: Context: Novel injectable calcium phosphate scaffold with homo periodontic ligament stem cell encapsulation in microbeads for bone re-formation Objects: 1) Develop a new concept of homo periodontic ligament stem cellular phones (hPDLSCs) capsulised in degradable alginate microbeads (DAMB) with homo platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Enquire the proliferation and osteogenic distinction of hPDLSCs in ICPC with hPL as a xeno-free postscript and sensual serum replenishment for bone weave designing lotions. The ICPC melted consisted of chitosan malate amalgamated with unfertile purified weewee at a chitosan/(chitosan + weewee) mass fraction of 7.5%. The ICPC pulverise was fused with the chitosan melted at a mass ratio of 2:1. Each glue was stationed into 3 mm 3 × 4 mm 3 × 25 mm 3 untainteds sword mildew to make flexural specimens. Each specimen was brooded with a chalks slip on each side, clamped, and procreated in a humidor with 100% comparative humidness at 37°C for 24 h. Load-bearing belongings hPDLSC ...
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Continue the text. Fix word choice.
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. The ICPC liquid consisted of chitosan malate mixed with sterile distilled water at a chitosan/(chitosan + water) mass fraction of 7.5%. The ICPC powder was mixed with the chitosan liquid at a mass ratio of 2:1. Each paste was placed into 3 mm 3 × 4 mm 3 × 25 mm 3 stainless steel molds to make flexural specimens. Each specimen was covered with a glass slide on each side, clamped, and incubated in a humidor with 100% relative humidity at 37°C for 24 h.
Load-bearing properties
hPDLSC isolation from extracted human teeth hPDLSCs were isolated from the PDL tissues on extracted human premolars collected from healthy patients. The procedures were approved by the Institutional Review Board of the University of Maryland Baltimore (HP-00052180). All patients or their guardians were informed with written consent. The PDL tissue was scraped off the middle third of the root surfaces (Chen et al., 2020). The scraped tissues were digested in a solution of 3 mg/ml collagenase I (Worthington Biochem, Freehold, NJ, United States) and 4 mg/ml dispase (Roche, Mannheim, Germany) for 1 h at 37°C in a humidified atmosphere with 5% CO 2 . Then the PDL samples were placed into culture dishes with growth medium. The medium consisted of Dulbecco's modified Eagle's medium (DMEM, GIBCO BRL, Grand Island, NY, United States) supplemented with 20% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, United States) and 1% penicillin/streptomycin (P. S, GIBCO BRL). The samples were incubated at 37°C with 5% CO 2 . Single cells were observed 3 days later, and cell colonies were formed at 7-10 days. The individual cell colonies were digested to a single cell suspension using filter paper (Whatman, TISCH Scientific, North Bend, Ohio, United States) with 0.25% Trypsin-EDTA (GIBCO BRL), and transferred to 24-well culture plates, and the cells were passaged when they reach to 80% confluency. It was shown in our previous study that the hPDLSCs were positive for STRO-1, CD146 + , and OCT4 and negative for CD34 and CD45 (Chen et al., 2020). Passage 3 cells were used in subsequent experiments.
hPDLSC encapsulation in alginate microbeads
Alginate was made degradable by oxidation at 7.5%, following a method described in a previous study Frontiers in Materials frontiersin.org 03 (Bouhadir et al., 2001). Three types of alginate-based microbeads were fabricated to encapsulate the hPDLSCs: 1) Alginate-fibrin microbeads (DAMB + fibrin); 2) Alginate-hPL microbeads (DAMB + hPL); 3) Alginate-fibrin-hPL microbeads (DAMB + fibrin + hPL). A 1.2% (mass fraction) sodium alginate solution was prepared by dissolving oxidized alginate in saline (155 mmol/L NaCl) containing 0.1% fibrinogen for preparing type-1 microbeads. For type 2 microbeads, oxidized alginate was dissolved by 50% dilution of hPL with saline. For type 3 microbeads, fibrinogen was added at a concentration of 0.1% to the alginate-hPL solution. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. The cells exhibited a polygonal morphology and a relatively high viability ( Figures 2F-H). At 14 days, the degradation of microbeads accelerated and most of the cells were released ( Figures 2J-L). The PLive increased with time due to proliferation in the ICPC + DAMB + fibrin + hPL, ICPC + DAMB + hPL, and ICPC + DAMB + fibrin + FBS. At 1 day, the PLive of hPDLSCs in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + hPL was slightly higher than that in the ICPC + DAMB + fibrin + FBS group, indicating that incorporation of hPL into the microbeads could better protect the hPDLSCs from the pH change and ion activities during the ICPC setting ( Figure 2M). In contrast, very few cells were observed to attach and spread in the ICPC + DAMB + fibrin + DMEM, the PLive of the hPDLSCs was lower, due to the lack of nutrition supplements.
The cell growth rate was evaluated via the CCK-8 assay ( Figure 2N). At 1 day and 3 days, the proliferation of hPDLSCs was low in the groups of ICPC + DAMB + fibrin, ICPC + DAMB + hPL, and ICPC + DAMB + fibrin + hPL. At 7 days, as more cells were released from the degradable microbeads, the cell growth was significantly enhanced. The proliferation rate in the ICPC + DAMB + fibrin and ICPC + DAMB + fibrin + hPL groups was higher than that of the ICPC + DAMB + hPL group (p < 0.05), indicating that the incorporation of fibrin accelerated the hPDLSCs migrated out from the microbeads. The absorbance of the hPDLSCs in the ICPC + DAMB + fibrin + DMEM group was lower over time, indicating that the DMEM did not support the proliferation of the hPDLSCs encapsulated in the microbeads.
FIGURE 1
Load-bearing property of scaffolds in ICPC + Chitosan group and control group: (A) flexural strength; (B) elastic modulus (mean ± sd; n = 4). Values for cancellous bone were obtained from the literature (Damien and Parsons, 1991).
Frontiers in Materials frontiersin.org 3A-F). The D Release of ICPC + DAMB + fibrin + hPL was significantly higher than that of ICPC + DAMB + hPL ( Figure 3G). This demonstrated that adding fibrin increased the degradation rate of the microbeads. As the microbeads degraded, cells were released, and the D Release of each group increased. There was no difference in D Release between each group at 14 days ( Figure 3G).
Alkaline phosphatase activity of hPDLSCs
Alizarin Red staining of minerals synthesized by hPDLSCs
At 21 days of osteogenic differentiation, the hPDLSCs from the microbeads exhibited a high viability and proliferation. Figures 6A,D,G show that the hPDLSCs released from the microbeads significantly proliferated and adhered to ICPC scaffold. This indicates that the ICPC scaffold was cytocompatible.
Representative ARS staining images of bone mineral secretion by hPDLSCs in the 12-well plate with ICPC are shown in Figures 6B,E,H. The red staining of mineralized nodules formed by the hPDLSCs was denser in the ICPC + DAMB + fibrin + hPL + osteo group than that of the ICPC + DAMB + fibrin + FBS + osteo group Figures 6C,F,I. The synthesized bone mineral in ICPC + DAMB + fibrin + FBS + osteo and ICPC + DAMB + fibrin + hPL + osteo increased with culture time from 1 to 21 days. The synthesized bone mineral amount in the ICPC + DAMB + fibrin + hPL + osteo was 6.9-fold and 13.2-fold that of the control group, at 14 days and 21 days, respectively (Figure 7). These results demonstrate that the hPLbased osteogenic medium significantly enhanced the osteogenic differentiation of the hPDLSCs.
Discussion
This study represents the first report to investigate degradable alginate hydrogel incorporated with hPL inside an
Frontiers in Materials
frontiersin.org 08 injectable calcium phosphate scaffold as a cell delivery system for bone regeneration. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
==
Domain: Biology Medicine Engineering<|endoftext|>Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. These host cells
FIGURE 7
Quantitative bone mineral synthesis by hPDLSCs. The hPDLSC-synthesized bone mineral in ICPC + DAMB + fibrin + hPL + osteo group and ICPC + DAMB + fibrin + FBS + osteo group was 13.2 folds and 11.1 folds that of the control group, respectively (mean ± sd; n = 6). Values with dissimilar letters are significantly different from each other (p < 0.05).
FIGURE 8
Schematic illustration showing the encapsulation of hPDLSCs in alginate microbeads incorporated with hPL and delivery with ICPC for bone regeneration.
Frontiers in Materials frontiersin.org included osteoblast progenitors, endothelial cells, and osteoclasts, which took over the responsibility of subsequent bone formation and remodeling (Wang et al., 2011;Wang J. et al., 2014). Interestingly, it has been revealed that MSCs could enhance bone repair by modulating the foreign body reaction to ICPC, attracting circulating monocytes, and inducing their differentiation into osteoclasts, thus favoring bone formation (Gamblin et al., 2014).
In the exhibit survey, the hPDLSCs in ICPC were gradually released from the degradable hydrogel microbeads and underwent differentiation into osteogenic lineage by the hPL-based medium, without exposure to the animal serum. Moreover, higher ALP activity, osteogenic expression, and bone mineralization were achieved in ICPC + DAMB + fibrin + hPL construct than in ICPC + DAMB + fibrin + FBS construct. Therefore, the novel hPDLSC-hPL-microbeads-ICPC construct is a highly promising xeno-free approach for bone regeneration. Future in vivo studies are needed to evaluate the bone regenerative capacity of the hPDLSC-hPL-microbeads-ICPC construct for the treatment of bone defects in animal models.
Conclusion
This study demonstrated for the first time the hPDLSCencapsulation in degradable alginate hydrogel microbeads with hPL inside an injectable calcium phosphate scaffold for bone regeneration. The ICPC scaffold was biocompatible, mechanically load-bearing, while supporting hPDLSC attachment, proliferation, and osteogenic differentiation with the hPL as xeno-free cell supplement. The microbeads incorporating with hPL protected the hPDLSCs from the setting reaction of ICPC. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. S, GIBCO BRL). The samples were incubated at 37°C with 5% CO 2 . Single cells were observed 3 days later, and cell colonies were formed at 7-10 days. The individual cell colonies were digested to a single cell suspension using filter paper (Whatman, TISCH Scientific, North Bend, Ohio, United States) with 0.25% Trypsin-EDTA (GIBCO BRL), and transferred to 24-well culture plates, and the cells were passaged when they reach to 80% confluency. It was shown in our previous study that the hPDLSCs were positive for STRO-1, CD146 + , and OCT4 and negative for CD34 and CD45 (Chen et al., 2020). Passage 3 cells were used in subsequent experiments.
hPDLSC encapsulation in alginate microbeads
Alginate was made degradable by oxidation at 7.5%, following a method described in a previous study Frontiers in Materials frontiersin.org 03 (Bouhadir et al., 2001). Three types of alginate-based microbeads were fabricated to encapsulate the hPDLSCs: 1) Alginate-fibrin microbeads (DAMB + fibrin); 2) Alginate-hPL microbeads (DAMB + hPL); 3) Alginate-fibrin-hPL microbeads (DAMB + fibrin + hPL). A 1.2% (mass fraction) sodium alginate solution was prepared by dissolving oxidized alginate in saline (155 mmol/L NaCl) containing 0.1% fibrinogen for preparing type-1 microbeads. For type 2 microbeads, oxidized alginate was dissolved by 50% dilution of hPL with saline. For type 3 microbeads, fibrinogen was added at a concentration of 0.1% to the alginate-hPL solution. hPDLSCs were encapsulated at a density of 100,000 cells/mL of alginate solution. The alginate-cell solution was loaded into a syringe that was connected to a bead-generating device (Var J). Nitrogen gas was fed to the gas inlet and a pressure of 8 psi was established to form a coaxial airflow to break up the alginate droplets (Zhou and Xu, 2011). For type 1 and type 3 microbeads, the droplets fell into a well of 6-well plate containing 8 ml of 200 mmol/L calcium chloride solution plus 1 NIH units/mL of thrombin (Millipore Sigma) and cross-linked for 20 min to form DAMB + fibrin and DAMB + fibrin + hPL. For DAMB + hPL, the droplets fell into a well of 200 mmol/L calcium chloride solution and crosslinked for 20 min to form microbeads. A microscope (Eclipse TE-2000S, Nikon, Melville, NY) was used to measure the size of the microbeads.
The ICPC paste was extruded through a sterile syringe into a 12 well-plate with 0.05 ml of paste per well. The cement was set in an incubator for 30 min. The hPDLSC-encapsulating microbeads were suspended in the culture medium and added into the 24 well-plate containing CPC for co-culture. The viability and proliferation of hPDLCSs were tested in four groups: 1. ICPC + DAMB + fibrin + DMEM group (Alginate-fibrin microbeads encapsulating 1×10 5 hPDLSCs with 0.05 ml ICPC scaffold in DMEM); 2. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
==
Domain: Biology Medicine Engineering<|endoftext|>Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. The number of the hPDLSCs with spindle-shape morphology was counted via Image-Pro Plus software. The density of the released hPDLSCs was calculated: D Release = N Release /A, where A is the area of the view field for D Release .
At 1, 7, and 14 days, cells were lysed in a 0.2% Triton X-100 (Millipore Sigma) solution. The ALP activity of the cell lysate was measured by using an ALP Assay kit (QuantiChrom, BioAssay Systems, Hayward, CA, United States) with p-Nitrophenylphosphate (pNPP) as a substrate. The ALP activity was determined by measuring the absorbance at an optical density of 405 nm using a microplate reader (SpectraMax M5). The protein of cell lysate was quantified using Protein Assay Kit (Pierce BCA, Thermo Scientific, Rockford, IL, United States) following the manufacturer's protocol. The ALP activity was normalized to the protein amount and reported as mU/mg protein (Zhao et al., 2019).
The total cellular RNA of the cells was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using a High-capacity cDNA Reverse Transcription kit (Applied Biosystems) in a thermal cycler (GenAmp PCR 2720, Applied Biosystems). RT 2 SYBR ® Green ROX qPCR Mastermix (Qiagen, Germantown, MD, United States) was used to quantify the transcript levels of glyceraldehyde 3phosphate dehydrogenase (GAPDH), alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), Collagen type 1 (COL1), and Osteogenicpontin (OPN). The human-specific primers were synthesized commercially (Millipore Sigma), and the sequences of the primers are listed in Table 1. The qPCR data collection and analyses were performed using a QuantStudio ™ 3 Real-Time PCR System (Thermo Scientific). Relative expressions were calculated using the 2 −ΔΔCT method and normalized by the C t value of the housekeeping gene GAPDH. The C t value of hPDLSCs in the control group at 1 day served as the calibrator.
At 1, 14, and 21 days, the hPDLSCs were fixed with 4% paraformaldehyde for 15 min and stained with ARS. After staining for 30 min, the ARS solution was removed and rinsed with distilled water to remove any loose ARS. The samples were imaged via microscopy (Eclipse TE-2000S, Nikon). For quantification, the stained mineralization was de-stained in 10% cetylpyridinium chloride (Millipore Sigma) for 30 min and the concentration was measured at an optical density of 652 nm using the microplate reader (SpectraMax M5). The ARS concentration of the control group at 1 day served as the calibrator.
Statistical analysis
Load-bearing properties
The flexural strength and elastic modulus of the ICPC scaffolds are plotted in Figure 1. Flexural strength and elastic modulus of ICPC scaffolds was 8.4 ± 0.91 MPa and 1.56 ± 0.1 GPa, significantly higher than 6.4 ± 0.43 MPa and 1.12 ± 0.23 GPa of the control group, respectively (p < 0.05). This demonstrates that the incorporation of chitosan improved the mechanical properties of the ICPC scaffold. Both of these values are higher than those of cancellous bone reported in the literature (Damien and Parsons, 1991).
Representative live/dead staining images from 1 to 14 days are shown in Figures 2(A-F). At 1 day, there were numerous live cells (stained green) and a few dead cells (stained red) in all groups (Figures 2A-E). At 7 days, the hPDLSCs were released from the degradable microbeads in the ICPC + DAMB + fibrin + hPL, ICPC + DAMB + hPL, and ICPC + DAMB + fibrin + FBS. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
==
Domain: Biology Medicine Engineering
|
[{"added": "2022-11-18T15:01:41.510Z", "created": "2022-11-16T00:00:00.000", "id": "253599070", "metadata": {"extfieldsofstudy": [], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmats.2022.977853/pdf", "pdf_hash": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "pdf_src": "Frontier", "provenance": "peS2o-0028.json.gz:575", "s2fieldsofstudy": ["Biology", "Medicine", "Engineering"], "sha1": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "year": 2022}, "source": "pes2o/s2orc", "version": "v3-fos-license"}, {"added": "2022-11-18T15:01:41.510Z", "created": "2022-11-16T00:00:00.000", "id": "253599070", "metadata": {"extfieldsofstudy": [], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmats.2022.977853/pdf", "pdf_hash": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "pdf_src": "Frontier", "provenance": "peS2o-0028.json.gz:575", "s2fieldsofstudy": ["Biology", "Medicine", "Engineering"], "sha1": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "year": 2022}, "source": "pes2o/s2orc", "version": "v3-fos-license"}]
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. In the exhibit survey, 2.5% hPL promoted the proliferation of hPDLSCs when compared with the 10% FBS. In a previous study, the use of 10% FBS showed a slightly higher proliferation of hPDLSCs as compared to 5% hPL. However, the difference in colony number was not statistically significant (Abuarqoub et al., 2019). In another study, hPDLSCs were cultured using a medium supplemented with 10% FBS, 5% PL + 5% FBS, or 10% PL. It was revealed that the media containing 5% PL + 5% FBS resulted in more significant stimulation of cell growth, when compared with those containing either 10% FBS or 10% PL. Furthermore, a tendency toward enhanced proliferation was exhibited in media containing 10% PL as compared with media containing 10% FBS (Wu et al., 2017). The differences of hPL concentration or hPDLSCs could be associated with the difference in the preparation method, the variability in the donors, and the storage conditions. Hence, the quality of hPL used by different laboratories reported in the literature may be somewhat different from each other (Christgau et al., 2006;Ogino et al., 2006;Schallmoser et al., 2007).
The implantation of ICPC scaffold with MSCs has achieved ectopic and orthotopic bone formation and critical-sized defect healing Xu et al., 2017). The seeded MSCs could directly deposit bone matrix minerals in the scaffold due to their osteogenic differentiation potential. The therapeutic benefit of the transplanted MSCs was associated with a paracrine mechanism that stimulated the recruitment of host cells. These host cells
FIGURE 7
Quantitative bone mineral synthesis by hPDLSCs. The hPDLSC-synthesized bone mineral in ICPC + DAMB + fibrin + hPL + osteo group and ICPC + DAMB + fibrin + FBS + osteo group was 13.2 folds and 11.1 folds that of the control group, respectively (mean ± sd; n = 6). Values with dissimilar letters are significantly different from each other (p < 0.05).
FIGURE 8
Schematic illustration showing the encapsulation of hPDLSCs in alginate microbeads incorporated with hPL and delivery with ICPC for bone regeneration.
Frontiers in Materials frontiersin.org included osteoblast progenitors, endothelial cells, and osteoclasts, which took over the responsibility of subsequent bone formation and remodeling (Wang et al., 2011;Wang J. et al., 2014). Interestingly, it has been revealed that MSCs could enhance bone repair by modulating the foreign body reaction to ICPC, attracting circulating monocytes, and inducing their differentiation into osteoclasts, thus favoring bone formation (Gamblin et al., 2014).
In the exhibit survey, the hPDLSCs in ICPC were gradually released from the degradable hydrogel microbeads and underwent differentiation into osteogenic lineage by the hPL-based medium, without exposure to the animal serum. Moreover, higher ALP activity, osteogenic expression, and bone mineralization were achieved in ICPC + DAMB + fibrin + hPL construct than in ICPC + DAMB + fibrin + FBS construct. Therefore, the novel hPDLSC-hPL-microbeads-ICPC construct is a highly promising xeno-free approach for bone regeneration. Future in vivo studies are needed to evaluate the bone regenerative capacity of the hPDLSC-hPL-microbeads-ICPC construct for the treatment of bone defects in animal models.
Conclusion
This study demonstrated for the first time the hPDLSCencapsulation in degradable alginate hydrogel microbeads with hPL inside an injectable calcium phosphate scaffold for bone regeneration. The ICPC scaffold was biocompatible, mechanically load-bearing, while supporting hPDLSC attachment, proliferation, and osteogenic differentiation with the hPL as xeno-free cell supplement. The microbeads incorporating with hPL protected the hPDLSCs from the setting reaction of ICPC. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
==
Domain: Biology Medicine Engineering
|
[{"added": "2022-11-18T15:01:41.510Z", "created": "2022-11-16T00:00:00.000", "id": "253599070", "metadata": {"extfieldsofstudy": [], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmats.2022.977853/pdf", "pdf_hash": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "pdf_src": "Frontier", "provenance": "peS2o-0028.json.gz:575", "s2fieldsofstudy": ["Biology", "Medicine", "Engineering"], "sha1": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "year": 2022}, "source": "pes2o/s2orc", "version": "v3-fos-license"}]
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This below document has 2 sentences that start with 'There has been no report',
2 sentences that end with 'tissue engineering applications'. It has approximately 831 words, 30 sentences, and 15 paragraph(s).
<<<<>>>>
Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. In addition, it has been reported that the incorporation of hPL could improve the viability, adhesion, and proliferation of a various types of cell in the alginate hydrogel (Sandri et al., 2015;Saporito et al., 2019).
However, there has been no report that investigated the alginate hydrogel incorporation of hPL as a cell delivery system with ICPC for bone tissue regeneration.
The excellent biocompatibility of ICPC renders it suitable for cell and growth factor delivery. hPL has been used as a cell nutrient supplement for cell expansion, as well as a source of growth factors for tissue engineering applications. To date, a literature search revealed no report on the development of injectable hPDLSC-hPL-CPC-microbead constructs for bone tissue engineering. There has been no report on hPLcontaining degradable microbeads as a vehicle to deliver hPDLSCs. There has been no report of investigating hPL as a cell growth supplement for hPDLSCs in culture with CPC, using the FBS-free and xeno-free cell culture medium alternative to avoid the risk of transmitting bovine infections and the initiation of xenogeneic immunological reactions.
Therefore, the objectives of the present study were to: 1) Develop a novel construct of hPDLSCs encapsulated in hydrogel microbeads with hPL and ICPC; 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in microbeads in ICPC with hPL as the xeno-free supplement for bone regeneration. The following hypotheses were tested: 1) Encapsulating hPDLSCs in alginate microbeads would protect the hPDLSCs during the setting reaction of ICPC paste; 2) The incorporation of hPL could improve the viability of hPDLSCs in the alginate microbeads; 3) The hPDLSCs released from the degradable alginate microbeads in ICPC would be able to proliferate and undergo osteogenic differentiate when using a xeno-free growth medium with hPL.
Fabrication of injectable calcium phosphate cement scaffold
The ICPC powder consisted of a mixture of TTCP and DCPA at a TTCP: DCPA molar ratio of 1:1 because this ratio was shown to produce apatite minerals (Xu and Simon, 2005). The TTCP powder was synthesized from a solid-state reaction between CaHPO 4 and CaCO 3 (Baker Chemical Company, NJ, United States) and then ground to obtain a median particle size of 17 μm. The DCPA powder (Baker Chemical) was ground to obtain a median particle size of 1 μm. Chitosan was used as cement liquid as previous studies have indicated that the addition of chitosan to CPC enhanced the strength and durability of ICPC (Weir and Xu, 2008). Two cement liquids at 0 and 7.5% chitosan were used. The ICPC liquid consisted of chitosan malate mixed with sterile distilled water at a chitosan/(chitosan + water) mass fraction of 7.5%. The ICPC powder was mixed with the chitosan liquid at a mass ratio of 2:1. Each paste was placed into 3 mm 3 × 4 mm 3 × 25 mm 3 stainless steel molds to make flexural specimens. Each specimen was covered with a glass slide on each side, clamped, and incubated in a humidor with 100% relative humidity at 37°C for 24 h.
Load-bearing properties
hPDLSC isolation from extracted human teeth hPDLSCs were isolated from the PDL tissues on extracted human premolars collected from healthy patients. The procedures were approved by the Institutional Review Board of the University of Maryland Baltimore (HP-00052180). All patients or their guardians were informed with written consent. The PDL tissue was scraped off the middle third of the root surfaces (Chen et al., 2020). The scraped tissues were digested in a solution of 3 mg/ml collagenase I (Worthington Biochem, Freehold, NJ, United States) and 4 mg/ml dispase (Roche, Mannheim, Germany) for 1 h at 37°C in a humidified atmosphere with 5% CO 2 . Then the PDL samples were placed into culture dishes with growth medium. The medium consisted of Dulbecco's modified Eagle's medium (DMEM, GIBCO BRL, Grand Island, NY, United States) supplemented with 20% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, United States) and 1% penicillin/streptomycin (P. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
==
Domain: Biology Medicine Engineering
|
[{"added": "2022-11-18T15:01:41.510Z", "created": "2022-11-16T00:00:00.000", "id": "253599070", "metadata": {"extfieldsofstudy": [], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmats.2022.977853/pdf", "pdf_hash": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "pdf_src": "Frontier", "provenance": "peS2o-0028.json.gz:575", "s2fieldsofstudy": ["Biology", "Medicine", "Engineering"], "sha1": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "year": 2022}, "source": "pes2o/s2orc", "version": "v3-fos-license"}]
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This below document has 2 sentences that start with 'A previous study', 2 sentences that end with 'et al., 2016)', 2 sentences that end with '(Chen et al., 2012)'. It has approximately 792 words, 28 sentences, and 11 paragraph(s).
<<<<>>>>
Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. As the setting reaction of ICPC paste is largely complete after 1 day, it would be desirable for the alginate microbeads to quickly degrade, thus releasing the cells from the microbeads for enhanced cell viability and proliferation. A previous study reported that alginate could be chemically modified using oxidizing agents such as sodium periodate to produce oxidized alginate that was hydrolytically degradable Frontiers in Materials frontiersin.org (Zhou and Xu, 2011). In addition, it has been reported that adding fibrin could improve the attachment and proliferation of the cells in the alginate hydrogel, as well as to accelerate the release of cells from the alginate-based microbeads (Liu et al., 2013b). Fibrinogen was converted to fibrin via the mediation of thrombin and self-assembles into fibrin mesh, providing cell binding sites for cell attachment, migration, and proliferation (Li et al., 2015). As the cells migrated out of the microbeads, the porosity of the hydrogel increased, thus accelerating the degradation of the hydrogel. A previous study showed that the cell-encapsulating-alginate-fibrin-microbeads had diameters of about 100-500 μm, and the microbeads started to degrade at day 4 and released the encapsulated hiPSCs (Wang et al., 2016). Another study encapsulated hUCMSCs into the oxidized alginate-fibrin microbeads of about 300 μm in sizes (Chen et al., 2012). The microbeads started to release the cells at 4 days and completely degraded at 21 days (Chen et al., 2012). Therefore, microbeads with sizes of several hundred microns were suitable for injection, and were quickly degraded to release the cells.
FBS is the most widely-used supplement for the cultivation and expansion of eukaryotic cells. FBS contains essential components for cell growth and maintenance such as hormones, vitamins, transport proteins, trace elements, spreading, and growth factors (Brunner et al., 2010), which is effective on most types of human and animal cells. However, FBS-expanded MSCs could evoke immune responses against xenogenic serum antigens in the human body (Spees et al., 2004;Tonti and Mannello, 2008). In addition, FBS is a potential source of microbial contaminants such as fungi, bacteria, viruses, or prions. Indeed, a 20%-50% contamination rate of the virus was reported for FBS (Even et al., 2006). Furthermore, the ingredients of FBS are not precisely defined, and lot-to-lot variability and unintended interactions with test substances can lead to unexpected or undesired outcomes in the clinical application (Jochems et al., 2002). Therefore, it is critically important and highly desirable to develop a FBS-free medium as a cell nutrition supplement for cell-based therapies.
The use of hPL for MSC expansion was first reported by Doucet et al., in 2005(Doucet et al., 2005. Since then, hPL has been proven as a viable alternative to FBS, enabling efficient propagation of MSC under animal serum-free conditions for clinical application. There are several advantages for hPL for cell growth supplement: 1) hPL can be easily obtained and produced hPDLSCs showed high proliferation in ICPC for 21 days (A,D,G). Representative ARS staining images of mineral synthesis by hPDLSCs with ICPC (B,E,H). Representative images of mineralized nodules formed by the hPDLSCs. More mineralized nodules were present in the ICPC + DAMB + fibrin + hPL + osteo group than in ICPC + DAMB + fibrin + FBS + osteo group and control group (C,F,I).
Frontiers in Materials frontiersin.org using freeze-thaw procedures (Dessels et al., 2016); 2) Because hPL is derived from humans, neither bovine viruses nor immune reactions against bovine proteins are a concern (Saury et al., 2018); 3) hPL can be used in autologous settings to minimize the risk of immunological reactions (Sánchez et al., 2003); 4) hPL is more efficient in terms of costs and proliferation rate than FBS for certain types of MSCs (Burnouf et al., 2016). Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
==
Domain: Biology Medicine Engineering
|
[{"added": "2022-11-18T15:01:41.510Z", "created": "2022-11-16T00:00:00.000", "id": "253599070", "metadata": {"extfieldsofstudy": [], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmats.2022.977853/pdf", "pdf_hash": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "pdf_src": "Frontier", "provenance": "peS2o-0028.json.gz:575", "s2fieldsofstudy": ["Biology", "Medicine", "Engineering"], "sha1": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "year": 2022}, "source": "pes2o/s2orc", "version": "v3-fos-license"}]
|
Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. hPDLSCs were encapsulated at a density of 100,000 cells/mL of alginate solution. The alginate-cell solution was loaded into a syringe that was connected to a bead-generating device (Var J). Nitrogen gas was fed to the gas inlet and a pressure of 8 psi was established to form a coaxial airflow to break up the alginate droplets (Zhou and Xu, 2011). For type 1 and type 3 microbeads, the droplets fell into a well of 6-well plate containing 8 ml of 200 mmol/L calcium chloride solution plus 1 NIH units/mL of thrombin (Millipore Sigma) and cross-linked for 20 min to form DAMB + fibrin and DAMB + fibrin + hPL. For DAMB + hPL, the droplets fell into a well of 200 mmol/L calcium chloride solution and crosslinked for 20 min to form microbeads. A microscope (Eclipse TE-2000S, Nikon, Melville, NY) was used to measure the size of the microbeads.
The ICPC paste was extruded through a sterile syringe into a 12 well-plate with 0.05 ml of paste per well. The cement was set in an incubator for 30 min. The hPDLSC-encapsulating microbeads were suspended in the culture medium and added into the 24 well-plate containing CPC for co-culture. The viability and proliferation of hPDLCSs were tested in four groups: 1. ICPC + DAMB + fibrin + DMEM group (Alginate-fibrin microbeads encapsulating 1×10 5 hPDLSCs with 0.05 ml ICPC scaffold in DMEM); 2. ICPC + DAMB + fibrin + FBS group (Alginate-fibrin microbeads encapsulating 1 × 10 5 hPDLSCs with 0.05 ml ICPC scaffold in growth medium containing 10% FBS); 3. ICPC + DAMB + hPL group (Alginate-hPL microbeads encapsulating with 1 × 10 5 hPDLSCs with 0.05 ml ICPC scaffold, cultured in DMEM for the first three days and then in growth medium containing 2.5% hPL); 4. ICPC + DAMB + fibrin + hPL group (Alginate-fibrin-hPL microbeads encapsulating with 1 × 10 5 hPDLSCs with 0.05 ml ICPC scaffold, cultured in DMEM for the first three days and then in growth medium containing 2.5% hPL).
After culturing for 1, 7, and 14 days, cells were stained with a live/dead kit (Invitrogen) and observed via epifluorescence microscopy (Eclipse TE-2000S, Nikon). Two images were taken at random locations for each sample, with four samples yielding 8 images at each time point for each group. Live and dead cells were counted via Image-Pro Plus software. The percentages of live cells (P Live ) were calculated. P Live = number of live cells/(number of live cells + number of dead cells).
The proliferation rate of hPDLSCs was investigated via Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan), following the manufacturer's protocol. At 1, 3, 7, and 14 days, the culture medium was carefully removed and placed with 500 µl DMEM containing 50 µl CCK-8 dye. After 2 h incubation at 37°C, a 200 µl aliquot from each well was placed in a 96-well plate and the absorbance at an optical density of 450 nm (OD450 nm) was measured with a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, United States).
To evaluate the number of hPDLSCs released from the degrading alginate microbeads, the samples were imaged via microscopy (Eclipse TE-2000S, Nikon). Two images were taken at random locations for each sample, with four samples yielding 8 images at each time point for each group. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
==
Domain: Biology Medicine Engineering
|
[{"added": "2022-11-18T15:01:41.510Z", "created": "2022-11-16T00:00:00.000", "id": "253599070", "metadata": {"extfieldsofstudy": [], "oa_license": "CCBY", "oa_status": "GOLD", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmats.2022.977853/pdf", "pdf_hash": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "pdf_src": "Frontier", "provenance": "peS2o-0028.json.gz:575", "s2fieldsofstudy": ["Biology", "Medicine", "Engineering"], "sha1": "f07726259424b63b6d06a5ee1fa3f4a5b3a5d614", "year": 2022}, "source": "pes2o/s2orc", "version": "v3-fos-license"}]
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This below document has
* 2 sentences that start with 'There has been no report',
* 2 sentences that end with 'tissue engineering applications'. It has approximately 842 words, 30 sentences, and 14 paragraph(s).
<<<<>>>>
Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. Seo et al. (2004) identified hPDLSCs in the periodontal ligament (PDL) and demonstrated that hPDLSCs implanted into nude mice generated cementum/PDL-like structures that resembled the native PDL. Compared with other MSCs, hPDLSCs have a marked ability to generate multiple tissue types, including alveolar bone, cementum, and Sharpey's fibers, making them an ideal source for cell-based dental therapy. However, there has no report that investigated the effect of hPL as a cell growth supplement on the proliferation and osteogenic differentiation of hPDLSCs in culture with ICPC.
In stem cell-based bone tissue engineering, the number of functioning cells in the bone injury site appears to be of critical importance. However, it was reported that few transplanted cells survived via local administration of cell suspensions. One study demonstrated that only 50% of initial donor BMSCs remained alive 48 h after implantation, and only 5% survived after 8 weeks (Giannoni et al., 2010). Another study showed that less than 1% of human multipotent stromal cells were detectable after 30 days post-implantation (Becquart et al., 2012). Thus the preferred approach was delivering a large number of cells to the target site via a carrier scaffold. Alginate hydrogel is highly hydrated with excellent biocompatibility and has shown great potential as a carrier for cell delivery (Izeia et al., 2020;Xu et al., 2021). Previous studies have reported that alginate microbeads are an ideal vehicle to localize the cells at the target defect site for cellbased therapy (Li et al., 2012;Pal et al., 2014). In addition, it has been reported that the incorporation of hPL could improve the viability, adhesion, and proliferation of a various types of cell in the alginate hydrogel (Sandri et al., 2015;Saporito et al., 2019).
However, there has been no report that investigated the alginate hydrogel incorporation of hPL as a cell delivery system with ICPC for bone tissue regeneration.
The excellent biocompatibility of ICPC renders it suitable for cell and growth factor delivery. hPL has been used as a cell nutrient supplement for cell expansion, as well as a source of growth factors for tissue engineering applications. To date, a literature search revealed no report on the development of injectable hPDLSC-hPL-CPC-microbead constructs for bone tissue engineering. There has been no report on hPLcontaining degradable microbeads as a vehicle to deliver hPDLSCs. There has been no report of investigating hPL as a cell growth supplement for hPDLSCs in culture with CPC, using the FBS-free and xeno-free cell culture medium alternative to avoid the risk of transmitting bovine infections and the initiation of xenogeneic immunological reactions.
Therefore, the objectives of the present study were to: 1) Develop a novel construct of hPDLSCs encapsulated in hydrogel microbeads with hPL and ICPC; 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in microbeads in ICPC with hPL as the xeno-free supplement for bone regeneration. The following hypotheses were tested: 1) Encapsulating hPDLSCs in alginate microbeads would protect the hPDLSCs during the setting reaction of ICPC paste; 2) The incorporation of hPL could improve the viability of hPDLSCs in the alginate microbeads; 3) The hPDLSCs released from the degradable alginate microbeads in ICPC would be able to proliferate and undergo osteogenic differentiate when using a xeno-free growth medium with hPL.
Fabrication of injectable calcium phosphate cement scaffold
The ICPC powder consisted of a mixture of TTCP and DCPA at a TTCP: DCPA molar ratio of 1:1 because this ratio was shown to produce apatite minerals (Xu and Simon, 2005). The TTCP powder was synthesized from a solid-state reaction between CaHPO 4 and CaCO 3 (Baker Chemical Company, NJ, United States) and then ground to obtain a median particle size of 17 μm. The DCPA powder (Baker Chemical) was ground to obtain a median particle size of 1 μm. Chitosan was used as cement liquid as previous studies have indicated that the addition of chitosan to CPC enhanced the strength and durability of ICPC (Weir and Xu, 2008). Two cement liquids at 0 and 7.5% chitosan were used. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. The C t value of hPDLSCs in the control group at 1 day served as the calibrator.
At 1, 14, and 21 days, the hPDLSCs were fixed with 4% paraformaldehyde for 15 min and stained with ARS. After staining for 30 min, the ARS solution was removed and rinsed with distilled water to remove any loose ARS. The samples were imaged via microscopy (Eclipse TE-2000S, Nikon). For quantification, the stained mineralization was de-stained in 10% cetylpyridinium chloride (Millipore Sigma) for 30 min and the concentration was measured at an optical density of 652 nm using the microplate reader (SpectraMax M5). The ARS concentration of the control group at 1 day served as the calibrator.
Statistical analysis
Load-bearing properties
The flexural strength and elastic modulus of the ICPC scaffolds are plotted in Figure 1. Flexural strength and elastic modulus of ICPC scaffolds was 8.4 ± 0.91 MPa and 1.56 ± 0.1 GPa, significantly higher than 6.4 ± 0.43 MPa and 1.12 ± 0.23 GPa of the control group, respectively (p < 0.05). This demonstrates that the incorporation of chitosan improved the mechanical properties of the ICPC scaffold. Both of these values are higher than those of cancellous bone reported in the literature (Damien and Parsons, 1991).
Representative live/dead staining images from 1 to 14 days are shown in Figures 2(A-F). At 1 day, there were numerous live cells (stained green) and a few dead cells (stained red) in all groups (Figures 2A-E). At 7 days, the hPDLSCs were released from the degradable microbeads in the ICPC + DAMB + fibrin + hPL, ICPC + DAMB + hPL, and ICPC + DAMB + fibrin + FBS. The cells exhibited a polygonal morphology and a relatively high viability ( Figures 2F-H). At 14 days, the degradation of microbeads accelerated and most of the cells were released ( Figures 2J-L). The PLive increased with time due to proliferation in the ICPC + DAMB + fibrin + hPL, ICPC + DAMB + hPL, and ICPC + DAMB + fibrin + FBS. At 1 day, the PLive of hPDLSCs in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + hPL was slightly higher than that in the ICPC + DAMB + fibrin + FBS group, indicating that incorporation of hPL into the microbeads could better protect the hPDLSCs from the pH change and ion activities during the ICPC setting ( Figure 2M). In contrast, very few cells were observed to attach and spread in the ICPC + DAMB + fibrin + DMEM, the PLive of the hPDLSCs was lower, due to the lack of nutrition supplements.
The cell growth rate was evaluated via the CCK-8 assay ( Figure 2N). At 1 day and 3 days, the proliferation of hPDLSCs was low in the groups of ICPC + DAMB + fibrin, ICPC + DAMB + hPL, and ICPC + DAMB + fibrin + hPL. At 7 days, as more cells were released from the degradable microbeads, the cell growth was significantly enhanced. The proliferation rate in the ICPC + DAMB + fibrin and ICPC + DAMB + fibrin + hPL groups was higher than that of the ICPC + DAMB + hPL group (p < 0.05), indicating that the incorporation of fibrin accelerated the hPDLSCs migrated out from the microbeads. The absorbance of the hPDLSCs in the ICPC + DAMB + fibrin + DMEM group was lower over time, indicating that the DMEM did not support the proliferation of the hPDLSCs encapsulated in the microbeads.
FIGURE 1
Load-bearing property of scaffolds in ICPC + Chitosan group and control group: (A) flexural strength; (B) elastic modulus (mean ± sd; n = 4). Values for cancellous bone were obtained from the literature (Damien and Parsons, 1991).
Frontiers in Materials frontiersin.org 3A-F). Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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This below document has 2 paragraphs that start with 'Objectives: 1) Develop a novel'. It has approximately 827 words, 29 sentences, and 13 paragraph(s).
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. Methods: hPDLSCs were encapsulated in alginate-fibrin microbeads (DAMB + fibrin), alginate-hPL degradable microbeads (DAMB + hPL), or alginate-fibrin-hPL microbeads (DAMB + fibrin + hPL). The proliferation and osteogenic differentiation of hPDLSCs were investigated in culturing with the ICPC scaffold. Results: Flexural strength of ICPC was 8.4 ± 0.91 MPa, and elastic modulus was 1.56 ± 0.1 GPa, exceeding those of cancellous bone. hPDLSCs had higher viability in DAMB + fibrin + hPL group than in DAMB + fibrin. ALP was 69.97 ± 16.96 mU/mg for ICPC + DAMB + fibrin + hPL group, higher than 30.68 ± 2.86 mU/mg of ICPC + DAMB + fibrin (p < 0.05) and 4.12 ± 1.65 mU/mg of control (p < 0.01). At 7 days, osteogenic gene expressions (ALP, RUNX2, COL1, and OPN) in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + fibrin were 4–11 folds that of control. At 21 days, the hPDLSC-synthesized bone mineral amounts in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + fibrin were 13.2 folds and 11.1 folds that of control group, respectively. Conclusion: The novel injectable CPC scaffold encapsulating hPDLSCs and hPL is promising to protect and deliver hPDLSCs. The hPL-based medium significantly enhanced the osteogenic differentiation of hPDLSCs in ICPC + DAMB + fibrin + hPL construct, suggesting a promising xeno-free approach for bone tissue regeneration applications.
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications.
Introduction
Bone tissue engineering involves the use of biomaterials, cells and bioactive factors and represents a promising approach for bone regeneration. Injectable calcium phosphate cement (ICPC) is used for bone repair due to their similarity to bone minerals (Szpalski et al., 2012a;Szpalski et al., 2012b;. The first ICPC was developed in 1986 and consisted of a mixture of tetracalcium phosphate [TTCP, Ca 4 (PO 4 ) 2 O] and dicalcium phosphate anhydrous (DCPA, CaHPO 4 ). ICPC was approved by the Food and Drug Administration (FDA) for the repair of craniofacial defects due to its excellent biocompatibility, osteoconductivity, resorbability, and injectability (Friedman et al., 1998).
In stem cell therapy, MSCs need to proliferate for an optimal therapeutic dose and differentiate into multiple cell lineages for effective tissue repair and clinical treatment. Therefore, the culture medium is essential to supply nutrients for cell metabolism, proliferation, and differentiation in vitro. Currently, cell culture medium supplements are based on animal serum, mostly fetal bovine serum (FBS), which consists of a large number of low and high molecular weight biomolecules with different physiological activities (Gstraunthaler, 2003;van der Valk et al., 2018). However, it has reported the FBS could evoke the xenogeneic immunological reactions and raise the risk of transmitting bovine infections (Brunner et al., 2010). As ICPC have been widely investigated in a number of clinical trials for bone regeneration, ICPC scaffolds seeding with MSCs represent a new strategy to promote prevascularization and enhance bone tissue engineering efficacy (Lin et al., 2019). Therefore, it is urgently needed to switch the culture medium to a FBS-free, xeno-free alternative, thus enable the MSCs seeding ICPC product not cause any potential infections, allergies or malignancies and ultimately facilitate the clinical application.
Human platelet lysate (hPL) is a valuable, non-xenogenic alternative to FBS in cell culture (Bernardo et al., 2007;Rauch et al., 2011). The advantages of hPL includes that it is humanderived, serum-free, and cost-effective. An increasing number of studies demonstrated that hPL showed a growth-promoting effect on a multitude of cell lines (Fernandez-Rebollo et al., 2017). Among the various types of MSCs, periodontal ligament stem cells (hPDLSCs) play a key role in dental and periodontal tissue regeneration. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. The first ICPC was developed in 1986 and consisted of a mixture of tetracalcium phosphate [TTCP, Ca 4 (PO 4 ) 2 O] and dicalcium phosphate anhydrous (DCPA, CaHPO 4 ). ICPC was approved by the Food and Drug Administration (FDA) for the repair of craniofacial defects due to its excellent biocompatibility, osteoconductivity, resorbability, and injectability (Friedman et al., 1998).
In stem cell therapy, MSCs need to proliferate for an optimal therapeutic dose and differentiate into multiple cell lineages for effective tissue repair and clinical treatment. Therefore, the culture medium is essential to supply nutrients for cell metabolism, proliferation, and differentiation in vitro. Currently, cell culture medium supplements are based on animal serum, mostly fetal bovine serum (FBS), which consists of a large number of low and high molecular weight biomolecules with different physiological activities (Gstraunthaler, 2003;van der Valk et al., 2018). However, it has reported the FBS could evoke the xenogeneic immunological reactions and raise the risk of transmitting bovine infections (Brunner et al., 2010). As ICPC have been widely investigated in a number of clinical trials for bone regeneration, ICPC scaffolds seeding with MSCs represent a new strategy to promote prevascularization and enhance bone tissue engineering efficacy (Lin et al., 2019). Therefore, it is urgently needed to switch the culture medium to a FBS-free, xeno-free alternative, thus enable the MSCs seeding ICPC product not cause any potential infections, allergies or malignancies and ultimately facilitate the clinical application.
Human platelet lysate (hPL) is a valuable, non-xenogenic alternative to FBS in cell culture (Bernardo et al., 2007;Rauch et al., 2011). The advantages of hPL includes that it is humanderived, serum-free, and cost-effective. An increasing number of studies demonstrated that hPL showed a growth-promoting effect on a multitude of cell lines (Fernandez-Rebollo et al., 2017). Among the various types of MSCs, periodontal ligament stem cells (hPDLSCs) play a key role in dental and periodontal tissue regeneration. Seo et al. (2004) identified hPDLSCs in the periodontal ligament (PDL) and demonstrated that hPDLSCs implanted into nude mice generated cementum/PDL-like structures that resembled the native PDL. Compared with other MSCs, hPDLSCs have a marked ability to generate multiple tissue types, including alveolar bone, cementum, and Sharpey's fibers, making them an ideal source for cell-based dental therapy. However, there has no report that investigated the effect of hPL as a cell growth supplement on the proliferation and osteogenic differentiation of hPDLSCs in culture with ICPC.
In stem cell-based bone tissue engineering, the number of functioning cells in the bone injury site appears to be of critical importance. However, it was reported that few transplanted cells survived via local administration of cell suspensions. One study demonstrated that only 50% of initial donor BMSCs remained alive 48 h after implantation, and only 5% survived after 8 weeks (Giannoni et al., 2010). Another study showed that less than 1% of human multipotent stromal cells were detectable after 30 days post-implantation (Becquart et al., 2012). Thus the preferred approach was delivering a large number of cells to the target site via a carrier scaffold. Alginate hydrogel is highly hydrated with excellent biocompatibility and has shown great potential as a carrier for cell delivery (Izeia et al., 2020;Xu et al., 2021). Previous studies have reported that alginate microbeads are an ideal vehicle to localize the cells at the target defect site for cellbased therapy (Li et al., 2012;Pal et al., 2014). Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering
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Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications. ICPC + DAMB + fibrin + FBS group (Alginate-fibrin microbeads encapsulating 1 × 10 5 hPDLSCs with 0.05 ml ICPC scaffold in growth medium containing 10% FBS); 3. ICPC + DAMB + hPL group (Alginate-hPL microbeads encapsulating with 1 × 10 5 hPDLSCs with 0.05 ml ICPC scaffold, cultured in DMEM for the first three days and then in growth medium containing 2.5% hPL); 4. ICPC + DAMB + fibrin + hPL group (Alginate-fibrin-hPL microbeads encapsulating with 1 × 10 5 hPDLSCs with 0.05 ml ICPC scaffold, cultured in DMEM for the first three days and then in growth medium containing 2.5% hPL).
After culturing for 1, 7, and 14 days, cells were stained with a live/dead kit (Invitrogen) and observed via epifluorescence microscopy (Eclipse TE-2000S, Nikon). Two images were taken at random locations for each sample, with four samples yielding 8 images at each time point for each group. Live and dead cells were counted via Image-Pro Plus software. The percentages of live cells (P Live ) were calculated. P Live = number of live cells/(number of live cells + number of dead cells).
The proliferation rate of hPDLSCs was investigated via Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan), following the manufacturer's protocol. At 1, 3, 7, and 14 days, the culture medium was carefully removed and placed with 500 µl DMEM containing 50 µl CCK-8 dye. After 2 h incubation at 37°C, a 200 µl aliquot from each well was placed in a 96-well plate and the absorbance at an optical density of 450 nm (OD450 nm) was measured with a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, United States).
To evaluate the number of hPDLSCs released from the degrading alginate microbeads, the samples were imaged via microscopy (Eclipse TE-2000S, Nikon). Two images were taken at random locations for each sample, with four samples yielding 8 images at each time point for each group. The number of the hPDLSCs with spindle-shape morphology was counted via Image-Pro Plus software. The density of the released hPDLSCs was calculated: D Release = N Release /A, where A is the area of the view field for D Release .
At 1, 7, and 14 days, cells were lysed in a 0.2% Triton X-100 (Millipore Sigma) solution. The ALP activity of the cell lysate was measured by using an ALP Assay kit (QuantiChrom, BioAssay Systems, Hayward, CA, United States) with p-Nitrophenylphosphate (pNPP) as a substrate. The ALP activity was determined by measuring the absorbance at an optical density of 405 nm using a microplate reader (SpectraMax M5). The protein of cell lysate was quantified using Protein Assay Kit (Pierce BCA, Thermo Scientific, Rockford, IL, United States) following the manufacturer's protocol. The ALP activity was normalized to the protein amount and reported as mU/mg protein (Zhao et al., 2019).
The total cellular RNA of the cells was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using a High-capacity cDNA Reverse Transcription kit (Applied Biosystems) in a thermal cycler (GenAmp PCR 2720, Applied Biosystems). RT 2 SYBR ® Green ROX qPCR Mastermix (Qiagen, Germantown, MD, United States) was used to quantify the transcript levels of glyceraldehyde 3phosphate dehydrogenase (GAPDH), alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), Collagen type 1 (COL1), and Osteogenicpontin (OPN). The human-specific primers were synthesized commercially (Millipore Sigma), and the sequences of the primers are listed in Table 1. The qPCR data collection and analyses were performed using a QuantStudio ™ 3 Real-Time PCR System (Thermo Scientific). Relative expressions were calculated using the 2 −ΔΔCT method and normalized by the C t value of the housekeeping gene GAPDH. Therefore, the novel hPDLSC-hPLmicrobeads-ICPC construct is highly promising for bone regeneration without the risk of infection from unknown pathogens by using animal-origin serums.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
Study conception and design: GQ, MH, JL, and HX Acquisition of data: GQ, TM, and DK Analysis and interpretation of data: GQ, TM, MW, AS, and TO Critical revision: GQ, PW, LZ, HX, and YX.
Funding
This study was supported by National Natural Science Foundation of China 31771051 (LZ) and National Institutes of Health grant R21 DE029611 (AS and HX).
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Domain: Biology Medicine Engineering<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. The allelic application will improve breeding efficiency, increase cultivar resistance to sheath blight in rice and ultimately secure food production worldwide.
Allelic analysis can only be applied in association mapping where large number of diversified genotypes are used and multiple alleles are involved at each associated marker locus in the mapping panel. Using this method, germplasm accessions that are identified in the association mapping strategy to possess multiple putative resistant alleles can be crossed with other accessions that have a different complement of putative resistant alleles. The selection of progeny possessing the most putative resistant alleles should be more effective than it is for resistant loci. In this regard, association mapping offers advantages for identifying parental material and specific alleles that can enhance breeding.
Putative resistant alleles and ancestry background for sheath blight
Jia et al. [58] reported 52 entries that are significantly more resistant to ShB than Jasmine 85. The resistant entries were identified from 1,794 entries of the USDA rice core collection that has 35% indica, 27% temperate japonica, 24% tropical japonica, 10% aus and 4% aromatic genotypes [52]. Based on the ancestry classification, there are 621 indica entries in the core and 45 of them are included in the resistant list, making a resistance frequency of 7.2% for indica germplasm. Accordingly, the resistance frequency is 2.8% for aromatic, 1.7% for aus, and 0.2% each for temperate japonica and tropical japonica. In a study conducted by Zuo et al. [23], japonica cultivars showed higher sheath blight severity than indica cultivars. They describe a general observation that japonica rice is more susceptible than indica rice. Furthermore, Jasmine 85, Tetep and Teqing, used as parents in many studies on mapping ShB resistance, all belong to indica.
This study demonstrated that: 1) a majority of the ShB putative resistant alleles existed in indica germplasm, 2) most of the resistant entries with a large number of putative resistant alleles were indica, conversely 3) only a very small portion of putative resistant alleles existed in japonica, and 4) the most susceptible entries with very few or no putative resistant alleles were japonica (Fig. 5 and Table S1). Entry GSOR 310389 is an example which had eight out of ten putative resistant alleles, showed a high level of resistance to ShB, and is indica. The results from association mapping match well with the phenotypic observation that most resistant genotypes are indica and resistant germplasm is rare in japonica.
ShB associated markers and QTL identification
Our genome-wide search found ten marker loci that were significantly associated with sheath blight resistance (Fig. 4). Both RM11229 (Chr1 at 22.7 Mb) and RM7203 (Chr11 at 1.1 Mb) are novel QTLs that have not been previously reported. RM11229 is approximately 5.0 Mb away from a ShB QTL reported by Channamallikarjuna et al. [16] and RM7203 resides about 3.3 Mb away from one QTL identified by Li et al. [9]. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. The core collection has been shown to be representative of the genetic diversity found in more than 18,000 accessions of the USDA rice whole collection [60]. The mini-core has proven to be an efficient platform for association mapping and has been successfully applied to mapping QTLs for improving grain yield [47]. We excluded fourteen entries of wild species to minimize interference due to different genetic structure [61] and replaced them with fourteen core entries known to have greater resistance to ShB than Jasmine 85, a common resistant check in the comprehensive evaluation of the core collection [58]. The replacement aimed to enhance detection of QTLs by increasing the frequency of putative resistant alleles in the panel.
Phenotyping
A complete set of 1,794 entries in the USDA rice core collection was evaluated in 2008, using the MCM with three replications, three plants in each replication following a randomized incomplete block design over time [58]. Rice cultivars, Lemont (susceptible) and Jasmine 85 (resistant), were included as repeated checks in each replicate to serve as standards for evaluation. Both the check cultivars have been used as standard checks in many other studies regarding ShB resistance [11,12,14,18]. In 2009, the 217 entries of the mini-core collection, plus those core entries that showed significantly more resistance than Jasmine 85, were re-evaluated using the same protocol. LSmean of ShB severity from six replications including 18 observations of each entry was used for association mapping.
The isolate RR0140-1 of R. solani was selected from 102 isolates collected state-wide from Arkansas rice fields due to its slow growing phenotype [51]. Slow growing isolates cause relatively consistent disease reaction and differentiate susceptible cultivars from moderately resistant ones better than fast growing isolates [51]. Field evaluations showed no differences in disease reactions between the slow growing isolates and the fast ones [51]. Further, the RR0140-1 isolate have been adapted by numerous studies [6,18,50]. Pathogen inoculum of RR0140-1 were grown by placing sclerotia in the centre of potato dextrose agar (PDA) plates (Sigma-Aldrich, St. Louis) containing 0.005% (wt/vol) tetracycline, and then transferred to a fresh PDA medium for 5-6 days at 27uC under darkness. Mycelium discs (7 mm in diameter) were excised from the outer growing area in the culture plate where the outer mycelia were mostly active. Rice seedlings were inoculated at the three-leaf stage.
In the greenhouse, each 12612 cm pot was filled with presterilized soil to ensure that the study was not confounded by the presence of soil borne R. solani inoculum. Pots with drainage holes were placed in flats filled with shallow water (,5 cm). Five seeds of each accession were planted in each pot and thinned to three uniform plants before pathogen inoculation. The three remaining plants in a given pot were referred to as one experimental unit or replicate. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Due to the high cost of cultural practices and the phytotoxic influence associated with the application of fungicides, the use of ShB resistant cultivars is considered the most economical and environmentally sound strategy in managing this disease. Understandings of genetic control will facilitate cultivar improvement for this disease and secure global food production.
The necrotrophic ShB pathogen has a broad host range and no complete resistance has been identified in either commercial rice cultivars or wild related species [3,4]. However, substantial differences in susceptibility to ShB among rice cultivars have been observed under field conditions [5,6]. Differential levels of resistance and the associated resistance genes have been studied among rice germplasm accessions [7]. Rice ShB resistance is believed to be controlled by multiple genes or quantitative trait loci (QTLs) [8]. Since Li et al. [9] first identified ShB QTLs using restricted fragment length polymorphism (RFLP) markers under field conditions, over 30 resistant ShB QTLs have been reported using various mapping populations, such as F 2 s [10][11][12][13][14], double haploid (DH) lines [15], recombinant inbred lines (RILs) [8,[16][17][18], near-isogenic introgression lines (NIL) [19] and backcross populations [20][21][22][23]. 'Teqing' and 'Jasmine 85' have been repeatedly involved in these studies as the ShB resistant parents. We are the first to map rice ShB QTLs using association mapping strategy in a global germplasm collection.
In association mapping, each identified marker usually has multiple alleles in the mapping panel and each allele in a marker locus contributes differently to the associated trait. Agrama and Yan [42] reported that three alleles at each of three associated loci (allele 87 of RM490, 105 of RM413 and 122 of RM277) and two alleles at another locus (182 and 183 of RM263) had significantly greater contribution to straighthead resistance than other counterparts. Li et al. [47] determined that allele 126 bp had the greatest effect on increasing grain yield, plant weight and grains/panicle branch among eight alleles of RM471. There is no study on allelic distribution for associated loci in a global rice germplasm collection.
Linkage disequilibrium (LD), defined as the non-random association of alleles at separate loci located on the same chromosome [24], is a prerequisite for association mapping. The distance at which LD declines with genetic or physical distance determines the marker density needed for achieving a reasonable mapping resolution. The extent of LD may vary among different genomic regions [48]. Numerous studies on global germplasm collections indicate 25 cM as a reasonable resolution for association mapping in rice [41,47,49]. In our study, we used 154 simple sequence repeat (SSR) markers plus an indel to provide coverage of 10 cM across the rice genome for sufficient mapping resolution.
Accurate phenotyping is essential for mapping, especially when the target trait is controlled by multiple genes or QTLs such as ShB resistance. All the previous studies phenotyped ShB resistance under field conditions with only one exception, Liu et al. (2009) [18], where a micro-chamber method (MCM) was adapted. The MCM described by Jia et al. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. (2007) [6] has proven to effectively minimize the confounding effects of environmental and morphological factors, thus generating more reliable data. Furthermore, numeric measurement of ShB in the MCM should be more accurate than the traditional visual scoring under field conditions. Because of these advantages, the MCM has been widely applied in studies of ShB resistance [18,50,51].
Using association mapping, our objectives were to 1) map QTLs associated with ShB resistance phenotyped with the MCM, 2) identify putative resistant alleles in a global germplasm collection, and 3) explore the use of ShB putative resistant alleles in a breeding program.
Variation of ShB severity ratings
The 217 sub-core entries in the mapping panel originated from fifteen geographic regions including 77 countries worldwide. India had the most entries (6.5%), followed by China (5.5%), Indonesia (4.1%), Japan (4.1%) and Taiwan (4.1%). Their name, origin, ShB severity rating, structure group and entry number in the Genetic Stocks Oryza (GSOR) collection ( [URL]/ Main/docs.htm?docid = 8318) are presented in (Table S1). The ShB severity ratings among the 217 entries were distributed normally, ranging from 0.25660.111 to 0.90960.096 with an average of 0.52160.008 (Fig. 1). The resistant check Jasmine 85 was rated 0.47260.021 and susceptible check Lemont was rated 0.94660.080. Twenty-four entries (11.1%) were significantly more resistant to ShB than Jasmine 85 at the 5% level of probability while 54 others (24.9%) had similar resistance. Population structure Structure analysis from Q1 to Q10 across twenty runs for the 217 sub-core entries genotyped with 155 genome-wide DNA markers using STRUCTURE demonstrated that when Q reached five, the Pr(Q) became more-or-less plateaued, so Q5 captured the major structure in our data. Thus, the mapping panel was divided into five subgroups and each entry was classified to an appropriate subgroup using STRUCTURE. Inferred by reference cultivars recommended by Agrama et al. [52,53], the five subgroups were denoted as temperate japonica (TEJ), aus (AUS), aromatic (ARO), indica (IND), and tropical japonica (TRJ) ( Fig. 2A). A similar structural pattern was seen with the PCA analysis with the first two axes explaining 75.03% of variation (Fig. 2B). Furthermore, the genetic distance based on cluster analysis also divided the mapping panel into five major clusters (Fig. 2C). All three approaches led to the same conclusion: a five-group structure could clearly and sufficiently explain the existing genetic diversity in the mapping panel. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. They describe a general observation that japonica rice is more susceptible than indica rice. Furthermore, Jasmine 85, Tetep and Teqing, used as parents in many studies on mapping ShB resistance, all belong to indica.
This study demonstrated that: 1) a majority of the ShB putative resistant alleles existed in indica germplasm, 2) most of the resistant entries with a large number of putative resistant alleles were indica, conversely 3) only a very small portion of putative resistant alleles existed in japonica, and 4) the most susceptible entries with very few or no putative resistant alleles were japonica (Fig. 5 and Table S1). Entry GSOR 310389 is an example which had eight out of ten putative resistant alleles, showed a high level of resistance to ShB, and is indica. The results from association mapping match well with the phenotypic observation that most resistant genotypes are indica and resistant germplasm is rare in japonica.
ShB associated markers and QTL identification
Our genome-wide search found ten marker loci that were significantly associated with sheath blight resistance (Fig. 4). Both RM11229 (Chr1 at 22.7 Mb) and RM7203 (Chr11 at 1.1 Mb) are novel QTLs that have not been previously reported. RM11229 is approximately 5.0 Mb away from a ShB QTL reported by Channamallikarjuna et al. [16] and RM7203 resides about 3.3 Mb away from one QTL identified by Li et al. [9]. The remaining eight QTLs identified in this study were either quite near (less than 1.4 Mb distant) or within the interval of previously identified QTLs. The ten associated markers identified in this study were located on seven chromosomes (Chr1, 2, 4, 5, 6, 8 and 11) (Fig. 4).
On Chr1 our study identified RM11229 and RM 237, which occur within 4.4 Mb of each other, as markers associated with ShB resistance. RM11229 explained the most phenotypic variation (9.5%) and its putative resistant allele 158 bp had the smallest average ShB score (0.414 in Table 2), indicating the greatest resistance among the ten putative resistant alleles. RM237 was the most significant marker for ShB resistance (p = 0.002). Therefore, the 4.4 Mb gap between RM11229 and RM237 on Chr1 should be a target area for fine-mapping ShB resistant genes in rice. RM237 at 26.8 Mb is near the ShB QTL region spanning 27.6 to 34 Mb found by Charnnamallikarjuna et al. [16].
On Chr4, three reports uniformly indicated ShB QTLs on the long arm at 29.8 to 33.6 Mb. This is a very narrow region of 3.8 Mb on the physical map, and corresponds to a small estimated cM distance in the mapping population developed from susceptible Lemont and resistant Teqing parents [8,9,59]. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Data were analyzed using GeneMapper v. 3.7 software (Applied Biosystems).
Population Structure Analysis
Analysis of population structure in the mapping panel was performed using STRUCTURE software [64,65]. Rare alleles, with frequency of less than 5% in the panel, were treated as missing data for structure analysis, principal components analysis (PCA), cluster analysis and association mapping. We implemented a model-based clustering method for inferring population structure using distinctive allele frequencies and assigning individuals into Q clusters. Twenty independent runs were performed for each value of Q, ranging from one to ten, using the admixture model with a burn-in of 50,000 iterations followed by 100,000 iterations during analysis. Subgroups were determined on the basis of the following criteria: (1) likelihood plot of these models; (2) stability of grouping patterns across twenty runs; and (3) germplasm information about the materials under study. To validate the population structure and compare the different models, PCA was conducted to obtain eigenvectors for further model testing and association analysis. Genetic distance was calculated with PowerMarker [66] using Nei's method [67]. The resulting unweighted pair-group method with arithmetic mean (UPGMA) tree was viewed using MEGA 4.0 [68].
Model comparisons and association analysis
The flexible mixed model [69] was used to control population structure. For the purpose of model comparisons, the phenotypic vector is modeled asy~X bzQvzZuze, where b is vector of marker effects to be estimated. The term Qv contains the coordinates of the individuals of p dimensions in Q matrix generated by STRUCTURE [64,65] and PCA matrix by NTSYSpc version 2.1 [70]; X and Z are the incidence matrices of 1 s and 0 s that relate y to b and u, respectively. u is a vector of polygene background effects; and e is a vector of residual. The phenotypic covariance matrix was assumed to have the form V~Z(2Ks 2 g )Z T zIs 2 e , where K is the K matrix including relative kinship coefficients defining the degree of genetic covariance between a pair of individuals [71], I is an n6n identity matrix, s 2 g is the genetic variance attributable to genome wide effects, and s 2 e is the residual variance.
When model K or Q+K or PCA+K were tested for a fit, we found little convergence because of the low level of relatedness among entries in the panel. Thus, a simple model (ignoring the effect of population structure), possible linear models of Q2-Q10 (considering different number of subgroups from two to ten) and PCA1-PCA10 (PCA matrix with different number of dimensions from one to ten) were compared for the best fit to ShB determined by Bayesian information criterion (BIC). In order to control false positive rates, the genomic control (GC) method [72] was further used for correcting population structure. The association mapping was conducted using the best fit model with TASSEL v.2.1 [73], followed by the GC. The associated markers with ShB resistance were claimed at the probability level of 0.05. The ShB severity ratings of germplasm entries that carried the same allele in an associated marker locus were averaged to estimate allelic effect on the ShB rating. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. The LSMEANS option was used to calculate the least-square means (LSMs) of each entry and the LSMs were used for the association mapping. The statistical differences of the accession to each check (Jasmine 85 and Lemont) were determined by a Dunnett's multiple comparison test, using the diff = control option.
Genotyping DNA was extracted from leaf tissue of five plants for each of the 217 entries using a rapid alkali extraction procedure [63] and genotyped with 154 SSR markers plus an indel. The 155 molecular markers covered the entire rice genome with an average genetic distance of 10 cM, described by Li et al. [47]. PCR amplifications were performed according to Agrama et al. (2007) [42]. For each marker, forward primers were labeled with either 6FAM, NED or Hex (Applied Biosystems, Foster City, CA, USA or Integrated DNA Technologies, Coralville, IA, USA). The amplifications were performed using MJ Research Tetrad thermal cyclers (Bio-Rad, Hercules, CA, USA). PCR products were pooled based on color and size range of amplified fragments (typically three markers per run along with ROX-labeled size standard), and the DNA was denatured by heating samples at 94uC for 5 min. The samples were separated on an ABI Prism 3730 DNA Analyzer according to the manufacturer's instructions (Applied Biosystems). Data were analyzed using GeneMapper v. 3.7 software (Applied Biosystems).
Population Structure Analysis
Analysis of population structure in the mapping panel was performed using STRUCTURE software [64,65]. Rare alleles, with frequency of less than 5% in the panel, were treated as missing data for structure analysis, principal components analysis (PCA), cluster analysis and association mapping. We implemented a model-based clustering method for inferring population structure using distinctive allele frequencies and assigning individuals into Q clusters. Twenty independent runs were performed for each value of Q, ranging from one to ten, using the admixture model with a burn-in of 50,000 iterations followed by 100,000 iterations during analysis. Subgroups were determined on the basis of the following criteria: (1) likelihood plot of these models; (2) stability of grouping patterns across twenty runs; and (3) germplasm information about the materials under study. To validate the population structure and compare the different models, PCA was conducted to obtain eigenvectors for further model testing and association analysis. Genetic distance was calculated with PowerMarker [66] using Nei's method [67]. The resulting unweighted pair-group method with arithmetic mean (UPGMA) tree was viewed using MEGA 4.0 [68].
Model comparisons and association analysis
The flexible mixed model [69] was used to control population structure. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Li et al. [47] determined that allele 126 bp had the greatest effect on increasing grain yield, plant weight and grains/panicle branch among eight alleles of RM471. There is no study on allelic distribution for associated loci in a global rice germplasm collection.
Linkage disequilibrium (LD), defined as the non-random association of alleles at separate loci located on the same chromosome [24], is a prerequisite for association mapping. The distance at which LD declines with genetic or physical distance determines the marker density needed for achieving a reasonable mapping resolution. The extent of LD may vary among different genomic regions [48]. Numerous studies on global germplasm collections indicate 25 cM as a reasonable resolution for association mapping in rice [41,47,49]. In our study, we used 154 simple sequence repeat (SSR) markers plus an indel to provide coverage of 10 cM across the rice genome for sufficient mapping resolution.
Accurate phenotyping is essential for mapping, especially when the target trait is controlled by multiple genes or QTLs such as ShB resistance. All the previous studies phenotyped ShB resistance under field conditions with only one exception, Liu et al. (2009) [18], where a micro-chamber method (MCM) was adapted. The MCM described by Jia et al. (2007) [6] has proven to effectively minimize the confounding effects of environmental and morphological factors, thus generating more reliable data. Furthermore, numeric measurement of ShB in the MCM should be more accurate than the traditional visual scoring under field conditions. Because of these advantages, the MCM has been widely applied in studies of ShB resistance [18,50,51].
Using association mapping, our objectives were to 1) map QTLs associated with ShB resistance phenotyped with the MCM, 2) identify putative resistant alleles in a global germplasm collection, and 3) explore the use of ShB putative resistant alleles in a breeding program.
Variation of ShB severity ratings
The 217 sub-core entries in the mapping panel originated from fifteen geographic regions including 77 countries worldwide. India had the most entries (6.5%), followed by China (5.5%), Indonesia (4.1%), Japan (4.1%) and Taiwan (4.1%). Their name, origin, ShB severity rating, structure group and entry number in the Genetic Stocks Oryza (GSOR) collection ( [URL]/ Main/docs.htm?docid = 8318) are presented in (Table S1). The ShB severity ratings among the 217 entries were distributed normally, ranging from 0.25660.111 to 0.90960.096 with an average of 0.52160.008 (Fig. 1). The resistant check Jasmine 85 was rated 0.47260.021 and susceptible check Lemont was rated 0.94660.080. Twenty-four entries (11.1%) were significantly more resistant to ShB than Jasmine 85 at the 5% level of probability while 54 others (24.9%) had similar resistance. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. The putative resistant alleles of both RM133 (allele 230 bp) and RM408 (allele 119 bp) were common with more than 45% of entries among the 217 subcore entries in the mapping panel. RM7203 on the short arm of Chr11 at 1.1 Mb was a novel ShB QTL that has not been reported. The putative resistant allele 88 of RM7203 existed in 55% of the 217 entries, so was the most common allele and had a ShB mean (0.470) similar to Jasmine 85 (0.472).
At the bottom of Chr11, two markers RM254 (at 23.7 Mb) and RM1233 (at 26.5 Mb) were identified, explaining relatively high phenotypic variation among ten markers, 5.3% and 5.1%, respectively. The RM1233 was one of the flanking markers for qSBR11-1 (26.5,27.2 Mb) reported by Channamallikarjuna et al. [16]. The putative resistant alleles, 169 bp of RM254 and 177 bp of RM1233, were in 12 and 35 entries, respectively. Their ShB ratings were lower than Jasmine 85 in average, indicating a stronger resistance.
Above comparisons confirm eight out of ten marker loci identified in our association mapping with two novel QTLs, RM11229 and RM7203, for sheath blight resistance. The confirmation of previously identified QTLs provides validation for the accuracy of QTLs identified in our study. Furthermore, the comparisons demonstrate that association mapping can locate many QTLs over the entire genome since the mapping panel includes a large number of diversified entries of germplasm. In biparental linkage mapping studies, fewer QTLs are typically identified and can only be located in a limited area in the genome where the two parents differ.
Germplasm panel
The rice mini-core collection of United States Department of Agriculture (USDA) contains 217 entries [53] derived from 1,794 entries of a core collection [60]. The core collection has been shown to be representative of the genetic diversity found in more than 18,000 accessions of the USDA rice whole collection [60]. The mini-core has proven to be an efficient platform for association mapping and has been successfully applied to mapping QTLs for improving grain yield [47]. We excluded fourteen entries of wild species to minimize interference due to different genetic structure [61] and replaced them with fourteen core entries known to have greater resistance to ShB than Jasmine 85, a common resistant check in the comprehensive evaluation of the core collection [58]. The replacement aimed to enhance detection of QTLs by increasing the frequency of putative resistant alleles in the panel.
Phenotyping
A complete set of 1,794 entries in the USDA rice core collection was evaluated in 2008, using the MCM with three replications, three plants in each replication following a randomized incomplete block design over time [58]. Rice cultivars, Lemont (susceptible) and Jasmine 85 (resistant), were included as repeated checks in each replicate to serve as standards for evaluation. Both the check cultivars have been used as standard checks in many other studies regarding ShB resistance [11,12,14,18]. In 2009, the 217 entries of the mini-core collection, plus those core entries that showed significantly more resistance than Jasmine 85, were re-evaluated using the same protocol. LSmean of ShB severity from six replications including 18 observations of each entry was used for association mapping.
The isolate RR0140-1 of R. solani was selected from 102 isolates collected state-wide from Arkansas rice fields due to its slow growing phenotype [51]. Slow growing isolates cause relatively consistent disease reaction and differentiate susceptible cultivars from moderately resistant ones better than fast growing isolates [51]. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Each of the three seedlings in a pot was individually inoculated with a round mycelium disc of RR0140-1 pathogen as described by Jia et al. [6] with modification. Each disk was pressed up to the base of the seedling stem, assuring that the mycelium was in contact with the plant. After inoculation, each pot was immediately covered with a 2-liter soft drink bottle with the bottom and cap removed. Relative humidity was maintained over 80% in the bottle, which favoured growth of the sheath blight pathogen on the plants. The greenhouse temperatures were set for day/night at 30/22uC, respectively with a 12 h photoperiod.
Plant response to the sheath blight pathogen was measured using the ratio between the height of the pathogen growing up the plant and the height of the leaf collar on the last emerged leaf. Because mature plant height varied from 70 to 202 cm in this collection [60], the ratio excluded possible interference of plant height in scoring disease response. Therefore, the smaller the ratio, the greater the resistance was for an entry. Measurements were taken when the ratio reached 1.0 for 75% of the susceptible check plants, Lemont, so that the maximum susceptibility was scored 1.0.
ShB rating data were analyzed using the GLIMMIX procedure in SAS version 9.1.3 [62]. The experimental design of randomized incomplete block formed the basis of the statistical model, where the accession is a fixed effect and block is treated as random effect. The LSMEANS option was used to calculate the least-square means (LSMs) of each entry and the LSMs were used for the association mapping. The statistical differences of the accession to each check (Jasmine 85 and Lemont) were determined by a Dunnett's multiple comparison test, using the diff = control option.
Genotyping DNA was extracted from leaf tissue of five plants for each of the 217 entries using a rapid alkali extraction procedure [63] and genotyped with 154 SSR markers plus an indel. The 155 molecular markers covered the entire rice genome with an average genetic distance of 10 cM, described by Li et al. [47]. PCR amplifications were performed according to Agrama et al. (2007) [42]. For each marker, forward primers were labeled with either 6FAM, NED or Hex (Applied Biosystems, Foster City, CA, USA or Integrated DNA Technologies, Coralville, IA, USA). The amplifications were performed using MJ Research Tetrad thermal cyclers (Bio-Rad, Hercules, CA, USA). PCR products were pooled based on color and size range of amplified fragments (typically three markers per run along with ROX-labeled size standard), and the DNA was denatured by heating samples at 94uC for 5 min. The samples were separated on an ABI Prism 3730 DNA Analyzer according to the manufacturer's instructions (Applied Biosystems). (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. The mean ShB ratings for entries containing three, two, one and zero putative resistant alleles were 0.483, 0.535, Table 1. Comparative analysis of different subgroups using structure (Q model) and different dimensions in principal components analysis (PCA) for association mapping of sheath blight resistance using 217 entries genotyped with 155 molecular markers. 0.582 and 0.598, respectively. There was a strong and negative correlation between the ShB severity rating and number of putative resistant alleles (r = 20.535, p,0.0001).
Our mapping results showed that most entries containing a large number of putative resistant alleles were IND (Fig. 5 and Table S1). All entries with six or more putative resistant alleles were IND with only one exception of AUS.
Discussion
Pyramiding putative resistant alleles for cultivar improvement R. solani is a soil-borne necrotrophic fungus and its group AG1-IA has a broad host range including rice, maize, wheat, sorghum, bean (Phaseolus spp.) and soybean [Glycine max (L.) Merr.] [54]. The pathogen's ability to persist in soil and on crop residues allows it to, survive in multiple ways and makes disease management difficult. There is no complete resistance to ShB in rice because the resistance is quantitatively controlled by numerous genes or quantitative trait loci (QTLs). More than 30 QTLs responsible for ShB have been reported in rice [8,[10][11][12][13][14][15][16][17][18][19][20][21][22][23]. However, all of these studies have been limited to conventional mapping populations from a small number of parents, which limits the alleles in the progeny to those present in the parental lines.
From our diverse mapping panel including 217 sub-core entries, we identified ten marker loci significantly associated with ShB resistance, each locus had numerous alleles, each allele contributed differently to ShB resistance, and the putative resistant allele of each locus contributed the most (Table 2). Highly significant correlation demonstrated that as more putative resistant alleles pyramided in a germplasm entry, the entry had greater resistance to ShB. Among 24 entries that were significantly more resistant than the resistant check Jasmine 85 that had three putative resistant alleles, 17 (71%) contained four or more putative resistant alleles. Among 54 entries that had similar resistance with Jasmine 85, 29 (54%) possessed three or more putative resistant alleles.
These findings suggest that marker-assisted breeding for ShB resistance can be conducted on an allelic level by pyramiding putative resistant alleles in a cultivar. This can be accomplished in a two pronged approach by combining parental lines based upon their combination of number of different ShB resistant loci and possessing the putative resistant alleles at these loci. For example, resistant entry GSOR 310389 that has eight putative resistant alleles could be crossed with a commercial cultivar having fewer putative resistant alleles, and the progeny would be selected based on those having the most highly putative resistant alleles at the most loci. The breeding program would end up with the selection of progeny containing the most putative resistant alleles, potentially having greater resistance to ShB than either parental line.
Pyramiding responsible genes has been successfully applied in rice breeding for disease resistance including bacterial blight (Xanthomonas oryzae pv.) and blast (Magnaporthe oryzae). [57] combined three major genes, Pi-d, Pi-z and Pi-k h , and bred blast resistant cultivar Jefferson. All the successful applications of gene pyramiding have been at the gene loci level. Our findings in this study will help enhance the application to allelic level in crop breeding. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Global attention has focused on examining individual mapping populations for quantitative trait loci (QTLs) for ShB resistance, but to date no study has taken advantage of association mapping to examine hundreds of lines for potentially novel QTLs. Our objective was to identify ShB QTLs via association representing in rice using 217 sub-core submissions from the USDA rice core collecting, which were phenotyped with a micro-chamber screening method and genotyped with 155 genome-wide markers. Structure analysis divided the mapping panel into five groups, and model comparison revealed that PCA5 with genomic control was the best model for association mapping of ShB. Ten marker loci on seven chromosomes were importantly associated with response to the ShB pathogen. Among multiple allelomorphs in each identified loci, the allele contributing the greatest effect to ShB resistance was named the putative resistant allele. Among 217 entries, entry GSOR 310389 contained the most putative resistant alleles, eight out of ten. The number of putative resistant alleles presented in an entry was highly and significantly correlated with the decrease of ShB rating (r = −0.535) or the increase of ShB resistance. Majority of the resistant entries that contained a large number of the putative resistant alleles belonged to indica, which is consistent with a general observation that most ShB resistant accessions are of indica origin. These findings demonstrate the potential to improve breeding efficiency by using marker-assisted selection to pyramid putative resistant alleles from various loci in a cultivar for enhanced ShB resistance in rice.
Introduction
Rice (Oryza sativa L.) feeds more than half of the world's population [1] and genetic improvement of this food crop can serve as a major component of sustainable food production. Rice sheath blight (ShB), caused by the soil-borne fungal pathogen Rhizoctonia solani Kühn, is a major disease of rice that greatly reduces yield and grain quality worldwide [2]. Due to the high cost of cultural practices and the phytotoxic influence associated with the application of fungicides, the use of ShB resistant cultivars is considered the most economical and environmentally sound strategy in managing this disease. Understandings of genetic control will facilitate cultivar improvement for this disease and secure global food production.
The necrotrophic ShB pathogen has a broad host range and no complete resistance has been identified in either commercial rice cultivars or wild related species [3,4]. However, substantial differences in susceptibility to ShB among rice cultivars have been observed under field conditions [5,6]. Differential levels of resistance and the associated resistance genes have been studied among rice germplasm accessions [7]. Rice ShB resistance is believed to be controlled by multiple genes or quantitative trait loci (QTLs) [8]. Since Li et al. [9] first identified ShB QTLs using restricted fragment length polymorphism (RFLP) markers under field conditions, over 30 resistant ShB QTLs have been reported using various mapping populations, such as F 2 s [10][11][12][13][14], double haploid (DH) lines [15], recombinant inbred lines (RILs) [8,[16][17][18], near-isogenic introgression lines (NIL) [19] and backcross populations [20][21][22][23]. 'Teqing' and 'Jasmine 85' have been repeatedly involved in these studies as the ShB resistant parents. We are the first to map rice ShB QTLs using association mapping strategy in a global germplasm collection.
In association mapping, each identified marker usually has multiple alleles in the mapping panel and each allele in a marker locus contributes differently to the associated trait. Agrama and Yan [42] reported that three alleles at each of three associated loci (allele 87 of RM490, 105 of RM413 and 122 of RM277) and two alleles at another locus (182 and 183 of RM263) had significantly greater contribution to straighthead resistance than other counterparts. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>This below document has 2 sentences that start with 'The putative resistant',
2 sentences that end with 'by Li et al'. It has approximately 460 words, 26 sentences, and 6 paragraph(s).
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Our identified marker RM8217 at 32.6 Mb was within the qSB-4-2 (30.6,33.6 Mb) by Pinson et al. [8] and Qsbr4a (31.7,33.6 Mb) by Li et al. [9], and in close proximity (within 0.6 Mb) to QRlh4 (29.8,32.0 Mb) by Xie et al. [59]. This small area confirmed by multiple studies strongly suggests a reliable location harboring ShB QTL.
On Chr5, 6 and 8, we identified one ShB associated marker from each chromosome. RM146 on Chr5 at 18.1 Mb is close to the Rsb 1 reported by Che et al. [10] between RM164 320 at 19.1 Mb and RM39 300 at 20.7 Mb near the centromere. RM133 (Chr6 at 0.2 Mb) and RM408 (Chr8 at 0.1 Mb) were close to the qShB6 (0.5,1.8 Mb) described by Liu et al. [18] and the QSbr8a (1.5,2.1 Mp) by Li et al. [9], respectively. The putative resistant alleles of both RM133 (allele 230 bp) and RM408 (allele 119 bp) were common with more than 45% of entries among the 217 subcore entries in the mapping panel. RM7203 on the short arm of Chr11 at 1.1 Mb was a novel ShB QTL that has not been reported. The putative resistant allele 88 of RM7203 existed in 55% of the 217 entries, so was the most common allele and had a ShB mean (0.470) similar to Jasmine 85 (0.472).
At the bottom of Chr11, two markers RM254 (at 23.7 Mb) and RM1233 (at 26.5 Mb) were identified, explaining relatively high phenotypic variation among ten markers, 5.3% and 5.1%, respectively. The RM1233 was one of the flanking markers for qSBR11-1 (26.5,27.2 Mb) reported by Channamallikarjuna et al. [16]. The putative resistant alleles, 169 bp of RM254 and 177 bp of RM1233, were in 12 and 35 entries, respectively. Their ShB ratings were lower than Jasmine 85 in average, indicating a stronger resistance.
Above comparisons confirm eight out of ten marker loci identified in our association mapping with two novel QTLs, RM11229 and RM7203, for sheath blight resistance. The confirmation of previously identified QTLs provides validation for the accuracy of QTLs identified in our study. Furthermore, the comparisons demonstrate that association mapping can locate many QTLs over the entire genome since the mapping panel includes a large number of diversified entries of germplasm. In biparental linkage mapping studies, fewer QTLs are typically identified and can only be located in a limited area in the genome where the two parents differ.
Germplasm panel
The rice mini-core collection of United States Department of Agriculture (USDA) contains 217 entries [53] derived from 1,794 entries of a core collection [60]. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Population structure Structure analysis from Q1 to Q10 across twenty runs for the 217 sub-core entries genotyped with 155 genome-wide DNA markers using STRUCTURE demonstrated that when Q reached five, the Pr(Q) became more-or-less plateaued, so Q5 captured the major structure in our data. Thus, the mapping panel was divided into five subgroups and each entry was classified to an appropriate subgroup using STRUCTURE. Inferred by reference cultivars recommended by Agrama et al. [52,53], the five subgroups were denoted as temperate japonica (TEJ), aus (AUS), aromatic (ARO), indica (IND), and tropical japonica (TRJ) ( Fig. 2A). A similar structural pattern was seen with the PCA analysis with the first two axes explaining 75.03% of variation (Fig. 2B). Furthermore, the genetic distance based on cluster analysis also divided the mapping panel into five major clusters (Fig. 2C). All three approaches led to the same conclusion: a five-group structure could clearly and sufficiently explain the existing genetic diversity in the mapping panel. In the mapping panel, IND had the most entries (86), followed by TRJ (49), AUS (39), TEJ (36), and ARO (7). Among 24 entries having greater resistance to ShB than the resistant check, Jasmine 85, 20 belonged to IND, two to AUS and one each to TRJ and admix (TRJ-AUS-IND).
Determination of the best fit model
From dimension 1 to 10 in the PCA and structure Q1 to Q10, PCA5 had the smallest Bayesian Information Criterion (BIC) value, indicating that PCA5 should be the best fit model to map ShB QTLs (Table 1). Hence, we tested each of 155 molecular markers for association with ShB resistance using PCA5, and plotted the observed versus expected -Log10(P) before and after correction using the genomic control (GC). The plots of the PCA5+GC were distributed more uniformly and was much closer to the expected -Log10(P) than PCA5 alone (Fig. 3). In other words, the PCA5+GC model showed better control for Type I errors. Therefore, the GC approach was applied to correct the biased estimation. The P values generated from the PCA5 model after the GC correction were used to present the significance level of each marker.
Marker loci and their alleles associated with sheath blight
Ten marker loci were identified to be significantly associated with ShB resistance at the probability level of 5% or lower, three on chromosome (Chr) 11, two on Chr1, and one each on Chr2, 4, 5, 6 and 8 ( Table 2, Fig. 4). RM237 on Chr1 at 27.1 Mb had the highest significance rating for ShB at the 0.002 level of probability. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. However, all of these studies have been limited to conventional mapping populations from a small number of parents, which limits the alleles in the progeny to those present in the parental lines.
From our diverse mapping panel including 217 sub-core entries, we identified ten marker loci significantly associated with ShB resistance, each locus had numerous alleles, each allele contributed differently to ShB resistance, and the putative resistant allele of each locus contributed the most (Table 2). Highly significant correlation demonstrated that as more putative resistant alleles pyramided in a germplasm entry, the entry had greater resistance to ShB. Among 24 entries that were significantly more resistant than the resistant check Jasmine 85 that had three putative resistant alleles, 17 (71%) contained four or more putative resistant alleles. Among 54 entries that had similar resistance with Jasmine 85, 29 (54%) possessed three or more putative resistant alleles.
These findings suggest that marker-assisted breeding for ShB resistance can be conducted on an allelic level by pyramiding putative resistant alleles in a cultivar. This can be accomplished in a two pronged approach by combining parental lines based upon their combination of number of different ShB resistant loci and possessing the putative resistant alleles at these loci. For example, resistant entry GSOR 310389 that has eight putative resistant alleles could be crossed with a commercial cultivar having fewer putative resistant alleles, and the progeny would be selected based on those having the most highly putative resistant alleles at the most loci. The breeding program would end up with the selection of progeny containing the most putative resistant alleles, potentially having greater resistance to ShB than either parental line.
Pyramiding responsible genes has been successfully applied in rice breeding for disease resistance including bacterial blight (Xanthomonas oryzae pv.) and blast (Magnaporthe oryzae). [57] combined three major genes, Pi-d, Pi-z and Pi-k h , and bred blast resistant cultivar Jefferson. All the successful applications of gene pyramiding have been at the gene loci level. Our findings in this study will help enhance the application to allelic level in crop breeding. The allelic application will improve breeding efficiency, increase cultivar resistance to sheath blight in rice and ultimately secure food production worldwide.
Allelic analysis can only be applied in association mapping where large number of diversified genotypes are used and multiple alleles are involved at each associated marker locus in the mapping panel. Using this method, germplasm accessions that are identified in the association mapping strategy to possess multiple putative resistant alleles can be crossed with other accessions that have a different complement of putative resistant alleles. The selection of progeny possessing the most putative resistant alleles should be more effective than it is for resistant loci. In this regard, association mapping offers advantages for identifying parental material and specific alleles that can enhance breeding.
Putative resistant alleles and ancestry background for sheath blight
Jia et al. [58] reported 52 entries that are significantly more resistant to ShB than Jasmine 85. The resistant entries were identified from 1,794 entries of the USDA rice core collection that has 35% indica, 27% temperate japonica, 24% tropical japonica, 10% aus and 4% aromatic genotypes [52]. Based on the ancestry classification, there are 621 indica entries in the core and 45 of them are included in the resistant list, making a resistance frequency of 7.2% for indica germplasm. Accordingly, the resistance frequency is 2.8% for aromatic, 1.7% for aus, and 0.2% each for temperate japonica and tropical japonica. In a study conducted by Zuo et al. [23], japonica cultivars showed higher sheath blight severity than indica cultivars. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. RM11229 on the long arm of Chr1 explained the most phenotypic variation (9.5%) with significance at the 0.044 level of probability. RM11229 and 1233 each had six alleles, the most among the 217 sub-core entries, followed by RM341 and 254 (five alleles), RM237, 8217,146 and 408 (four), RM133 (three) and RM7203 (two) ( Table 2).
Among the six alleles of RM11229, allele 158 was present in 18 entries that had the lowest average ShB rating (0.414), and thus, it was designated as the 'putative resistant allele' of this marker locus. Accordingly, ten alleles, one each from the ten associated marker loci, were noted as the putative resistant allele in Table 2 because they had the greatest effect to decrease ShB among all the alleles for their respective loci ( Table 2). ShB rating was the smallest for putative resistant allele 158 of RM11229 among the ten putative resistant alleles. Of the other five putative resistant alleles, 139 of RM341 (present in 17 entries), 340 of RM146 (28 entries), 88 of RM7203 (120 entries), 169 of RM254 (12 entries) and 177 of RM1233 (35 entries), had lower ShB means ranging 0.447-0.470 than the resistant check Jasmine 85 (0.472), suggesting a stronger effect for resistance to ShB than Jasmine 85. The remaining four putative resistant alleles had similar ShB ratings with Jasmine 85, suggesting a similar effect for the level of ShB control.
Number of putative resistant alleles and sheath blight resistance
As the number of putative resistant alleles in the germplasm increased, so did germplasm resistance to ShB (Table S1). GSOR 310389 from Korea contained the most putative resistant alleles, eight out of ten, and had a ShB rating of 0.351 which was significantly more resistant than the resistant check Jasmine 85 which contained three putative resistant alleles and had a ShB rating of 0.472. Among seven entries containing six putative resistant alleles with a mean of 0.386 ShB, GSOR 310475 and 311475 were more resistant than Jasmine 85 and had ShB ratings of 0.324 and 0.336, respectively. Among 28 entries having five putative resistant alleles with a mean ShB rating of 0.444, seven were significantly more resistant than Jasmine 85. Seven, out of 35 entries which carried four putative resistant alleles and had a mean ShB 0.466, were identified to be significantly more resistant than Jasmine 85. The mean ShB ratings for entries containing three, two, one and zero putative resistant alleles were 0.483, 0.535, Table 1. Comparative analysis of different subgroups using structure (Q model) and different dimensions in principal components analysis (PCA) for association mapping of sheath blight resistance using 217 entries genotyped with 155 molecular markers. 0.582 and 0.598, respectively. There was a strong and negative correlation between the ShB severity rating and number of putative resistant alleles (r = 20.535, p,0.0001).
Our mapping results showed that most entries containing a large number of putative resistant alleles were IND (Fig. 5 and Table S1). All entries with six or more putative resistant alleles were IND with only one exception of AUS.
Discussion
Pyramiding putative resistant alleles for cultivar improvement R. solani is a soil-borne necrotrophic fungus and its group AG1-IA has a broad host range including rice, maize, wheat, sorghum, bean (Phaseolus spp.) and soybean [Glycine max (L.) Merr.] [54]. The pathogen's ability to persist in soil and on crop residues allows it to, survive in multiple ways and makes disease management difficult. There is no complete resistance to ShB in rice because the resistance is quantitatively controlled by numerous genes or quantitative trait loci (QTLs). More than 30 QTLs responsible for ShB have been reported in rice [8,[10][11][12][13][14][15][16][17][18][19][20][21][22][23]. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. The remaining eight QTLs identified in this study were either quite near (less than 1.4 Mb distant) or within the interval of previously identified QTLs. The ten associated markers identified in this study were located on seven chromosomes (Chr1, 2, 4, 5, 6, 8 and 11) (Fig. 4).
On Chr1 our study identified RM11229 and RM 237, which occur within 4.4 Mb of each other, as markers associated with ShB resistance. RM11229 explained the most phenotypic variation (9.5%) and its putative resistant allele 158 bp had the smallest average ShB score (0.414 in Table 2), indicating the greatest resistance among the ten putative resistant alleles. RM237 was the most significant marker for ShB resistance (p = 0.002). Therefore, the 4.4 Mb gap between RM11229 and RM237 on Chr1 should be a target area for fine-mapping ShB resistant genes in rice. RM237 at 26.8 Mb is near the ShB QTL region spanning 27.6 to 34 Mb found by Charnnamallikarjuna et al. [16].
On Chr4, three reports uniformly indicated ShB QTLs on the long arm at 29.8 to 33.6 Mb. This is a very narrow region of 3.8 Mb on the physical map, and corresponds to a small estimated cM distance in the mapping population developed from susceptible Lemont and resistant Teqing parents [8,9,59]. Our identified marker RM8217 at 32.6 Mb was within the qSB-4-2 (30.6,33.6 Mb) by Pinson et al. [8] and Qsbr4a (31.7,33.6 Mb) by Li et al. [9], and in close proximity (within 0.6 Mb) to QRlh4 (29.8,32.0 Mb) by Xie et al. [59]. This small area confirmed by multiple studies strongly suggests a reliable location harboring ShB QTL.
On Chr5, 6 and 8, we identified one ShB associated marker from each chromosome. RM146 on Chr5 at 18.1 Mb is close to the Rsb 1 reported by Che et al. [10] between RM164 320 at 19.1 Mb and RM39 300 at 20.7 Mb near the centromere. RM133 (Chr6 at 0.2 Mb) and RM408 (Chr8 at 0.1 Mb) were close to the qShB6 (0.5,1.8 Mb) described by Liu et al. [18] and the QSbr8a (1.5,2.1 Mp) by Li et al. [9], respectively. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. In the mapping panel, IND had the most entries (86), followed by TRJ (49), AUS (39), TEJ (36), and ARO (7). Among 24 entries having greater resistance to ShB than the resistant check, Jasmine 85, 20 belonged to IND, two to AUS and one each to TRJ and admix (TRJ-AUS-IND).
Determination of the best fit model
From dimension 1 to 10 in the PCA and structure Q1 to Q10, PCA5 had the smallest Bayesian Information Criterion (BIC) value, indicating that PCA5 should be the best fit model to map ShB QTLs (Table 1). Hence, we tested each of 155 molecular markers for association with ShB resistance using PCA5, and plotted the observed versus expected -Log10(P) before and after correction using the genomic control (GC). The plots of the PCA5+GC were distributed more uniformly and was much closer to the expected -Log10(P) than PCA5 alone (Fig. 3). In other words, the PCA5+GC model showed better control for Type I errors. Therefore, the GC approach was applied to correct the biased estimation. The P values generated from the PCA5 model after the GC correction were used to present the significance level of each marker.
Marker loci and their alleles associated with sheath blight
Ten marker loci were identified to be significantly associated with ShB resistance at the probability level of 5% or lower, three on chromosome (Chr) 11, two on Chr1, and one each on Chr2, 4, 5, 6 and 8 ( Table 2, Fig. 4). RM237 on Chr1 at 27.1 Mb had the highest significance rating for ShB at the 0.002 level of probability. RM11229 on the long arm of Chr1 explained the most phenotypic variation (9.5%) with significance at the 0.044 level of probability. RM11229 and 1233 each had six alleles, the most among the 217 sub-core entries, followed by RM341 and 254 (five alleles), RM237, 8217,146 and 408 (four), RM133 (three) and RM7203 (two) ( Table 2).
Among the six alleles of RM11229, allele 158 was present in 18 entries that had the lowest average ShB rating (0.414), and thus, it was designated as the 'putative resistant allele' of this marker locus. Accordingly, ten alleles, one each from the ten associated marker loci, were noted as the putative resistant allele in Table 2 because they had the greatest effect to decrease ShB among all the alleles for their respective loci ( Table 2). ShB rating was the smallest for putative resistant allele 158 of RM11229 among the ten putative resistant alleles. Of the other five putative resistant alleles, 139 of RM341 (present in 17 entries), 340 of RM146 (28 entries), 88 of RM7203 (120 entries), 169 of RM254 (12 entries) and 177 of RM1233 (35 entries), had lower ShB means ranging 0.447-0.470 than the resistant check Jasmine 85 (0.472), suggesting a stronger effect for resistance to ShB than Jasmine 85. The remaining four putative resistant alleles had similar ShB ratings with Jasmine 85, suggesting a similar effect for the level of ShB control.
Number of putative resistant alleles and sheath blight resistance
As the number of putative resistant alleles in the germplasm increased, so did germplasm resistance to ShB (Table S1). GSOR 310389 from Korea contained the most putative resistant alleles, eight out of ten, and had a ShB rating of 0.351 which was significantly more resistant than the resistant check Jasmine 85 which contained three putative resistant alleles and had a ShB rating of 0.472. Among seven entries containing six putative resistant alleles with a mean of 0.386 ShB, GSOR 310475 and 311475 were more resistant than Jasmine 85 and had ShB ratings of 0.324 and 0.336, respectively. Among 28 entries having five putative resistant alleles with a mean ShB rating of 0.444, seven were significantly more resistant than Jasmine 85. Seven, out of 35 entries which carried four putative resistant alleles and had a mean ShB 0.466, were identified to be significantly more resistant than Jasmine 85. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. Field evaluations showed no differences in disease reactions between the slow growing isolates and the fast ones [51]. Further, the RR0140-1 isolate have been adapted by numerous studies [6,18,50]. Pathogen inoculum of RR0140-1 were grown by placing sclerotia in the centre of potato dextrose agar (PDA) plates (Sigma-Aldrich, St. Louis) containing 0.005% (wt/vol) tetracycline, and then transferred to a fresh PDA medium for 5-6 days at 27uC under darkness. Mycelium discs (7 mm in diameter) were excised from the outer growing area in the culture plate where the outer mycelia were mostly active. Rice seedlings were inoculated at the three-leaf stage.
In the greenhouse, each 12612 cm pot was filled with presterilized soil to ensure that the study was not confounded by the presence of soil borne R. solani inoculum. Pots with drainage holes were placed in flats filled with shallow water (,5 cm). Five seeds of each accession were planted in each pot and thinned to three uniform plants before pathogen inoculation. The three remaining plants in a given pot were referred to as one experimental unit or replicate. Each of the three seedlings in a pot was individually inoculated with a round mycelium disc of RR0140-1 pathogen as described by Jia et al. [6] with modification. Each disk was pressed up to the base of the seedling stem, assuring that the mycelium was in contact with the plant. After inoculation, each pot was immediately covered with a 2-liter soft drink bottle with the bottom and cap removed. Relative humidity was maintained over 80% in the bottle, which favoured growth of the sheath blight pathogen on the plants. The greenhouse temperatures were set for day/night at 30/22uC, respectively with a 12 h photoperiod.
Plant response to the sheath blight pathogen was measured using the ratio between the height of the pathogen growing up the plant and the height of the leaf collar on the last emerged leaf. Because mature plant height varied from 70 to 202 cm in this collection [60], the ratio excluded possible interference of plant height in scoring disease response. Therefore, the smaller the ratio, the greater the resistance was for an entry. Measurements were taken when the ratio reached 1.0 for 75% of the susceptible check plants, Lemont, so that the maximum susceptibility was scored 1.0.
ShB rating data were analyzed using the GLIMMIX procedure in SAS version 9.1.3 [62]. The experimental design of randomized incomplete block formed the basis of the statistical model, where the accession is a fixed effect and block is treated as random effect. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences<|endoftext|>Allelic Analysis of Sheath Blight Resistance with Association Mapping in Rice Sheath blight (ShB) caused by the soil-borne pathogen Rhizoctonia solani is one of the most devastating diseases in rice world-wide. For the purpose of model comparisons, the phenotypic vector is modeled asy~X bzQvzZuze, where b is vector of marker effects to be estimated. The term Qv contains the coordinates of the individuals of p dimensions in Q matrix generated by STRUCTURE [64,65] and PCA matrix by NTSYSpc version 2.1 [70]; X and Z are the incidence matrices of 1 s and 0 s that relate y to b and u, respectively. u is a vector of polygene background effects; and e is a vector of residual. The phenotypic covariance matrix was assumed to have the form V~Z(2Ks 2 g )Z T zIs 2 e , where K is the K matrix including relative kinship coefficients defining the degree of genetic covariance between a pair of individuals [71], I is an n6n identity matrix, s 2 g is the genetic variance attributable to genome wide effects, and s 2 e is the residual variance.
When model K or Q+K or PCA+K were tested for a fit, we found little convergence because of the low level of relatedness among entries in the panel. Thus, a simple model (ignoring the effect of population structure), possible linear models of Q2-Q10 (considering different number of subgroups from two to ten) and PCA1-PCA10 (PCA matrix with different number of dimensions from one to ten) were compared for the best fit to ShB determined by Bayesian information criterion (BIC). In order to control false positive rates, the genomic control (GC) method [72] was further used for correcting population structure. The association mapping was conducted using the best fit model with TASSEL v.2.1 [73], followed by the GC. The associated markers with ShB resistance were claimed at the probability level of 0.05. The ShB severity ratings of germplasm entries that carried the same allele in an associated marker locus were averaged to estimate allelic effect on the ShB rating. (DOC)
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Domain: Biology Medicine Agricultural And Food Sciences
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Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. Likewise, the enzyme addition to the NC2 diet significantly improved (P < 0.05) energy, DM, CP, EE, ash and P retention, but didn't restore to the level of the PC (P < 0.05). Supplementing the enzyme complex to the NC1 diet increased AME by 60 kcal/kg, and in NC2 diet by 117 kcal/kg. Meng et al. [2] found improved digestibility of the NSP (from 6.3% without enzymes to 14.9% with enzymes) and AMEn (+2.3%) in a corn-soybean meal diet containing 9% total NSP. The function of phytase resulted in improvements in digestibility of dietary P and other minerals [5,26,29]. Carbohydrases can partially break-down cell wall matrix thus liberate more nutrients, and facilitate contact between endogenous digestive enzymes and their substrates, improving the overall utilization of nutrients in feedstuff [30]. This study showed that NSPdegrading enzymes and phytase were able to improve availability of energy, CP, EE, P, Ca and ash, thus boosting bird performance. This was well in line with Cowieson et al. [25,] and Cowieson and Adeola [10], who demonstrated that the performance of broiler chicks fed on low nutrient density corn-based diets returned to the level of birds fed on nutritionally adequate diet after supplementing with exogenous xylanase, amylase, protease and phytase by improving the digestibility of energy, DM, N, lipids, amino acids, Ca and P when broiler chicks fed on corn-soybean mealbased diets.
Ca and P metabolism
Bone mineral concentration may be more sensitive than performance in estimating mineral deficiency. The requirement of avP and Ca for maximum P, Ca and ash deposition in tibia was greater than for adequate growth performance [15,26]. Similar results were observed in the current study, in which the enzyme supplementation increased bone mineralization of ducklings fed on both NC1 and NC2 diets, with a greater response observed from the birds fed on the most avP and Ca inadequate diets. With reduction on avP and Ca contents (avP 1.5 g/kg and Ca 1.2 g/kg), supplemental enzymes restored performance in full, but insufficient to restore bone mineralization to that of the PC. Francesch et al. [15], who investigated the same multiple-enzyme complex in corn-soybean meal diets for broilers, found significantly Means in the same row, values with the same lowercase letter superscripts mean no significant difference (P > 0.05), different lowercase letter mean significant difference (P < 0.05). 1 Data are means of 7 ducks per treatment. Using negative control treatments with or without enzyme supplementation. 5 NR = Apparent metabolizable energy, digestible amino acid, available P and Ca reduction. Using negative control treatments with or without enzyme supplementation. 5 NR = Apparent metabolizable energy, digestible amino acid, available P and Ca reduction.
differences in bone ash mineralization at 43 d between the NC and PC groups, as well as with the NC diet with the multiple-enzyme complex. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
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Domain: Agricultural and Food Sciences Biology Medicine<|endoftext|>Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. Duplicate proximate analyses were performed on diets and excreta samples. Dry matter(DM) analysis of samples was conducted by drying the samples in an oven at 105°C for 24 h. Energy content of the samples was determined by the adiabatic oxygen bomb calorimeter. Nitrogen, ester extract (EE), ash, Ca and total P contents of the samples were determined according to AOAC [21].
Statistical analysis
To establish differences between PC and reduced nutrient diets, with and without enzyme supplementation, data were analyzed as a randomized complete block design with a one-way ANOVA using the GLM procedures of SAS [22]. To determine the main effects of level of nutrient reduction and enzyme supplementation response and their interaction, data without the PC were analyzed by a 2 × 2 factorial analysis of the variance. Statistical significance was set at P ≤ 0.05.
Growth performance
The results of growth performance are shown in Table 2. Ducklings fed on NC2 diet had the lowest (P < 0.05) BW (d 14 and 35) and FI (d 35) compared to those fed on PC, NC1 with the enzyme and NC2 with the enzyme. The addition of the enzyme complex significantly increased (P < 0.05) BW and FI at days 14 and 35 to the level of PC diet, but no effect on F/G. Feed to gain ratio in NC1, NC1 with enzyme, NC2 and NC2 with enzyme diets was marked higher (P < 0.05) than that of the PC diet. Interaction between reduced dietary nutrient level and enzyme supplementation was detected (P <0.05) for BW at 14 d of age and FI during the whole experiment. These results suggest that NC1 nutrient levels (1 to 14 d, AME 2,730 kcal/kg, avP 0.32%, Ca 0.75%; 15 to 35 d, AME 2,830 kcal/kg, avP 0.27%, Ca 0.66%) were able to meet the growth requirement of the meat ducks; but NC2 nutrient level (1 to 14 d, ME 2,700 kcal/kg, avP 0.27%, Ca 0.73%; 15 to 35 d, ME 2,800 kcal/kg, avP 0.23%, Ca 0.64%) showed a negative effect on performance. Xie et al. [23] and Fan et al. [24] studied energy requirement of White Pekin ducklings from posthatching to 21 d or 15 to 42 d by comparing 5 dietary energy levels (2,455, 2,603, 2,751, 2,902, 3,057 kcal/kg) or by 6 dietary energy levels (2,600, 2,700, 2,800, 2,900, 3,000, 3,100 kcal/kg), respectively, the results revealed that BW and FI seemed insensitive to dietary ME change within a range of 0 to 100 kcal/kg, whilst feed conversion did. We consider the poor performance of the ducks fed on NC2 diet in this study was resulted from avP deficiency as peviously reported by several authors [15,25,26].
Nutrient utilization
The effects of enzyme supplementation on nutrition utilization are summarized in Table 3. The down-spec of nutrients significantly decreased (P < 0.01) availability of CP and EE compared with the PC diet. Feeding NC2 diet reduced (P < 0.01) availability of DM, energy, CP, EE, ash, Ca and P compared to feeding PC and NC1 diet. The addition of the enzyme to the NC diets significantly improved (P < 0.05) energy, CP, EE, ash, Ca and P availability. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
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Domain: Agricultural and Food Sciences Biology Medicine
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Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. Interaction between reduced dietary nutrient level and enzyme supplementation was detected (P <0.05) for BW at 14 d of age and FI during the whole experiment. These results suggest that NC1 nutrient levels (1 to 14 d, AME 2,730 kcal/kg, avP 0.32%, Ca 0.75%; 15 to 35 d, AME 2,830 kcal/kg, avP 0.27%, Ca 0.66%) were able to meet the growth requirement of the meat ducks; but NC2 nutrient level (1 to 14 d, ME 2,700 kcal/kg, avP 0.27%, Ca 0.73%; 15 to 35 d, ME 2,800 kcal/kg, avP 0.23%, Ca 0.64%) showed a negative effect on performance. Xie et al. [23] and Fan et al. [24] studied energy requirement of White Pekin ducklings from posthatching to 21 d or 15 to 42 d by comparing 5 dietary energy levels (2,455, 2,603, 2,751, 2,902, 3,057 kcal/kg) or by 6 dietary energy levels (2,600, 2,700, 2,800, 2,900, 3,000, 3,100 kcal/kg), respectively, the results revealed that BW and FI seemed insensitive to dietary ME change within a range of 0 to 100 kcal/kg, whilst feed conversion did. We consider the poor performance of the ducks fed on NC2 diet in this study was resulted from avP deficiency as peviously reported by several authors [15,25,26].
Nutrient utilization
The effects of enzyme supplementation on nutrition utilization are summarized in Table 3. The down-spec of nutrients significantly decreased (P < 0.01) availability of CP and EE compared with the PC diet. Feeding NC2 diet reduced (P < 0.01) availability of DM, energy, CP, EE, ash, Ca and P compared to feeding PC and NC1 diet. The addition of the enzyme to the NC diets significantly improved (P < 0.05) energy, CP, EE, ash, Ca and P availability. Likewise, the enzyme addition to the NC2 diet significantly improved (P < 0.05) energy, DM, CP, EE, ash and P retention, but didn't restore to the level of the PC (P < 0.05). Supplementing the enzyme complex to the NC1 diet increased AME by 60 kcal/kg, and in NC2 diet by 117 kcal/kg. Meng et al. [2] found improved digestibility of the NSP (from 6.3% without enzymes to 14.9% with enzymes) and AMEn (+2.3%) in a corn-soybean meal diet containing 9% total NSP. The function of phytase resulted in improvements in digestibility of dietary P and other minerals [5,26,29]. Carbohydrases can partially break-down cell wall matrix thus liberate more nutrients, and facilitate contact between endogenous digestive enzymes and their substrates, improving the overall utilization of nutrients in feedstuff [30]. This study showed that NSPdegrading enzymes and phytase were able to improve availability of energy, CP, EE, P, Ca and ash, thus boosting bird performance. This was well in line with Cowieson et al. [25,] and Cowieson and Adeola [10], who demonstrated that the performance of broiler chicks fed on low nutrient density corn-based diets returned to the level of birds fed on nutritionally adequate diet after supplementing with exogenous xylanase, amylase, protease and phytase by improving the digestibility of energy, DM, N, lipids, amino acids, Ca and P when broiler chicks fed on corn-soybean mealbased diets.
Ca and P metabolism
Bone mineral concentration may be more sensitive than performance in estimating mineral deficiency. The requirement of avP and Ca for maximum P, Ca and ash deposition in tibia was greater than for adequate growth performance [15,26]. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
==
Domain: Agricultural and Food Sciences Biology Medicine<|endoftext|>Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. Similar results were observed in the current study, in which the enzyme supplementation increased bone mineralization of ducklings fed on both NC1 and NC2 diets, with a greater response observed from the birds fed on the most avP and Ca inadequate diets. With reduction on avP and Ca contents (avP 1.5 g/kg and Ca 1.2 g/kg), supplemental enzymes restored performance in full, but insufficient to restore bone mineralization to that of the PC. Francesch et al. [15], who investigated the same multiple-enzyme complex in corn-soybean meal diets for broilers, found significantly Means in the same row, values with the same lowercase letter superscripts mean no significant difference (P > 0.05), different lowercase letter mean significant difference (P < 0.05). 1 Data are means of 7 ducks per treatment. Using negative control treatments with or without enzyme supplementation. 5 NR = Apparent metabolizable energy, digestible amino acid, available P and Ca reduction. Using negative control treatments with or without enzyme supplementation. 5 NR = Apparent metabolizable energy, digestible amino acid, available P and Ca reduction.
differences in bone ash mineralization at 43 d between the NC and PC groups, as well as with the NC diet with the multiple-enzyme complex. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
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Domain: Agricultural and Food Sciences Biology Medicine
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Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. China is the largest country for duck production, accounting for 3/4 of the global duck husbandry. How to improve feed efficiency of diets containing high levels of by-products remains on the top of priority in duck husbandry. The present study was designed to investigate a multi-enzyme complex containing NSP enzymes and phytase on the growth performance, nutrient utilization and bone mineralization of meat ducklings fed on diets consisting in corn, soybean meal and byproducts.
Methods
Experimental design, birds and diets
Exp. 1, the study was a 2 × 2 + 1 factorial design consisting in five treatments [2 levels of AME, DAA, Ca and avP reduction, with or without enzyme supplementation, plus a positive control]. The positive control (PC) diet was formulated to be adequate or to exceed requirements of NRC (1994) [17], NC1 (down-spec AME 70 kcal/kg, DAA 2.0%, avP 1.0 g/kg and Ca 1.0 g/kg) and NC2 (down-spec AME 100 kcal/kg, DAA 2.5%, avP 1.5 g/kg and Ca 1.2 g/kg). From days 1 to 14 and 15 to 35 of age, the PC diet provided avP 0.42% and 0.38%, NC1 provided 0.32% and 0.28%, and NC2 provided 0.27% and 0.23%, respectively, without or with the addition of an enzyme complex (Rovabio® Max LC, a concentrated liquid solution containing xylanase, β-glucanase and phytase as main activities, extracted from fermentation broths of Penicillium funiculosum and Schizosaccharomyces pombe, Adisseo France SAS). The liquid enzyme was applied after feed pelleting at a dose of 200 mL/ 1,000 kg feed to provide a minimum 1,100 visco-units of endo-β-1,4-xylanase, 100 units of endo-1,3(4)-β-glucanase, and 500 phytase units (FTU)/kg feeds. There were five dietary treatments replicated 7 times each of 16 ducks per replicate and allocated at random by blocks, according to the location in the experimental house. The feeding program consisted of 2 diets, starter diet supplied from d 1 to d 14, followed by a grower diet from d 15 to d 35 (Table 1). All diets were formulated following the amino acid profile established by our laboratory, with digestible lysine: digestible methionine : digestible threonine: digestible trytophan = 100:48:66:27 in starter diets; and 100:44:67:23; for grower diets [18]. Bentonite was used as diluent. Feeds were supplied in pelleted form with diameter 2 mm for the starter and 3 mm for the grower. The enzyme was sprayed in liquid form after pelleting on to cooled pellets and at a dilution rate of 1/ 20.
Exp. 2 was a subsequent metabolizable study of Exp. 1. At day 35, six ducklings were selected in line with the mean body weight of each treatment. A total of 30 ducklings (1 duckling per cage and 6 cages per treatment) were placed into individual metabolic cages (50 cm × 50 cm) in a room with temperature maintained at 22-25°C with continuous lighting. At day 39, each duckling was surgically fitted with a retainer ring [19] and fed on corresponding diets until restoration of feed intake and excretion, followed by feed withdrawal for 12 h. All ducklings were then provided with their respective corresponding diets and their faecal collection bags were installed. Excreta samples were collected for a period of 48 h. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
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Domain: Agricultural and Food Sciences Biology Medicine<|endoftext|>Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. Bentonite was used as diluent. Feeds were supplied in pelleted form with diameter 2 mm for the starter and 3 mm for the grower. The enzyme was sprayed in liquid form after pelleting on to cooled pellets and at a dilution rate of 1/ 20.
Exp. 2 was a subsequent metabolizable study of Exp. 1. At day 35, six ducklings were selected in line with the mean body weight of each treatment. A total of 30 ducklings (1 duckling per cage and 6 cages per treatment) were placed into individual metabolic cages (50 cm × 50 cm) in a room with temperature maintained at 22-25°C with continuous lighting. At day 39, each duckling was surgically fitted with a retainer ring [19] and fed on corresponding diets until restoration of feed intake and excretion, followed by feed withdrawal for 12 h. All ducklings were then provided with their respective corresponding diets and their faecal collection bags were installed. Excreta samples were collected for a period of 48 h. The excreta collected were immediately frozen at -20°C.
Bird housing and management
Five hundred and sixty one-day-old male Cherry Valley ducks were used and distributed into 35 cages of 16 ducks per cage. All ducks were reared in cages (2.2 m × 1.2 m × 0.9 m) in a temperature and humidity controlled room with a 24 h constant light schedule and had free access to water and feed throughout the experimental period.
At the age of d 14 and d 35, birds and feed consumption were weighed by cage, body weight (BW) gain, feed intake (FI), feed to gain ratio (F/G) were then calculated. Seven ducklings per treatment (1 bird per pen) were randomly selected for blood sampling via the jugular vein. Serum was obtained by centrifuge at 3,000 rpm, 4°C for 10 min. All serum samples were stored -20°C. Contents of Ca, P and alkaline phosphatase (ALP) activity were measured by automated hematology analyzer (Yellow Springs Instrument Co., Inc., Yellow Springs, OH). Once blood was collected, birds were euthanized by cervical dislocation. Their left tibia was removed and stored at -20°C. After boiling/extraction, tibia ash, Ca and P contents were determined, according to the procedures of AOAC [20,21].
Chemical analysis
Diet and dried excreta samples were ground to pass through 0.5 mm screen using a mill grinder. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
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Domain: Agricultural and Food Sciences Biology Medicine
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Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. The excreta collected were immediately frozen at -20°C.
Bird housing and management
Five hundred and sixty one-day-old male Cherry Valley ducks were used and distributed into 35 cages of 16 ducks per cage. All ducks were reared in cages (2.2 m × 1.2 m × 0.9 m) in a temperature and humidity controlled room with a 24 h constant light schedule and had free access to water and feed throughout the experimental period.
At the age of d 14 and d 35, birds and feed consumption were weighed by cage, body weight (BW) gain, feed intake (FI), feed to gain ratio (F/G) were then calculated. Seven ducklings per treatment (1 bird per pen) were randomly selected for blood sampling via the jugular vein. Serum was obtained by centrifuge at 3,000 rpm, 4°C for 10 min. All serum samples were stored -20°C. Contents of Ca, P and alkaline phosphatase (ALP) activity were measured by automated hematology analyzer (Yellow Springs Instrument Co., Inc., Yellow Springs, OH). Once blood was collected, birds were euthanized by cervical dislocation. Their left tibia was removed and stored at -20°C. After boiling/extraction, tibia ash, Ca and P contents were determined, according to the procedures of AOAC [20,21].
Chemical analysis
Diet and dried excreta samples were ground to pass through 0.5 mm screen using a mill grinder. Duplicate proximate analyses were performed on diets and excreta samples. Dry matter(DM) analysis of samples was conducted by drying the samples in an oven at 105°C for 24 h. Energy content of the samples was determined by the adiabatic oxygen bomb calorimeter. Nitrogen, ester extract (EE), ash, Ca and total P contents of the samples were determined according to AOAC [21].
Statistical analysis
To establish differences between PC and reduced nutrient diets, with and without enzyme supplementation, data were analyzed as a randomized complete block design with a one-way ANOVA using the GLM procedures of SAS [22]. To determine the main effects of level of nutrient reduction and enzyme supplementation response and their interaction, data without the PC were analyzed by a 2 × 2 factorial analysis of the variance. Statistical significance was set at P ≤ 0.05.
Growth performance
The results of growth performance are shown in Table 2. Ducklings fed on NC2 diet had the lowest (P < 0.05) BW (d 14 and 35) and FI (d 35) compared to those fed on PC, NC1 with the enzyme and NC2 with the enzyme. The addition of the enzyme complex significantly increased (P < 0.05) BW and FI at days 14 and 35 to the level of PC diet, but no effect on F/G. Feed to gain ratio in NC1, NC1 with enzyme, NC2 and NC2 with enzyme diets was marked higher (P < 0.05) than that of the PC diet. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
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Domain: Agricultural and Food Sciences Biology Medicine<|endoftext|>Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck
Background
Previous studies demonstrated single or multiple enzyme complex such as carbohydrases and phytase can improve utilization of dietary nutrients, thus decrease the cost of feeds in poultry production [1]. Carbohydrases can hydrolyze non-starch polysaccharides (NSP), and release the encapsulated nutrients from the cell walls, and reduce digesta viscosity [2,3]. Phytase is effective to release phosphorous through hydrolyzing phytate phosphorous compounds. The addition of 500, 1,000, and 1,500 units of phytase to basal diet for 7-d-old ducks released 0.453, 0.847, and 1.242 g inorganic P/kg of diet after 10 d, respectively [4]. Similarly, previous reports showed supplementation of phytase exerted positive effect on the utilization of energy, nitrogen (N) and amino acids (AA) in poultry diets [5][6][7]. A study showed that carbohydrases may facilitate phytase to access phytate [8]. Other studies also demonstrated that phytase and carbohydrases may exert synergistic effects in wheat-based diets [5,9] or an additive effect [10,11] in corn-based diets based on performance parameters. The response to a multi-enzyme complex seems to depend on several factors, including the type of diet, level of nutrients in the diet, dose of enzymes, age of birds, and even the genetic strain of the birds [12][13][14].
Francesch and Geraert [15] reported that the supplementation with a multi-enzyme complex containing NSP degrading enzymes and phytase is very efficient in compensating the down-spec from reduction of 2.0 g/kg available phosphorus (avP), 1.6 g/kg calcium (Ca), 85 kcal/kg apparent metabolizable energy (AME), and 3.0% digestible amino acids (DAA) of corn-soybean meal diets in broilers. However, there are studies showing significant differences between duck and broiler in terms of nutrients utilization [16]. By far, most enzyme efficiency studies were conducted with broilers, whereas information regarding ducks is scarce. China is the largest country for duck production, accounting for 3/4 of the global duck husbandry. How to improve feed efficiency of diets containing high levels of by-products remains on the top of priority in duck husbandry. The present study was designed to investigate a multi-enzyme complex containing NSP enzymes and phytase on the growth performance, nutrient utilization and bone mineralization of meat ducklings fed on diets consisting in corn, soybean meal and byproducts.
Methods
Experimental design, birds and diets
Exp. 1, the study was a 2 × 2 + 1 factorial design consisting in five treatments [2 levels of AME, DAA, Ca and avP reduction, with or without enzyme supplementation, plus a positive control]. The positive control (PC) diet was formulated to be adequate or to exceed requirements of NRC (1994) [17], NC1 (down-spec AME 70 kcal/kg, DAA 2.0%, avP 1.0 g/kg and Ca 1.0 g/kg) and NC2 (down-spec AME 100 kcal/kg, DAA 2.5%, avP 1.5 g/kg and Ca 1.2 g/kg). From days 1 to 14 and 15 to 35 of age, the PC diet provided avP 0.42% and 0.38%, NC1 provided 0.32% and 0.28%, and NC2 provided 0.27% and 0.23%, respectively, without or with the addition of an enzyme complex (Rovabio® Max LC, a concentrated liquid solution containing xylanase, β-glucanase and phytase as main activities, extracted from fermentation broths of Penicillium funiculosum and Schizosaccharomyces pombe, Adisseo France SAS). The liquid enzyme was applied after feed pelleting at a dose of 200 mL/ 1,000 kg feed to provide a minimum 1,100 visco-units of endo-β-1,4-xylanase, 100 units of endo-1,3(4)-β-glucanase, and 500 phytase units (FTU)/kg feeds. There were five dietary treatments replicated 7 times each of 16 ducks per replicate and allocated at random by blocks, according to the location in the experimental house. The feeding program consisted of 2 diets, starter diet supplied from d 1 to d 14, followed by a grower diet from d 15 to d 35 (Table 1). All diets were formulated following the amino acid profile established by our laboratory, with digestible lysine: digestible methionine : digestible threonine: digestible trytophan = 100:48:66:27 in starter diets; and 100:44:67:23; for grower diets [18]. [29] found that overall tibia ash improved with phytase addition (P < 0.05) during a 42-d period in which broiler chickens were fed on an NC diet (with the starter and grower diets containing 2.4 and 1.8 g/kg non-phytate P, respectively).
In conclusion, results of the present experiment indicate that the lower nutrient density diets resulted in a reduction in growth performance, nutrient utilization and bone mineralization of meat ducklings which can be restored by supplementation of exogenous enzyme complex, and that the multi-enzyme complex has the potential to compensate AME 100 kcal/kg, DAA 2.5%, av P 1.5 g/kg and Ca 1.2 g/kg in basal diet consisting in corn, soybean meal and by-products.
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Domain: Agricultural and Food Sciences Biology Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Components of the same signal transduction pathway may also be shared by various stress factors such as drought, salt and cold. Although there are multiple pathways of signal-transduction systems operating at the cellular level for gene regulation.
In past, it has been reported that genes induced during stress conditions function not only in protecting cells from stress by producing important metabolic proteins, but also in regulating genes for signal transduction in the stress response [52]. Several stress induced cor genes such as rd29A, cor15A, kin1 and cor6.6 are triggered in response to cold treatment, ABA and water deficit stress in the early stages of the osmotic stress response.
Several stress induced cor genes such as rd29A, cor15A, kin1 and cor6.6 are triggered in response to cold treatment, ABA and water deficit stress in the early stages of the osmotic stress response. Similarly, a cis-acting element, dehydration responsive element (DRE) identified in A. thaliana, is also involved in ABA-independent gene expression under drought, low temperature and high salt stress conditions in many dehydration responsive genes like rd29A that are responsible for dehydration and cold-induced gene expression. Thus, clearly, the overexpression of some droughtresponsive transcription factors can lead to the expression of downstream genes and the enhancement of abiotic stress tolerance in plants. Thus, these gene products are classified into two groups [53,54]. The first group of proteins that probably function in stress tolerance includes chaperones, LEA proteins, osmotin, antifreeze proteins, mRNA-binding proteins, some key enzymes for osmolyte biosynthesis (like proline, water channel proteins, sugar and proline transporters, detoxification enzymes), enzymes for fatty acid metabolism (proteinase inhibitors, ferritin) and lipid-transfer proteins [58]. It has been reported that some of these stressinducible genes specially encoding proteins (such as enzymes for osmolyte biosynthesis, LEA proteins and detoxification enzymes) have been overexpressed in transgenic plants and produce stress-tolerant phenotypes in the transgenic plants [51,56]. These results indicate that the gene products of the stress-inducible genes really function in stress tolerance. The second group contains protein factors involved in regulation of signal transduction and gene expression that probably function during stress response [59]. This group includes various transcription factors that regulate different stress-inducible genes collectively or separately, and may constitute gene networks. Seki et al. [59] reported that some of these regulatory pathways are also involved in drought-, cold-, or high-salinity stress responses. Though, the clear cut functions of most of these genes are not fully understood. Functional analysis of these stressinducible transcription factors will provide precise information on the complex regulatory gene networks that are involved in responses to drought, cold, and high-salinity stresses [60,25]. Some of these stress-inducible regulatory genes that encode proteins (transcription factors) have been overexpressed in transgenic plants and generate stresstolerant phenotypes in them [61,62].
COLD TOLERANCE USING TRANSGENIC AP-PROACHES
When a plant is subjected to abiotic stress, a number of genes are turned on, resulting in increased levels of several metabolites and proteins, some of which may be responsible for conferring a certain degree of protection to these stresses.
A key to progress towards breeding better crops under stress has been to understand the changes in cellular, biochemical and molecular machinery that occur in response to stress. A key to progress towards breeding better crops under stress has been to understand the changes in cellular, biochemical and molecular machinery that occur in response to stress which in turn provides new tools and strategies to improve the environmental stress tolerance of crops. Since freezing tolerance is a multigenic trait [63], transformation of a single functional gene appears to have a limited effect on crop freezing tolerance [64]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Early research indicates that the invertase gene is regulated by the hormone abscisic acid (ABA). Oliver et al. [24] has experimented with injecting plants with ABA -the resulting rice plants are sterile, just like that if they experienced a cold snap. Also, ABA levels increase when conventional rice is exposed to cold, but they remain the same in the cold tolerant variety. Recent studies have indicated that the difference between cold-sensitive and tolerant rice is due to a different ability to control ABA levels [25]. It has also been shown that this mechanism may require interactions with other plant hormones like auxins [26]. Further, Zhao et al. [25] also reported that low temperature turns off the genes responsible for sugar transport into the pollen grains and therefore starch cannot be produced in the pollen in cold conditions. Cold did not cause repression of sugar delivery in cold tolerant Chinese rice and fertile pollen was still produced following cold treatment. The sugar metabolism genes also continued to function normally during cold treatment of cold tolerant rice. Ample genetic variation for cold tolerance is available in well adapted breeding population. Germplasm collected from high altitude and low temperature areas, cold tolerant mutant, somaclonal variants and wild species can be exploited for breeding improved cold tolerant genotypes in hilly areas [15].
MECHANISMS FOR UNDERSTANDING TOLER-ANCE TO COLD INJURY
Low temperature has a huge impact on the survival and geographical distribution of plants. It affects a range of cellular metabolisms in plant system depending on the intensity and duration of the stress. When exposed to low temperatures, plants respond with changes in their pattern of gene expression and protein products. Different studies have indicated that the membrane systems of the cell are the primary site of freezing injury in plants [2,27]. In addition, it is well established that freeze-induced membrane damage results primarily from the severe dehydration associated with freezing [27,28]. Many species of tropical or subtropical origin are injured or killed by nonfreezing low temperatures, and exhibit various symptoms of chilling injury such as chlorosis, necrosis, or growth retardation. In contrast, chillingtolerant species are able to grow at such cold temperatures.
However, multiple forms of membrane damage can occur as a consequence of freeze induced cellular dehydration including expansion-induced-lysis, lamellar-to-hexagonal-II phase transitions, and fracture jump lesions [28]. Thus, a key function of cold acclimation should be to stabilize membranes against freezing injury. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. The genes encoding protein factors that regulate gene expression and signal transduction, that function in stress responses may be useful for improving the cold tolerance of plants by gene transfer as they can regulate many stress-inducible genes involved in cold stress tolerance.
The CBF genes represent one of the most significant discoveries in the field of low temperature adaptation and signal transduction. All important crops and few vegetables species have contained this gene [11]. Various low temperatureinducible genes have been isolated from plants that appear to be involved in tolerance to cold stress [71,72], and the expression of some of them is regulated by C-repeat binding factor/dehydration-responsive element binding (CBF/ DREB1) transcription factors. Three CBF/DREB1 genes (CBF3/DREB1a, CBF1/DREB1b, and CBF2/DREB1c) belonging to the AP2/DREBP family of DNA-binding proteins have been identified in Arabidopsis [73,74,75]. Transgenic Arabidopsis plants constitutively over-expressing a cold inducible transcription factor (CBF1; CRT/DRE binding protein) showed tolerance to freezing without any negative effect on the development and growth characteristics [76]. Overexpression of Arabidopsis CBF1 (CRT/DRE binding protein) has been shown to activate cor homologous genes at non-acclimating temperatures [77]. The CBF1 cDNA when introduced into tomato (Solanum lycopersicum) under the control of a CaMV35S promoter improved tolerance to chilling, drought and salt stress but exhibited dwarf phenotype and reduction in fruit set and seed number per fruit [11].
The expression of related cold shock proteins (CSPs) from bacteria, CspA from Escherichia coli and CspB from Bacillus subtilis, promotes stress adaptation in multiple plant species [78]. Transgenic rice plants expressing CspA and CspB manifest improved stress tolerance for a number of abiotic stresses, including cold, heat, and water deficits. It has long been established that changes in gene expression occur upon exposure to cold acclimation. Number of COR genes isolated from Arabidopsis that encode polypeptides thought to have protective roles against dehydration. Expression profile experiments in Arabidopsis demonstrated that extensive changes in gene expression occur during cold acclimation and that a substantial number of the genes that are up-regulated by the cold response are involved in metabo-lism. Hajela et al. [79] reported that a set of genes that encode a related family of cold-regulated (COR) proteins, which are massively induced during cold acclimation [80]. These COR genes were used by different researchers to identify a family of Arabidopsis transcription factors known as either C-repeat binding factors (CBF) (CBF1, CBF2 and CBF3) or dehydration responsive element-binding factors (DREB) (DREB1B, DREB1C and DREB1A). In Arabidopsis (A. thaliana) and rice, the CBF/DREB1-dependent cold response pathway has been shown to play a predominant role in freezing tolerance through the process of cold acclimation. These CBFs/DREBs are upstream transcription factors that bind to promotor cis element CRT/DRE and activate the expression of these cold responsive genes [71]. Overexpression of CBF1/DREB1b and CBF3/DREB1a enhances cold tolerance by inducing COR (cold regulated) genes [81,76,75,5]. Furthermore, its overexpression causes many biochemical changes, such as the accumulation of sugar and proline [5]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Thus, the CBF/DREB1 genes are thought to be activators that integrate several components of the cold acclimation response by which plants increase their tolerance to low temperatures after exposure to nonfreezing conditions [5]. Several stress induced COR genes such as rd29A, COR15A, kin1 and COR6.6 are triggered in response to cold treatment, ABA and water deficit stress [4].
There have been numerous efforts in enhancing tolerance towards multiple stresses such as cold, drought and salt stress in crops other than the model plants like Arabidopsis, tobacco and alfalfa. An increased tolerance to freezing and drought in Arabidopsis was achieved by overexpressing CBF4, a close CBF/ DREB1 homolog whose expression is rapidly induced during drought stress and by ABA treatment, but not by cold [82]. Subsequently, the overexpression of DREB1A has been shown to improve the drought-and lowtemperature stress tolerance in tobacco, wheat and groundnut [83,84]. The use of stress inducible rd29A promoter minimized the negative effects on plant growth in these crop species. Further, Gilmour et al. [80] have shown that ectopic transgenic overexpression of CBF1/DREB1B, CBF2/ DREB1C or CBF3/DREB1A in Arabidopsis activates a suite of CBF/DREB target genes at warm temperatures and results in increased freezing and drought tolerance. CBF transcripts begin accumulating within 15 minutes of plants being exposed to low temperature strongly suggests that the low temperature "Thermometer" and "Signal Transducer" are present at warm non-inducing temperatures. Two cDNA clones that encode DRE-binding proteins, DREB1A/CBF3 and DREB2A, have been isolated by using yeast one-hybrid screening. cDNA clones encoding two DREB1A homologs (named DREB1B/CBF1 and DREB1C/CBF2) and one DREB2A homolog (DREB2B) were also isolated and Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its homolog was induced by dehydration. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as transacting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.
However, Agarwal et al. [85] showed that CBFs are negatively regulated by an upstream transcription factor, ThpI Thermal hysteresis proteins (Anti freeze protein) Transcription factor Arabidopsis Enhanced low temperature tolerance in transgenic plants was observed by changes of electrolyte leakage activity, malonyldialdehyde and proline contents [133] CBF1 CRT/DRE binding factor 1 Transcription factor Solanum Lycopersicum Detection of higher activity of superoxide dismutase (SOD), higher non-photochemical quenching (NPQ), and lower malondialdehyde (MDA) content in transgenic tomato leaves suggest that CBF1 protein plays an important role in protection of PSII and PSI during low temperature stress at low irradiance [108] MYB15 (an R2R3-MYB family protein) in Arabidopsis. MYB15 is expressed even in the absence of cold stress, and MYB15 can bind to MYB recognition elements (MYBRS) in the promoters of CBFs. MYB15 mutant plants show enhanced expression of CBFs during cold acclimation and enhanced freezing tolerance, whereas, transgenic Arabidopsis overexpressing MYB15 showed a decreased expression of CBFs and a reduction in freezing tolerance. Thus, MYB15 is an upstream transcription factor that negatively regulates the expression of CBFs.
Transcriptome analysis of ZAT12-overexpressing Arabidopsis revealed that the ZAT12 regulon consists of at least 24 cold standard set (COS) genes, of which nine are coldinduced and 15 are cold-repressed genes [55]. Constitutive overexpression of Arabidopsis DREB1A improved drought and low-temperature stress tolerance in tobacco, and regulation of transgene expression via the stress-inducible RD29A promoter minimized the negative effects on plant growth [83]. Similarly, the Arabidopsis DREB1A gene was placed under control of the RD29A promoter and transferred via biolistic transformation into bread wheat [84]. However, constitutive overexpression of the CBF genes using the cauliflower mosaic virus 35S promoter can result in undesirable agronomic traits. In Arabidopsis, CBF overexpression can cause a ''stunted'' growth phenotype, a decrease in seed yield and a delay in flowering [81,5]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Because many aspects of cold adaptation process are under transcriptional control, many transcription regulatory factors were chosen, hence, genetic engineering for introgression of such genes that are known to be involved in stress response and putative tolerance, might prove to be a faster track towards improving crop varieties for enhanced cold tolerance.
Low-temperature limitations have been overcome by the identification of cold-tolerant genes for applications in genetically transformed crops. In transgenic tobacco (Nicotiana tabacum), chilling tolerance at 1 ºC for 7 d was achieved by the over expression of a gene encoding chloroplast w3 fatty acid desaturase [65]. Furthermore, tolerance at 1 ºC for 11 d was conferred using a gene encoding a non-specific cyanobacterial desaturase, and the resultant transgenic tobacco plants showed a reduction in saturated fatty acid content in membrane lipids [66]. The over expression of glycerol-3phosphate acyl transferase altered the unsaturation of fatty acids and conferred chilling tolerance in transgenic plants [67][68][69]. Hence, modifications in lipid composition that stabilize cell membranes and prevent cellular leakage lead to cold tolerance.
Transgenic technology has opened up many exciting possibilities to improve cold stress in plants by introduction or removal of gene or genes that regulate a specific trait [70]. It also offers uncommon opportunities for improvement in genetic potential of plants in the form of development of spe-cific crop varieties that are more resistant to biotic and cold stresses with enhanced nutritional level.
During last two decades, advancement in plant biotechnology has led to the identification and isolation of a number of transcription factor(s) related to cold stress tolerance. A good number of genes which have been identified in different studies ( Table 1) raise the question of exactly which genes are most central to increasing cold/ freezing tolerance. The genes selected for transformation should be involved in encoding enzymes that are required for the biosynthesis of various osmoprotectants. Other classes of genes that selected for transformation include those that encoded enzymes for modifying membrane lipids, LEA protein, and detoxification enzymes. In these studies, either a single gene for a protective protein or an enzyme was overexpressed under the control of the constitutive 35S cauliflower mosaic virus (CaMV) promoter in transgenic plants, although several genes have been shown to function in environmental stress tolerance and response [56]. The genes encoding protein factors that regulate gene expression and signal transduction, that function in stress responses may be useful for improving the cold tolerance of plants by gene transfer as they can regulate many stress-inducible genes involved in cold stress tolerance.
The CBF genes represent one of the most significant discoveries in the field of low temperature adaptation and signal transduction. All important crops and few vegetables species have contained this gene [11]. Various low temperatureinducible genes have been isolated from plants that appear to be involved in tolerance to cold stress [71,72], and the expression of some of them is regulated by C-repeat binding factor/dehydration-responsive element binding (CBF/ DREB1) transcription factors. Three CBF/DREB1 genes (CBF3/DREB1a, CBF1/DREB1b, and CBF2/DREB1c) belonging to the AP2/DREBP family of DNA-binding proteins have been identified in Arabidopsis [73,74,75]. Transgenic Arabidopsis plants constitutively over-expressing a cold inducible transcription factor (CBF1; CRT/DRE binding protein) showed tolerance to freezing without any negative effect on the development and growth characteristics [76]. Overexpression of Arabidopsis CBF1 (CRT/DRE binding protein) has been shown to activate cor homologous genes at non-acclimating temperatures [77]. The CBF1 cDNA when introduced into tomato (Solanum lycopersicum) under the control of a CaMV35S promoter improved tolerance to chilling, drought and salt stress but exhibited dwarf phenotype and reduction in fruit set and seed number per fruit [11].
The expression of related cold shock proteins (CSPs) from bacteria, CspA from Escherichia coli and CspB from Bacillus subtilis, promotes stress adaptation in multiple plant species [78]. Transgenic rice plants expressing CspA and CspB manifest improved stress tolerance for a number of abiotic stresses, including cold, heat, and water deficits. It has long been established that changes in gene expression occur upon exposure to cold acclimation. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. The second group contains protein factors involved in regulation of signal transduction and gene expression that probably function during stress response [59]. This group includes various transcription factors that regulate different stress-inducible genes collectively or separately, and may constitute gene networks. Seki et al. [59] reported that some of these regulatory pathways are also involved in drought-, cold-, or high-salinity stress responses. Though, the clear cut functions of most of these genes are not fully understood. Functional analysis of these stressinducible transcription factors will provide precise information on the complex regulatory gene networks that are involved in responses to drought, cold, and high-salinity stresses [60,25]. Some of these stress-inducible regulatory genes that encode proteins (transcription factors) have been overexpressed in transgenic plants and generate stresstolerant phenotypes in them [61,62].
COLD TOLERANCE USING TRANSGENIC AP-PROACHES
When a plant is subjected to abiotic stress, a number of genes are turned on, resulting in increased levels of several metabolites and proteins, some of which may be responsible for conferring a certain degree of protection to these stresses.
A key to progress towards breeding better crops under stress has been to understand the changes in cellular, biochemical and molecular machinery that occur in response to stress. A key to progress towards breeding better crops under stress has been to understand the changes in cellular, biochemical and molecular machinery that occur in response to stress which in turn provides new tools and strategies to improve the environmental stress tolerance of crops. Since freezing tolerance is a multigenic trait [63], transformation of a single functional gene appears to have a limited effect on crop freezing tolerance [64]. Because many aspects of cold adaptation process are under transcriptional control, many transcription regulatory factors were chosen, hence, genetic engineering for introgression of such genes that are known to be involved in stress response and putative tolerance, might prove to be a faster track towards improving crop varieties for enhanced cold tolerance.
Low-temperature limitations have been overcome by the identification of cold-tolerant genes for applications in genetically transformed crops. In transgenic tobacco (Nicotiana tabacum), chilling tolerance at 1 ºC for 7 d was achieved by the over expression of a gene encoding chloroplast w3 fatty acid desaturase [65]. Furthermore, tolerance at 1 ºC for 11 d was conferred using a gene encoding a non-specific cyanobacterial desaturase, and the resultant transgenic tobacco plants showed a reduction in saturated fatty acid content in membrane lipids [66]. The over expression of glycerol-3phosphate acyl transferase altered the unsaturation of fatty acids and conferred chilling tolerance in transgenic plants [67][68][69]. Hence, modifications in lipid composition that stabilize cell membranes and prevent cellular leakage lead to cold tolerance.
Transgenic technology has opened up many exciting possibilities to improve cold stress in plants by introduction or removal of gene or genes that regulate a specific trait [70]. It also offers uncommon opportunities for improvement in genetic potential of plants in the form of development of spe-cific crop varieties that are more resistant to biotic and cold stresses with enhanced nutritional level.
During last two decades, advancement in plant biotechnology has led to the identification and isolation of a number of transcription factor(s) related to cold stress tolerance. A good number of genes which have been identified in different studies ( Table 1) raise the question of exactly which genes are most central to increasing cold/ freezing tolerance. The genes selected for transformation should be involved in encoding enzymes that are required for the biosynthesis of various osmoprotectants. Other classes of genes that selected for transformation include those that encoded enzymes for modifying membrane lipids, LEA protein, and detoxification enzymes. In these studies, either a single gene for a protective protein or an enzyme was overexpressed under the control of the constitutive 35S cauliflower mosaic virus (CaMV) promoter in transgenic plants, although several genes have been shown to function in environmental stress tolerance and response [56]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures.
In the last decade, extensive research efforts have been undertaken to identify and characterize cold-responsive (COR) genes and a number of homologous components of the Arabidopsis CBF cold response pathway in many plants have been found [6]. Many of these putative orthologs have been structured, analyzed and functionally tested. The expression patterns of the CBFs and CORs in response to low temperature are similar in a variety of plants species, involving rapid cold-induced expression of the CBFs followed by expression of CBF-targeted genes that increase freezing tolerance. Moreover, constitutive overexpression of the Arabidopsis CBF genes in other plants resulted in increased freezing tolerance that have been successfully used to engineer cold stress tolerance in several crop species [6].
Transgenic attempts with other structural genes have also been made with fair degree of success. Genetically engineered tobacco plants over-expressing chloroplast glycerol-3-phosphate acyltransferase (GPAT) gene (involved in phosphatidyl glycerol fatty acid desaturation) from squash (Cucurbita maxima) and A. thaliana [86] showed an increase in the number of unsaturated fatty acids present in the plant cell wall, which enhance the cold tolerance to the plants during cold stress. Expression of a plant phosphatase (At PP2CA) in transgenic A. thaliana can accelerate the development of cold acclimation and increase freezing tolerance. It has also shown that transgenic plants expressing a constitutively active kinase NPK1, is more tolerant to chilling and other abiotic stresses [87]. Pennycooke et al. [88] reported that downregulating -Gal ( -Galactosidase) in transgenic petunia resulted in an increase in freezing tolerance suggesting that engineering raffinose metabolism by transformation with -Gal provides an additional method for improving the freezing tolerance of plants. The overexpression of genes encoding LEA proteins can improve the stress tolerance of transgenic plants. Expression of the citrus gene encoding a LEA protein, CuCOR19 increased the cold tolerance of transgenic tobacco [89]. Likewise, the freezing tolerance of Arabidopsis was increased by the ectopic expression of the wheat gene WCS19 [47], the Arabidopsis gene COR15A [64], and the co-expression of the genes RAB18 and COR47 and XERO2 and ERD10 [90]. The freezing tolerance of strawberry leaves was enhanced by expression of the wheat dehydrin gene WCOR410 [72]. On the other hand, the expression of two cold-induced LEA proteins from spinach (Kaye et al., 1998) [91] and three desiccation-induced LEA proteins from C. plantagineum [92] in tobacco did not induce any significant changes in the freezing or drought tolerance of the respective transgenic plants. This may indicate either that not all LEA proteins make a significant contribution to plant stress tolerance, or that they need a particular background to function in, as suggested for transgenic strawberry plants [72]. Kim et al. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures.
Forward genetic analysis in Arabidopsis identified two transcription factors, high expression of osmotically responsive genes, HOS9 and HOS10, which are required for basal freezing tolerance [103,104]. Similarly, microarray analysis led to the identification of the cold-stress-inducible AP2 family transcription factor gene related to ABI3/VP1 (RAV1) [52,55] that might regulate plant growth under cold stress. RAV1 is down regulated by epibrassinolide and transgenic Arabidopsis overexpressing RAV1 exhibits a retardation of lateral root and rosette-leaf development. Further, the importance of CBF-independent pathways is also supported by analysis of mutants that have increased freezing tolerance, for example, mutations in eskimo1 (ESK1), a protein of unknown function, result in constitutive freezing tolerance. The Eskimo1 mutation was first identified as a mutation conferring frost survival without an acclimation period [105]. Later on, Bouchabke-Coussa et al. [106] clearly showed that ES-KIMO1 mutants are more tolerant to freezing but only after acclimation. The genes that are affected by the ESK1 mutation are distinct from those of the CBF regulon [107]. Recently, a CBF 1 gene has been introduced into tomato that resulted in transgenic plants showing higher activity of superoxide dismutase (SOD), higher non-photochemical quenching (NPQ), and lower malondialdehyde (MDA) content . Thus suggesting that CBF1 protein plays an important role in protection of PSII and PSI during low temperature stress at low irradiance [108].
Aforementioned studies clearly demonstrate the complex and interactive relationships among different pathways regulated by cold acclimation. Hence, elucidation of the mechanism regulating cold-regulated genes is an important goal in achieving a full understanding of cold acclimation. With the advent of molecular genetics and biotechnology, it is now possible to genetically engineer plants to be more tolerant to low temperature. The technologies can be so developed/ modified that will significantly improve breeding efficiency, resulting in rapid and accurate incorporation of cold tolerant genes into crop plants.
PROSPECTS OF COLD TOLERANT GENES IN HILL AGRICULTURE
Cold is an environmental factor that limits the geographical distribution and growing season of many plant species, and it often adversely affects crop quality and productivity [71]. Most temperate plants can acquire tolerance to freezing temperature by a prior exposure to low non-freezing temperature, a process known as cold acclimatization [39,109,110]. Plants of tropical and subtropical origins are sensitive to chilling temperature (0 o C-10 o C) and are incapable of cold acclimation. Genes induced by stress can be roughly classified into two groups: genes coding for regulatory proteins, mainly transcription factors, and genes encoding proteins involved directly in response mechanisms; genes from both classes are of interest. Variations in the expression of regulators could lead to a protective status before the emergence of stress and have multiple effects. Genes involved in protection or repair mechanisms could be new targets for the improvement of plant plasticity and adaptive responses to stress [111]. The unraveling of general stress responses in the model species Arabidopsis thaliana helped to identify potential targets for plant breeding. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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This below document has 2 sentences that end with 'improved tolerance to cold stress'. It has approximately 598 words, 25 sentences, and 5 paragraph(s).
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. The conventional breeding approaches are limited by the complexity of stress tolerance traits, low genetic variance of yield components under stress condition and lack of efficient selection criteria. It is important, therefore, to look for alternative strategies to develop cold stress tolerant crops.
Biotechnology offers new strategies that can be used to develop transgenic crop plants with improved tolerance to cold stress. Rapid advance in recombinant DNA technology and development of precise and efficient gene transfer protocols have resulted in efficient transformation and generation of transgenic lines in a number of crop species [8][9][10] (Fig. 1). A number of genes have been isolated and characterized that are responsive to freezing stress. Many studies have suggested that cold regulated gene expression is critical in plants for both chilling tolerance [11] and cold acclimation [12,13]. Advent of molecular tools has made it possible to select directly at the gene label without waiting for the phenotype notype to show up. Transgenic approach is being pursued actively throughout the world to improve traits including tolerance to biotic and abiotic stresses in a number of crops [14]. As compared to other stresses, plant responses to cold stress are complex, so the prospects of improving cold tolerance in crops seem not to be very bright. Despite this, efforts have been made during the last two decades to generate transgenic lines of different crops, which have shown improved tolerance to cold stress. Therefore, it is important to use most appropriate tools that help in reaching the goals. The genotype designed should be better than the available ones and must reach the farmers. An attempt has been made in this article to review the various mechanisms and genes involved in cold acclimatization and the possibilities where transgenic technology has been explored for breeding cold tolerance in crop plants.
MORPHO-PHYSIOLOGICAL BASIS OF COLD TOLERANCE
A large number of studies have evaluated different plant species tolerant to different stresses such as drought, salinity and cold. However, less detail is given with regard to the methods used to evaluate the stress response; these studies may bring about some misleading conclusions from an agronomic or physiology perspective [15]. This is particularly important, in order to closely mimic the life span of most crops under cycles of stress, rather than short exposure to very severe stresses; although we agree that short exposures to stress are certainly adequate if the purpose is to assess gene expression only. In this section, we focus on the agronomic/physiological perspective and do not mean to challenge the quality of the work done to assess gene expression. Our intention is to try to reconcile both approaches (agronomic and molecular) toward a common focus: breeding cold tolerance. Though precise details about the protocols used to evaluate the performance of plants to any given stress are very essential to assess the performance of materials.
The temperate and cool regions are those where altitudes ranged from 1600-2500 m amsl (above mean sea level) and temperature during crop growth period ranged from 5-20 0 C [16]. In temperate regions, low temperature is the primary abiotic stress which limits the crop productivity. The low temperature at seedling and reproductive stages is the major problem, results in slow establishment and low seed set which leads to poor yield of the crop [15]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Key to the tolerance of plants to abiotic stresses is a complex network of transcription factors and other regulatory genes that control multiple defense enzymes, proteins and pathways [51]. The discovery of gene expression change during cold acclimation was the starting of exploration of antifreezing molecular mechanisms. In this context, Zhao et al. [25] reported that gene expression profiling using DNA chips indicated that large numbers of genes were differentially expressed under cold stress. They identified 242 unique genes that are expressed differentially between cold sensitive and cold tolerant rice. These genes are involved in processes such as senescence, cell death, male sterility and plant hormone response. Similarly, global transcript profiling analyses indicated that > 10% of genes in the Arabidopsis genome were regulated during cold acclimation [52][53][54][55]. Transcriptome analysis using microarray technology is a powerful technique, which has proven very useful for discovering many stress-inducible genes involved in stress response and tolerance [25,[55][56][57]. Genes involved in stress signal sensing and a cascade of stress-signaling in A. thaliana has been of recent research interest. Components of the same signal transduction pathway may also be shared by various stress factors such as drought, salt and cold. Although there are multiple pathways of signal-transduction systems operating at the cellular level for gene regulation.
In past, it has been reported that genes induced during stress conditions function not only in protecting cells from stress by producing important metabolic proteins, but also in regulating genes for signal transduction in the stress response [52]. Several stress induced cor genes such as rd29A, cor15A, kin1 and cor6.6 are triggered in response to cold treatment, ABA and water deficit stress in the early stages of the osmotic stress response.
Several stress induced cor genes such as rd29A, cor15A, kin1 and cor6.6 are triggered in response to cold treatment, ABA and water deficit stress in the early stages of the osmotic stress response. Similarly, a cis-acting element, dehydration responsive element (DRE) identified in A. thaliana, is also involved in ABA-independent gene expression under drought, low temperature and high salt stress conditions in many dehydration responsive genes like rd29A that are responsible for dehydration and cold-induced gene expression. Thus, clearly, the overexpression of some droughtresponsive transcription factors can lead to the expression of downstream genes and the enhancement of abiotic stress tolerance in plants. Thus, these gene products are classified into two groups [53,54]. The first group of proteins that probably function in stress tolerance includes chaperones, LEA proteins, osmotin, antifreeze proteins, mRNA-binding proteins, some key enzymes for osmolyte biosynthesis (like proline, water channel proteins, sugar and proline transporters, detoxification enzymes), enzymes for fatty acid metabolism (proteinase inhibitors, ferritin) and lipid-transfer proteins [58]. It has been reported that some of these stressinducible genes specially encoding proteins (such as enzymes for osmolyte biosynthesis, LEA proteins and detoxification enzymes) have been overexpressed in transgenic plants and produce stress-tolerant phenotypes in the transgenic plants [51,56]. These results indicate that the gene products of the stress-inducible genes really function in stress tolerance. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. This information is necessary for our understanding of the complex network of molecular changes that are important for chilling and freezing tolerance in crop plants. A well focused approach combining the molecular, physiological and metabolic aspects of cold stress tolerance is required for bridging the knowledge gaps between short-and long-term effects of the genes and their products, and between the molecular or cellular expression of the genes and the whole plant phenotype under stress. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. STIFDB (available at [URL]. ) is a database of stress-related genes, which are upregulated in abiotic stress-related microarray experiments. STIFDB provides a platform to understand the stress-regulome of abiotic stress responsive genes in plants. STIFDB will be a highly useful resource for a researcher working on abiotic stress responses in plants [114].
Low temperature stress is a major environmental factor that not only limits where crops can be grown but also reduces yields depending on the weather in a particular growing season. In addition to exceptionally stressful years that cause significant yield reductions, less extreme stress almost certainly causes smaller losses over large areas to produce comparable yield reductions every year. Even in cases when freezing stress does not result in yield loses; it often results in crop quality reduction. Each year, worldwide losses in crop production due to low temperature damage amount to approximately $ 2 Billion. Some of the major losses include the 1995 early fall frosts in the US which caused losses of over $1 billion to corn and soybeans. The occasional freezes in Florida have shifted the citrus belt further south, and California sustained $650M of damage in 1998 to the citrus crop due to a winter freeze. The inherent cold hardiness of the crop determines in which agricultural areas it can be grown.
Crops that are more resistant to freezing stress would allow some geographical regions to grow more profitable and productive crops with less environmental risks. However despite continued efforts, traditional breeding has had only limited success in imparting crop plants with better freezing tolerance due to very little was known about the mechanisms that regulate chilling and freezing tolerance. With the advent of molecular genetics and biotechnology, it is now possible to genetically engineer plants to be more tolerant to many environmental adversities, including low temperature. Molecular studies have shown that several genes with various functions are induced by environmental stresses such as drought, high-salinity and low temperature in plants. Most of the dehydration responsive genes are induced by the plant hormone abscisic acid (ABA), but others are not. Expression analyses of dehydration-responsive genes have provided at least four independent regulatory systems (regulons) for gene expression in a model plant Arabidopsis thaliana. The cis-acting elements in the promoters of some genes that have a typical stress-inducible expression profile and the transcription factors that affect the expression of these genes have been analyzed [115].
FUTURE OUTLOOK AND CONCLUSION
The development of genetically engineered plants by the introduction and/or overexpression of selected genes seems to be a viable option to hasten the breeding of ''improved'' plants. Intuitively, genetic engineering would be a faster way to insert beneficial genes than through conventional or molecular breeding. Also, it would be the only option when genes of interest originate from cross barrier species, distant relatives, or from non-plant sources. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Therefore, it is important to use most appropriate tools that help in reaching the goals. The genotype designed should be better than the available ones and must reach the farmers. An attempt has been made in this article to review the various mechanisms and genes involved in cold acclimatization and the possibilities where transgenic technology has been explored for breeding cold tolerance in crop plants.
MORPHO-PHYSIOLOGICAL BASIS OF COLD TOLERANCE
A large number of studies have evaluated different plant species tolerant to different stresses such as drought, salinity and cold. However, less detail is given with regard to the methods used to evaluate the stress response; these studies may bring about some misleading conclusions from an agronomic or physiology perspective [15]. This is particularly important, in order to closely mimic the life span of most crops under cycles of stress, rather than short exposure to very severe stresses; although we agree that short exposures to stress are certainly adequate if the purpose is to assess gene expression only. In this section, we focus on the agronomic/physiological perspective and do not mean to challenge the quality of the work done to assess gene expression. Our intention is to try to reconcile both approaches (agronomic and molecular) toward a common focus: breeding cold tolerance. Though precise details about the protocols used to evaluate the performance of plants to any given stress are very essential to assess the performance of materials.
The temperate and cool regions are those where altitudes ranged from 1600-2500 m amsl (above mean sea level) and temperature during crop growth period ranged from 5-20 0 C [16]. In temperate regions, low temperature is the primary abiotic stress which limits the crop productivity. The low temperature at seedling and reproductive stages is the major problem, results in slow establishment and low seed set which leads to poor yield of the crop [15]. The low temperature limits the crop productivity when temperatures remain above freezing that is > 0 o C, it is called as chilling stress. Chilling sensitive cultivars are typically tropical genotypes. There is wide range of cold stress in temperate areas differing in both timing and intensity of low temperature. Yield losses are more severe when cold stress occurs during reproductive stage/ anthesis in rice which lead to high spikelet sterility [17]. Ability of crop genotypes / lines to survive / perform better under low temperature than other genotypes is called as cold tolerance. Ordinarily, it is the consequence of cold hardening that is an earlier exposure to a low temperature for a specific period as a result of which chilling tolerance of the concerned plants increases. Cold tolerance involves increased chlorophyll accumulation, reduced sensitivity of photosynthesis, improved germination, pollen fertility and seed set which are desirable as:
INCREASED CHLOROPHYLL ACCUMULATION
Low temperature inhibits chlorophyll accumulations in actively growing leaves. In rice, cold tolerant lines, for example, japonica accumulates more chlorophyll under cold stress than do cold sensitive line, for example of indica rice [18]. Rasolofo [19] evaluated 181 accessions to identify donor and outstanding cold tolerant lines using leaf discolouration score (1-3) and found 19 remained green (dark) after 10 days in the 12 o C cold water tank. Sanghera et al. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures.
Aforementioned studies clearly demonstrate the complex and interactive relationships among different pathways regulated by cold acclimation. Hence, elucidation of the mechanism regulating cold-regulated genes is an important goal in achieving a full understanding of cold acclimation. With the advent of molecular genetics and biotechnology, it is now possible to genetically engineer plants to be more tolerant to low temperature. The technologies can be so developed/ modified that will significantly improve breeding efficiency, resulting in rapid and accurate incorporation of cold tolerant genes into crop plants.
PROSPECTS OF COLD TOLERANT GENES IN HILL AGRICULTURE
Cold is an environmental factor that limits the geographical distribution and growing season of many plant species, and it often adversely affects crop quality and productivity [71]. Most temperate plants can acquire tolerance to freezing temperature by a prior exposure to low non-freezing temperature, a process known as cold acclimatization [39,109,110]. Plants of tropical and subtropical origins are sensitive to chilling temperature (0 o C-10 o C) and are incapable of cold acclimation. Genes induced by stress can be roughly classified into two groups: genes coding for regulatory proteins, mainly transcription factors, and genes encoding proteins involved directly in response mechanisms; genes from both classes are of interest. Variations in the expression of regulators could lead to a protective status before the emergence of stress and have multiple effects. Genes involved in protection or repair mechanisms could be new targets for the improvement of plant plasticity and adaptive responses to stress [111]. The unraveling of general stress responses in the model species Arabidopsis thaliana helped to identify potential targets for plant breeding. Arabidopsis genes involved in tolerance to abiotic stress were transferred, by genetic engineering, to many crops and tolerance was successfully conferred in the field, despite the complexity of plant responses to environmental stress [11,61]. Thus, finding new key genes responsible for abiotic stress tolerance phenotypes is of great importance not only for a better understanding of stress responses, but also for promising future crop improvement.
Many studies have suggested that cold regulated gene expression is critical in plants for both chilling tolerance [47,11] and cold acclimation [12,71,13,112]. Cold responsive genes encode a diverse array of proteins such as enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids and antioxidants: molecular chaperones, antifreeze proteins, and others with a presumed function in tolerance to dehydration caused by freezing [113,39,71]). The change in the gene expression occur in plant during cold acclimatization a developmental process that results in increased tolerance [28]. Since then, it has repeatedly been speculated that certain COR (cold regulated) genes might have role in freezing tolerance. To test this notion investigators have turned to isolating the characterizing genes that are expressed in response to low temperature. These efforts have led to the identification of a number of genes such as the COR 15a KINI, LTI 78, fad 7etc. of A. thaliana. The generic trends of the genes and transcription factors are available in STIFDB (Stress responsive Transcription Factor Database). We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Applications of genomic approaches and gene knockout strategies are progressing to accelerate efforts to assess systematically and understand complex quantitative traits such as acquired tolerance to temperature extremes. By using genetic and molecular approaches, a number of relevant genes have been identified and new information continually emerges to enrich the CBF cold-responsive pathway. Thus, the CBF/DREB1 genes are thought to be activators that integrate several components of the cold acclimation response by which plants increase their tolerance to low temperatures after exposure to non-freezing conditions. The DREB1/CBF genes have been successfully used to improve abiotic stress tolerance in a number of different crop plants. Studies on the other transcription factors associated with stress response are in progress.
However, the results of the transcriptome study demonstrate the highly complex nature of plant adaptation to low temperature. To overcome this problem a transgenic approach to promoting cold tolerance has been widely adopted, with some success. For example, increasing the accumulation of two compatible solutes, that is, glycinebetaine and trehalose, in transgenic rice by overexpressing either E. coli choline oxidase, or trehalose-6-phosphate synthase fused to trehalose-6-phosphate phosphatase, enhanced tolerance to both salt and cold. In fact a large number of genes identified in different studies have currently annotated with ''unknown function'' and involve new genes and new pathways indicates that our knowledge of the transcriptional control of the low temperature response is limited, and the regulation of these transcriptional responses is far more complex than pre-viously believed. Information on the low-temperature transcriptome, proteome and metabolome is expected to continue to increase in the near future. This information is necessary for our understanding of the complex network of molecular changes that are important for chilling and freezing tolerance in crop plants. A well focused approach combining the molecular, physiological and metabolic aspects of cold stress tolerance is required for bridging the knowledge gaps between short-and long-term effects of the genes and their products, and between the molecular or cellular expression of the genes and the whole plant phenotype under stress. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. And without pollen, pollination cannot occur, thereby, no grain is produced. CSIRO has found that all the ingredients for starch are present but they are not getting into the pollen grain where they are needed. A cell layer surrounding the pollen, called the 'tapetum', is responsible for feeding the pollen with sugar. The tapetum is only active for 1-2 days -so if a cold snap occurs at this time, then there is no further chance for pollen growth. But the sugar cannot freely move into the tapetum and pass through it to the pollen. Instead the sugar has to be broken down then transported in bits to the pollen. 'Invertase' is the catalyst that helps in breakdown of the sugar molecule to transport it into the tapetum before it is transported to the pollen [23]. Quantities of invertase are decreased in conventional rice when it is exposed to cold temperatures, but they remain at normal levels in a cold tolerant variety when it experiences cold. By comparing a cold tolerant strain of rice with conventional rice, CSIRO has found that the gene responsible for invertase looks exactly the same in the cold tolerant variety as it does in conventional rice. So the invertase gene itself does not make the rice plant cold tolerant -but instead a mechanism that regulates the invertase gene is different. Early research indicates that the invertase gene is regulated by the hormone abscisic acid (ABA). Oliver et al. [24] has experimented with injecting plants with ABA -the resulting rice plants are sterile, just like that if they experienced a cold snap. Also, ABA levels increase when conventional rice is exposed to cold, but they remain the same in the cold tolerant variety. Recent studies have indicated that the difference between cold-sensitive and tolerant rice is due to a different ability to control ABA levels [25]. It has also been shown that this mechanism may require interactions with other plant hormones like auxins [26]. Further, Zhao et al. [25] also reported that low temperature turns off the genes responsible for sugar transport into the pollen grains and therefore starch cannot be produced in the pollen in cold conditions. Cold did not cause repression of sugar delivery in cold tolerant Chinese rice and fertile pollen was still produced following cold treatment. The sugar metabolism genes also continued to function normally during cold treatment of cold tolerant rice. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. [44] engineered tobacco with ring zinc finger protein (RDCPt) from hot pepper (Capsicum annuum) and reported that expression of this gene resulted in improved cold tolerance in transgenic plants as compared to wild type. Dai et al. [93] reported that overexpression of OsMYB3R-2 in transgenic Arabidopsis increased tolerance to freezing when exposed to -8 ºC for 10 h. They found that survival after 6 d at normal conditions was 26.8% for the wild-type and 84.5% for transgenic lines. Phenotypically, most transgenic seedlings were green and could regrow as compared with the wild type; whereas most wild-type seedlings became white and did not regrow after removed to normal conditions. The survival percentage under different low temperatures also showed dramatic difference between the transgenic plants and wild-type plants.
Pramanik and Imai [94] reported that TPP (trehalose-6phosphate phosphatase) genes expressed in rice and their expression is induced by cold. Trehalose accumulates rapidly and transiently, which follows the transient induction of TPP activity, in rice tissues during chilling stress [94]. Overexpression of TPS (trehalose-6-phosphate synthase) and TPP genes enhanced the accumulation of trehalose and tolerance to cold stress in transgenic tobacco and rice [95][96][97][98]. However, the regulatory mechanism of TPPs by cold or other stresses is unclear. In another study, Su et al. [99] observed that MYBS3 plays a critical role in cold adaptation in rice and necessary for enhancing cold tolerance. They reported that transgenic rice constitutively overexpressing MYBS3 tolerated 4 ºC for at least 1 week and exhibited no yield penalty in normal field conditions. Based on previous studies it has been established that the CBF cold responsive pathway is an integral component of the cold acclimation response [100,63,6]. However, the transcriptome data showed that additional cold-regulatory pathways also exist [52,53,101]. Transcriptome comparisons indicated that only 12% of the cold-responsive genes are certain members of the CBF regulon. Moreover, at least 28% of the cold-responsive genes were not regulated by the CBF transcription factors, including 15 encoding known or putative transcription factors, indicating that these coldresponsive genes are members of different low-temperature regulons [55].
When overexpressed in Arabidopsis and tobacco, the soybean gene SCOF-1 (encodes a zinc-finger protein) can activate COR gene expression and increase freezing tolerance in non-acclimated transgenic plants, although the SCOF-1 protein does not directly bind to either the DRE/CRT or the ABRE elements [102]. SCOF-1 interacts with another G-box binding bZIP protein, SGBF-1. SGBF-1 can activate ABRE-driven reporter gene expression in Arabidopsis leaf protoplasts. Thus, SCOF-1 may regulate the activity of SGBF-1 as a transcription factor in inducing COR gene expression [102]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Ample genetic variation for cold tolerance is available in well adapted breeding population. Germplasm collected from high altitude and low temperature areas, cold tolerant mutant, somaclonal variants and wild species can be exploited for breeding improved cold tolerant genotypes in hilly areas [15].
MECHANISMS FOR UNDERSTANDING TOLER-ANCE TO COLD INJURY
Low temperature has a huge impact on the survival and geographical distribution of plants. It affects a range of cellular metabolisms in plant system depending on the intensity and duration of the stress. When exposed to low temperatures, plants respond with changes in their pattern of gene expression and protein products. Different studies have indicated that the membrane systems of the cell are the primary site of freezing injury in plants [2,27]. In addition, it is well established that freeze-induced membrane damage results primarily from the severe dehydration associated with freezing [27,28]. Many species of tropical or subtropical origin are injured or killed by nonfreezing low temperatures, and exhibit various symptoms of chilling injury such as chlorosis, necrosis, or growth retardation. In contrast, chillingtolerant species are able to grow at such cold temperatures.
However, multiple forms of membrane damage can occur as a consequence of freeze induced cellular dehydration including expansion-induced-lysis, lamellar-to-hexagonal-II phase transitions, and fracture jump lesions [28]. Thus, a key function of cold acclimation should be to stabilize membranes against freezing injury. Indeed, cold acclimation pre-vents expansion-induced-lyses and the formation of hexagonal II phase lipids in rye and other plants [28]. This ability to adapt has an impact on the distribution and survival of the plant, and on crop yields. Multiple mechanisms appear to be involved in this stabilization. The best documented are changes in lipid composition [28]. Secondly, temperature induced change in membrane fluidity is another consequences in plants during temperature stresses and might represent a potential site of perception and/or injury [29,30]. Adaptation of living cells to chilling temperatures is a function of alteration in the membrane lipid composition by increased fatty acid unsaturation. Genetically engineered tobacco plants over-expressing chloroplast glycerol-3phosphate acyltransferase (GPAT) gene (involved in phosphatidyl glycerol fatty acid desaturation) from squash (Cucurbita maxima) and A. thaliana showed an increase in the number of unsaturated fatty acids and a corresponding decrease in the chilling sensitivity. At low temperature, greater membrane lipid unsaturation appears to be crucial for optimum membrane function. An Arabidopsis fatty acid biosynthesis (FAB1) mutant with more saturated membranes showed decreased quantum efficiency of photosystem II (PSII), chlorophyll content and the amount of chloroplast glycerolipids after prolonged exposure to low temperature [31]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Number of COR genes isolated from Arabidopsis that encode polypeptides thought to have protective roles against dehydration. Expression profile experiments in Arabidopsis demonstrated that extensive changes in gene expression occur during cold acclimation and that a substantial number of the genes that are up-regulated by the cold response are involved in metabo-lism. Hajela et al. [79] reported that a set of genes that encode a related family of cold-regulated (COR) proteins, which are massively induced during cold acclimation [80]. These COR genes were used by different researchers to identify a family of Arabidopsis transcription factors known as either C-repeat binding factors (CBF) (CBF1, CBF2 and CBF3) or dehydration responsive element-binding factors (DREB) (DREB1B, DREB1C and DREB1A). In Arabidopsis (A. thaliana) and rice, the CBF/DREB1-dependent cold response pathway has been shown to play a predominant role in freezing tolerance through the process of cold acclimation. These CBFs/DREBs are upstream transcription factors that bind to promotor cis element CRT/DRE and activate the expression of these cold responsive genes [71]. Overexpression of CBF1/DREB1b and CBF3/DREB1a enhances cold tolerance by inducing COR (cold regulated) genes [81,76,75,5]. Furthermore, its overexpression causes many biochemical changes, such as the accumulation of sugar and proline [5]. Thus, the CBF/DREB1 genes are thought to be activators that integrate several components of the cold acclimation response by which plants increase their tolerance to low temperatures after exposure to nonfreezing conditions [5]. Several stress induced COR genes such as rd29A, COR15A, kin1 and COR6.6 are triggered in response to cold treatment, ABA and water deficit stress [4].
There have been numerous efforts in enhancing tolerance towards multiple stresses such as cold, drought and salt stress in crops other than the model plants like Arabidopsis, tobacco and alfalfa. An increased tolerance to freezing and drought in Arabidopsis was achieved by overexpressing CBF4, a close CBF/ DREB1 homolog whose expression is rapidly induced during drought stress and by ABA treatment, but not by cold [82]. Subsequently, the overexpression of DREB1A has been shown to improve the drought-and lowtemperature stress tolerance in tobacco, wheat and groundnut [83,84]. The use of stress inducible rd29A promoter minimized the negative effects on plant growth in these crop species. Further, Gilmour et al. [80] have shown that ectopic transgenic overexpression of CBF1/DREB1B, CBF2/ DREB1C or CBF3/DREB1A in Arabidopsis activates a suite of CBF/DREB target genes at warm temperatures and results in increased freezing and drought tolerance. CBF transcripts begin accumulating within 15 minutes of plants being exposed to low temperature strongly suggests that the low temperature "Thermometer" and "Signal Transducer" are present at warm non-inducing temperatures. Two cDNA clones that encode DRE-binding proteins, DREB1A/CBF3 and DREB2A, have been isolated by using yeast one-hybrid screening. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Some CSP-domain-containing proteins in plants are upregulated by cold stress, and might function as RNA chaperones in the regulation of translation [43,44]. A different cold-responsive nucleic-acid-binding protein, a zincfinger-containing glycine-rich RNA-binding protein from Arabidopsis designated atRZ-1a, is also upregulated by cold stress, and genetic analysis supports its function in freezing tolerance [45]. Compared to other organisms, plants have the largest number of DEAD-box RNA helicase genes [46]. One of these helicases, which is encoded by the Arabidopsis low expression of osmotically responsive genes4 (LOS4) gene, is essential for plant tolerance of chilling and freezing stress [47]. LOS4 is required for efficient export of RNA from the nucleus to the cytoplasm [48]. The Arabidopsis nucleoporin AtNUP160 suppressor of auxin resistance1 (SAR1) also controls RNA export, and is crucial for chilling and freezing tolerance [49]. Both LOS4 and AtNUP160 proteins are enriched at the nuclear rim [47,49]. Defects in the nucleocytoplasmic transport of RNA seem to affect cold tolerance preferentially, because the LOS4 and AtNUP160 mutant plants do not have severe growth or developmental phenotypes, nor are they strongly altered in the tolerance of other abiotic stresses.
KEY PLAYERS INVOLVED IN COLD RESPONSIVE PATHWAYS
Cold tolerance is the result of complex physiological mechanisms involving many cell and plant traits. Earlier studies have shown that the genetic control of cold tolerance is complex and can be regarded as polygenic [50] and the mechanism of how these genes controlled cold tolerance is still not fully clear. Therefore, the foundation for a better molecular and genetic understanding of the cold responsive pathways will improve our knowledge and pave the way for the development of improved methodologies for cold tolerance screening. Key to the tolerance of plants to abiotic stresses is a complex network of transcription factors and other regulatory genes that control multiple defense enzymes, proteins and pathways [51]. The discovery of gene expression change during cold acclimation was the starting of exploration of antifreezing molecular mechanisms. In this context, Zhao et al. [25] reported that gene expression profiling using DNA chips indicated that large numbers of genes were differentially expressed under cold stress. They identified 242 unique genes that are expressed differentially between cold sensitive and cold tolerant rice. These genes are involved in processes such as senescence, cell death, male sterility and plant hormone response. Similarly, global transcript profiling analyses indicated that > 10% of genes in the Arabidopsis genome were regulated during cold acclimation [52][53][54][55]. Transcriptome analysis using microarray technology is a powerful technique, which has proven very useful for discovering many stress-inducible genes involved in stress response and tolerance [25,[55][56][57]. Genes involved in stress signal sensing and a cascade of stress-signaling in A. thaliana has been of recent research interest. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. [17] found 18 cold tolerance IRCTN rice genotypes based on dark green colour and high spikelet fertility (>90%) under temperate conditions.
REDUCED SENSITIVITY OF PHOTOSYNTHESIS
Chloroplast and photosynthesis is major site of cold injury. Tolerance in these aspects is expressed in native vegetation adapted to growing under cool conditions. The reduced sensitivity of photosynthesis to cold has been observed in maize inbreds adapted to low temperature which is partly related to specific enzymes of the process [20].
Improved Pollen Fertility and Seed Set
Cold tolerance at reproductive stage is expressed as improved seed set and pollen fertility. It is largely a function of floral structure and function under stress. Lia et al. [22] reported plant cold tolerance in rice is associated with anther size, number of pollen grain, diameter of fertile pollen grains at booting stage. However, Sanghera et al. [17] reported that cold tolerance is associated with high spikelet fertility (>90%) and well panicle exsertion under temperate conditions.
Cold snaps cause a reaction in the plant that prevents sugar getting to the pollen. Without sugar there is no starch build-up which provides energy for pollen germination. And without pollen, pollination cannot occur, thereby, no grain is produced. CSIRO has found that all the ingredients for starch are present but they are not getting into the pollen grain where they are needed. A cell layer surrounding the pollen, called the 'tapetum', is responsible for feeding the pollen with sugar. The tapetum is only active for 1-2 days -so if a cold snap occurs at this time, then there is no further chance for pollen growth. But the sugar cannot freely move into the tapetum and pass through it to the pollen. Instead the sugar has to be broken down then transported in bits to the pollen. 'Invertase' is the catalyst that helps in breakdown of the sugar molecule to transport it into the tapetum before it is transported to the pollen [23]. Quantities of invertase are decreased in conventional rice when it is exposed to cold temperatures, but they remain at normal levels in a cold tolerant variety when it experiences cold. By comparing a cold tolerant strain of rice with conventional rice, CSIRO has found that the gene responsible for invertase looks exactly the same in the cold tolerant variety as it does in conventional rice. So the invertase gene itself does not make the rice plant cold tolerant -but instead a mechanism that regulates the invertase gene is different. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. [99] observed that MYBS3 plays a critical role in cold adaptation in rice and necessary for enhancing cold tolerance. They reported that transgenic rice constitutively overexpressing MYBS3 tolerated 4 ºC for at least 1 week and exhibited no yield penalty in normal field conditions. Based on previous studies it has been established that the CBF cold responsive pathway is an integral component of the cold acclimation response [100,63,6]. However, the transcriptome data showed that additional cold-regulatory pathways also exist [52,53,101]. Transcriptome comparisons indicated that only 12% of the cold-responsive genes are certain members of the CBF regulon. Moreover, at least 28% of the cold-responsive genes were not regulated by the CBF transcription factors, including 15 encoding known or putative transcription factors, indicating that these coldresponsive genes are members of different low-temperature regulons [55].
When overexpressed in Arabidopsis and tobacco, the soybean gene SCOF-1 (encodes a zinc-finger protein) can activate COR gene expression and increase freezing tolerance in non-acclimated transgenic plants, although the SCOF-1 protein does not directly bind to either the DRE/CRT or the ABRE elements [102]. SCOF-1 interacts with another G-box binding bZIP protein, SGBF-1. SGBF-1 can activate ABRE-driven reporter gene expression in Arabidopsis leaf protoplasts. Thus, SCOF-1 may regulate the activity of SGBF-1 as a transcription factor in inducing COR gene expression [102].
Forward genetic analysis in Arabidopsis identified two transcription factors, high expression of osmotically responsive genes, HOS9 and HOS10, which are required for basal freezing tolerance [103,104]. Similarly, microarray analysis led to the identification of the cold-stress-inducible AP2 family transcription factor gene related to ABI3/VP1 (RAV1) [52,55] that might regulate plant growth under cold stress. RAV1 is down regulated by epibrassinolide and transgenic Arabidopsis overexpressing RAV1 exhibits a retardation of lateral root and rosette-leaf development. Further, the importance of CBF-independent pathways is also supported by analysis of mutants that have increased freezing tolerance, for example, mutations in eskimo1 (ESK1), a protein of unknown function, result in constitutive freezing tolerance. The Eskimo1 mutation was first identified as a mutation conferring frost survival without an acclimation period [105]. Later on, Bouchabke-Coussa et al. [106] clearly showed that ES-KIMO1 mutants are more tolerant to freezing but only after acclimation. The genes that are affected by the ESK1 mutation are distinct from those of the CBF regulon [107]. Recently, a CBF 1 gene has been introduced into tomato that resulted in transgenic plants showing higher activity of superoxide dismutase (SOD), higher non-photochemical quenching (NPQ), and lower malondialdehyde (MDA) content . Thus suggesting that CBF1 protein plays an important role in protection of PSII and PSI during low temperature stress at low irradiance [108]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. The low temperature limits the crop productivity when temperatures remain above freezing that is > 0 o C, it is called as chilling stress. Chilling sensitive cultivars are typically tropical genotypes. There is wide range of cold stress in temperate areas differing in both timing and intensity of low temperature. Yield losses are more severe when cold stress occurs during reproductive stage/ anthesis in rice which lead to high spikelet sterility [17]. Ability of crop genotypes / lines to survive / perform better under low temperature than other genotypes is called as cold tolerance. Ordinarily, it is the consequence of cold hardening that is an earlier exposure to a low temperature for a specific period as a result of which chilling tolerance of the concerned plants increases. Cold tolerance involves increased chlorophyll accumulation, reduced sensitivity of photosynthesis, improved germination, pollen fertility and seed set which are desirable as:
INCREASED CHLOROPHYLL ACCUMULATION
Low temperature inhibits chlorophyll accumulations in actively growing leaves. In rice, cold tolerant lines, for example, japonica accumulates more chlorophyll under cold stress than do cold sensitive line, for example of indica rice [18]. Rasolofo [19] evaluated 181 accessions to identify donor and outstanding cold tolerant lines using leaf discolouration score (1-3) and found 19 remained green (dark) after 10 days in the 12 o C cold water tank. Sanghera et al. [17] found 18 cold tolerance IRCTN rice genotypes based on dark green colour and high spikelet fertility (>90%) under temperate conditions.
REDUCED SENSITIVITY OF PHOTOSYNTHESIS
Chloroplast and photosynthesis is major site of cold injury. Tolerance in these aspects is expressed in native vegetation adapted to growing under cool conditions. The reduced sensitivity of photosynthesis to cold has been observed in maize inbreds adapted to low temperature which is partly related to specific enzymes of the process [20].
Improved Pollen Fertility and Seed Set
Cold tolerance at reproductive stage is expressed as improved seed set and pollen fertility. It is largely a function of floral structure and function under stress. Lia et al. [22] reported plant cold tolerance in rice is associated with anther size, number of pollen grain, diameter of fertile pollen grains at booting stage. However, Sanghera et al. [17] reported that cold tolerance is associated with high spikelet fertility (>90%) and well panicle exsertion under temperate conditions.
Cold snaps cause a reaction in the plant that prevents sugar getting to the pollen. Without sugar there is no starch build-up which provides energy for pollen germination. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Arabidopsis genes involved in tolerance to abiotic stress were transferred, by genetic engineering, to many crops and tolerance was successfully conferred in the field, despite the complexity of plant responses to environmental stress [11,61]. Thus, finding new key genes responsible for abiotic stress tolerance phenotypes is of great importance not only for a better understanding of stress responses, but also for promising future crop improvement.
Many studies have suggested that cold regulated gene expression is critical in plants for both chilling tolerance [47,11] and cold acclimation [12,71,13,112]. Cold responsive genes encode a diverse array of proteins such as enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids and antioxidants: molecular chaperones, antifreeze proteins, and others with a presumed function in tolerance to dehydration caused by freezing [113,39,71]). The change in the gene expression occur in plant during cold acclimatization a developmental process that results in increased tolerance [28]. Since then, it has repeatedly been speculated that certain COR (cold regulated) genes might have role in freezing tolerance. To test this notion investigators have turned to isolating the characterizing genes that are expressed in response to low temperature. These efforts have led to the identification of a number of genes such as the COR 15a KINI, LTI 78, fad 7etc. of A. thaliana. The generic trends of the genes and transcription factors are available in STIFDB (Stress responsive Transcription Factor Database). STIFDB (available at [URL]. ) is a database of stress-related genes, which are upregulated in abiotic stress-related microarray experiments. STIFDB provides a platform to understand the stress-regulome of abiotic stress responsive genes in plants. STIFDB will be a highly useful resource for a researcher working on abiotic stress responses in plants [114].
Low temperature stress is a major environmental factor that not only limits where crops can be grown but also reduces yields depending on the weather in a particular growing season. In addition to exceptionally stressful years that cause significant yield reductions, less extreme stress almost certainly causes smaller losses over large areas to produce comparable yield reductions every year. Even in cases when freezing stress does not result in yield loses; it often results in crop quality reduction. Each year, worldwide losses in crop production due to low temperature damage amount to approximately $ 2 Billion. Some of the major losses include the 1995 early fall frosts in the US which caused losses of over $1 billion to corn and soybeans. The occasional freezes in Florida have shifted the citrus belt further south, and California sustained $650M of damage in 1998 to the citrus crop due to a winter freeze. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. In these cases, the enhancement of antioxidative mechanisms [40], increased levels of sugars in the apoplastic space [41], and the induction of genes encoding molecular chaperones [39], respectively, could have protective effects.
In cold stress-tolerant plants, many genes involved in the synthesis of osmoprotectants-organic compounds such as amino acids (e.g. proline), quaternary and other amines (e.g. glycinebetaine and polyamines) and a variety of sugars and sugar alcohols (e.g. mannitol, trehalose and galactinol) that accumulate during osmotic adjustment-have been used. Both cold-stress-induced transcripts and constitutively expressed transcripts need to be processed, exported to the cytoplasm and kept in conformations that are competent for translation. RNA can fold into extensive secondary structures that could interfere with its function, and cold temperatures exacerbate this interference. Many genes that respond to multiple stresses like dehydration and low temperature at the transcriptional level are also induced which protects the cell from dehydration and chilling. In order to restore the cellular function and make plants more tolerant to stress, transferring a single gene encoding a single specific stress protein may not be sufficient to reach the required tolerance levels. To overcome such constraints, enhancing tolerance towards multiple stresses by a gene encoding a stress inducible transcription factor that regulates a number of other genes is a promising approach. In bacteria, nucleic-acidbinding cold shock proteins (CSPs) accumulate at cold temperatures and function as transcription antiterminators or translational enhancers by destabilizing RNA secondary structure [42]. Some CSP-domain-containing proteins in plants are upregulated by cold stress, and might function as RNA chaperones in the regulation of translation [43,44]. A different cold-responsive nucleic-acid-binding protein, a zincfinger-containing glycine-rich RNA-binding protein from Arabidopsis designated atRZ-1a, is also upregulated by cold stress, and genetic analysis supports its function in freezing tolerance [45]. Compared to other organisms, plants have the largest number of DEAD-box RNA helicase genes [46]. One of these helicases, which is encoded by the Arabidopsis low expression of osmotically responsive genes4 (LOS4) gene, is essential for plant tolerance of chilling and freezing stress [47]. LOS4 is required for efficient export of RNA from the nucleus to the cytoplasm [48]. The Arabidopsis nucleoporin AtNUP160 suppressor of auxin resistance1 (SAR1) also controls RNA export, and is crucial for chilling and freezing tolerance [49]. Both LOS4 and AtNUP160 proteins are enriched at the nuclear rim [47,49]. Defects in the nucleocytoplasmic transport of RNA seem to affect cold tolerance preferentially, because the LOS4 and AtNUP160 mutant plants do not have severe growth or developmental phenotypes, nor are they strongly altered in the tolerance of other abiotic stresses.
KEY PLAYERS INVOLVED IN COLD RESPONSIVE PATHWAYS
Cold tolerance is the result of complex physiological mechanisms involving many cell and plant traits. Earlier studies have shown that the genetic control of cold tolerance is complex and can be regarded as polygenic [50] and the mechanism of how these genes controlled cold tolerance is still not fully clear. Therefore, the foundation for a better molecular and genetic understanding of the cold responsive pathways will improve our knowledge and pave the way for the development of improved methodologies for cold tolerance screening. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Indeed, cold acclimation pre-vents expansion-induced-lyses and the formation of hexagonal II phase lipids in rye and other plants [28]. This ability to adapt has an impact on the distribution and survival of the plant, and on crop yields. Multiple mechanisms appear to be involved in this stabilization. The best documented are changes in lipid composition [28]. Secondly, temperature induced change in membrane fluidity is another consequences in plants during temperature stresses and might represent a potential site of perception and/or injury [29,30]. Adaptation of living cells to chilling temperatures is a function of alteration in the membrane lipid composition by increased fatty acid unsaturation. Genetically engineered tobacco plants over-expressing chloroplast glycerol-3phosphate acyltransferase (GPAT) gene (involved in phosphatidyl glycerol fatty acid desaturation) from squash (Cucurbita maxima) and A. thaliana showed an increase in the number of unsaturated fatty acids and a corresponding decrease in the chilling sensitivity. At low temperature, greater membrane lipid unsaturation appears to be crucial for optimum membrane function. An Arabidopsis fatty acid biosynthesis (FAB1) mutant with more saturated membranes showed decreased quantum efficiency of photosystem II (PSII), chlorophyll content and the amount of chloroplast glycerolipids after prolonged exposure to low temperature [31]. A triple mutant fatty acid desaturation (fad3-2 fad7-2 fad8) devoid of trienoic fatty acids (18:3 or 16:3) produced a phenotype similar to FAB1, when plants were subjected to prolonged low temperature exposure [32]. Similarly, fad5 and fad6 mutants with more saturated membranes became chlorotic and showed growth retardation during low temperature incubation [33]. In addition to membrane unsaturation, it appears that lipid asymmetry in the membrane also contributes to membrane physical structure at low temperature [34]. The accumulation of sucrose and other simple sugars that typically occurs with cold acclimation also seems likely to contribute to the stabilization of membranes as these molecules can protect membranes against freezeinduced damage in vitro [35,36].
Besides, protective chaperone like function of LEA proteins acting against cellular damage has been proposed, indicating the role of LEA proteins in anti aggregation of enzymes under desiccation and freezing stresses. There is emerging evidence that certain novel hydrophilic and late embryogenesis abundant (LEA) polypeptides also participate in the stabilization of membranes against freeze-induced injury. These hydrophilic and late embryogenesis abundant polypeptides are predicted to contain regions capable of forming amphipathic -helices which are shown to have strong effect on intrinsic curvature of monolayers and their propensity to form hexagonal II phase. They are said to defer their formation at lower temperatures [37]. There is another evidence that freeze-induced production of reactive oxygen species contributes to membrane damage and that intercellular ice can form adhesions with cell walls and membranes and cause cell rupture [38]. Further, there is evidence that protein denaturation occurs in plants at low temperature [39] which could potentially result in cellular damage. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. A triple mutant fatty acid desaturation (fad3-2 fad7-2 fad8) devoid of trienoic fatty acids (18:3 or 16:3) produced a phenotype similar to FAB1, when plants were subjected to prolonged low temperature exposure [32]. Similarly, fad5 and fad6 mutants with more saturated membranes became chlorotic and showed growth retardation during low temperature incubation [33]. In addition to membrane unsaturation, it appears that lipid asymmetry in the membrane also contributes to membrane physical structure at low temperature [34]. The accumulation of sucrose and other simple sugars that typically occurs with cold acclimation also seems likely to contribute to the stabilization of membranes as these molecules can protect membranes against freezeinduced damage in vitro [35,36].
Besides, protective chaperone like function of LEA proteins acting against cellular damage has been proposed, indicating the role of LEA proteins in anti aggregation of enzymes under desiccation and freezing stresses. There is emerging evidence that certain novel hydrophilic and late embryogenesis abundant (LEA) polypeptides also participate in the stabilization of membranes against freeze-induced injury. These hydrophilic and late embryogenesis abundant polypeptides are predicted to contain regions capable of forming amphipathic -helices which are shown to have strong effect on intrinsic curvature of monolayers and their propensity to form hexagonal II phase. They are said to defer their formation at lower temperatures [37]. There is another evidence that freeze-induced production of reactive oxygen species contributes to membrane damage and that intercellular ice can form adhesions with cell walls and membranes and cause cell rupture [38]. Further, there is evidence that protein denaturation occurs in plants at low temperature [39] which could potentially result in cellular damage. In these cases, the enhancement of antioxidative mechanisms [40], increased levels of sugars in the apoplastic space [41], and the induction of genes encoding molecular chaperones [39], respectively, could have protective effects.
In cold stress-tolerant plants, many genes involved in the synthesis of osmoprotectants-organic compounds such as amino acids (e.g. proline), quaternary and other amines (e.g. glycinebetaine and polyamines) and a variety of sugars and sugar alcohols (e.g. mannitol, trehalose and galactinol) that accumulate during osmotic adjustment-have been used. Both cold-stress-induced transcripts and constitutively expressed transcripts need to be processed, exported to the cytoplasm and kept in conformations that are competent for translation. RNA can fold into extensive secondary structures that could interfere with its function, and cold temperatures exacerbate this interference. Many genes that respond to multiple stresses like dehydration and low temperature at the transcriptional level are also induced which protects the cell from dehydration and chilling. In order to restore the cellular function and make plants more tolerant to stress, transferring a single gene encoding a single specific stress protein may not be sufficient to reach the required tolerance levels. To overcome such constraints, enhancing tolerance towards multiple stresses by a gene encoding a stress inducible transcription factor that regulates a number of other genes is a promising approach. In bacteria, nucleic-acidbinding cold shock proteins (CSPs) accumulate at cold temperatures and function as transcription antiterminators or translational enhancers by destabilizing RNA secondary structure [42]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Pennycooke et al. [88] reported that downregulating -Gal ( -Galactosidase) in transgenic petunia resulted in an increase in freezing tolerance suggesting that engineering raffinose metabolism by transformation with -Gal provides an additional method for improving the freezing tolerance of plants. The overexpression of genes encoding LEA proteins can improve the stress tolerance of transgenic plants. Expression of the citrus gene encoding a LEA protein, CuCOR19 increased the cold tolerance of transgenic tobacco [89]. Likewise, the freezing tolerance of Arabidopsis was increased by the ectopic expression of the wheat gene WCS19 [47], the Arabidopsis gene COR15A [64], and the co-expression of the genes RAB18 and COR47 and XERO2 and ERD10 [90]. The freezing tolerance of strawberry leaves was enhanced by expression of the wheat dehydrin gene WCOR410 [72]. On the other hand, the expression of two cold-induced LEA proteins from spinach (Kaye et al., 1998) [91] and three desiccation-induced LEA proteins from C. plantagineum [92] in tobacco did not induce any significant changes in the freezing or drought tolerance of the respective transgenic plants. This may indicate either that not all LEA proteins make a significant contribution to plant stress tolerance, or that they need a particular background to function in, as suggested for transgenic strawberry plants [72]. Kim et al. [44] engineered tobacco with ring zinc finger protein (RDCPt) from hot pepper (Capsicum annuum) and reported that expression of this gene resulted in improved cold tolerance in transgenic plants as compared to wild type. Dai et al. [93] reported that overexpression of OsMYB3R-2 in transgenic Arabidopsis increased tolerance to freezing when exposed to -8 ºC for 10 h. They found that survival after 6 d at normal conditions was 26.8% for the wild-type and 84.5% for transgenic lines. Phenotypically, most transgenic seedlings were green and could regrow as compared with the wild type; whereas most wild-type seedlings became white and did not regrow after removed to normal conditions. The survival percentage under different low temperatures also showed dramatic difference between the transgenic plants and wild-type plants.
Pramanik and Imai [94] reported that TPP (trehalose-6phosphate phosphatase) genes expressed in rice and their expression is induced by cold. Trehalose accumulates rapidly and transiently, which follows the transient induction of TPP activity, in rice tissues during chilling stress [94]. Overexpression of TPS (trehalose-6-phosphate synthase) and TPP genes enhanced the accumulation of trehalose and tolerance to cold stress in transgenic tobacco and rice [95][96][97][98]. However, the regulatory mechanism of TPPs by cold or other stresses is unclear. In another study, Su et al. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. The inherent cold hardiness of the crop determines in which agricultural areas it can be grown.
Crops that are more resistant to freezing stress would allow some geographical regions to grow more profitable and productive crops with less environmental risks. However despite continued efforts, traditional breeding has had only limited success in imparting crop plants with better freezing tolerance due to very little was known about the mechanisms that regulate chilling and freezing tolerance. With the advent of molecular genetics and biotechnology, it is now possible to genetically engineer plants to be more tolerant to many environmental adversities, including low temperature. Molecular studies have shown that several genes with various functions are induced by environmental stresses such as drought, high-salinity and low temperature in plants. Most of the dehydration responsive genes are induced by the plant hormone abscisic acid (ABA), but others are not. Expression analyses of dehydration-responsive genes have provided at least four independent regulatory systems (regulons) for gene expression in a model plant Arabidopsis thaliana. The cis-acting elements in the promoters of some genes that have a typical stress-inducible expression profile and the transcription factors that affect the expression of these genes have been analyzed [115].
FUTURE OUTLOOK AND CONCLUSION
The development of genetically engineered plants by the introduction and/or overexpression of selected genes seems to be a viable option to hasten the breeding of ''improved'' plants. Intuitively, genetic engineering would be a faster way to insert beneficial genes than through conventional or molecular breeding. Also, it would be the only option when genes of interest originate from cross barrier species, distant relatives, or from non-plant sources. Applications of genomic approaches and gene knockout strategies are progressing to accelerate efforts to assess systematically and understand complex quantitative traits such as acquired tolerance to temperature extremes. By using genetic and molecular approaches, a number of relevant genes have been identified and new information continually emerges to enrich the CBF cold-responsive pathway. Thus, the CBF/DREB1 genes are thought to be activators that integrate several components of the cold acclimation response by which plants increase their tolerance to low temperatures after exposure to non-freezing conditions. The DREB1/CBF genes have been successfully used to improve abiotic stress tolerance in a number of different crop plants. Studies on the other transcription factors associated with stress response are in progress.
However, the results of the transcriptome study demonstrate the highly complex nature of plant adaptation to low temperature. To overcome this problem a transgenic approach to promoting cold tolerance has been widely adopted, with some success. For example, increasing the accumulation of two compatible solutes, that is, glycinebetaine and trehalose, in transgenic rice by overexpressing either E. coli choline oxidase, or trehalose-6-phosphate synthase fused to trehalose-6-phosphate phosphatase, enhanced tolerance to both salt and cold. In fact a large number of genes identified in different studies have currently annotated with ''unknown function'' and involve new genes and new pathways indicates that our knowledge of the transcriptional control of the low temperature response is limited, and the regulation of these transcriptional responses is far more complex than pre-viously believed. Information on the low-temperature transcriptome, proteome and metabolome is expected to continue to increase in the near future. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. cDNA clones encoding two DREB1A homologs (named DREB1B/CBF1 and DREB1C/CBF2) and one DREB2A homolog (DREB2B) were also isolated and Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its homolog was induced by dehydration. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as transacting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.
However, Agarwal et al. [85] showed that CBFs are negatively regulated by an upstream transcription factor, ThpI Thermal hysteresis proteins (Anti freeze protein) Transcription factor Arabidopsis Enhanced low temperature tolerance in transgenic plants was observed by changes of electrolyte leakage activity, malonyldialdehyde and proline contents [133] CBF1 CRT/DRE binding factor 1 Transcription factor Solanum Lycopersicum Detection of higher activity of superoxide dismutase (SOD), higher non-photochemical quenching (NPQ), and lower malondialdehyde (MDA) content in transgenic tomato leaves suggest that CBF1 protein plays an important role in protection of PSII and PSI during low temperature stress at low irradiance [108] MYB15 (an R2R3-MYB family protein) in Arabidopsis. MYB15 is expressed even in the absence of cold stress, and MYB15 can bind to MYB recognition elements (MYBRS) in the promoters of CBFs. MYB15 mutant plants show enhanced expression of CBFs during cold acclimation and enhanced freezing tolerance, whereas, transgenic Arabidopsis overexpressing MYB15 showed a decreased expression of CBFs and a reduction in freezing tolerance. Thus, MYB15 is an upstream transcription factor that negatively regulates the expression of CBFs.
Transcriptome analysis of ZAT12-overexpressing Arabidopsis revealed that the ZAT12 regulon consists of at least 24 cold standard set (COS) genes, of which nine are coldinduced and 15 are cold-repressed genes [55]. Constitutive overexpression of Arabidopsis DREB1A improved drought and low-temperature stress tolerance in tobacco, and regulation of transgene expression via the stress-inducible RD29A promoter minimized the negative effects on plant growth [83]. Similarly, the Arabidopsis DREB1A gene was placed under control of the RD29A promoter and transferred via biolistic transformation into bread wheat [84]. However, constitutive overexpression of the CBF genes using the cauliflower mosaic virus 35S promoter can result in undesirable agronomic traits. In Arabidopsis, CBF overexpression can cause a ''stunted'' growth phenotype, a decrease in seed yield and a delay in flowering [81,5].
In the last decade, extensive research efforts have been undertaken to identify and characterize cold-responsive (COR) genes and a number of homologous components of the Arabidopsis CBF cold response pathway in many plants have been found [6]. Many of these putative orthologs have been structured, analyzed and functionally tested. The expression patterns of the CBFs and CORs in response to low temperature are similar in a variety of plants species, involving rapid cold-induced expression of the CBFs followed by expression of CBF-targeted genes that increase freezing tolerance. Moreover, constitutive overexpression of the Arabidopsis CBF genes in other plants resulted in increased freezing tolerance that have been successfully used to engineer cold stress tolerance in several crop species [6].
Transgenic attempts with other structural genes have also been made with fair degree of success. Genetically engineered tobacco plants over-expressing chloroplast glycerol-3-phosphate acyltransferase (GPAT) gene (involved in phosphatidyl glycerol fatty acid desaturation) from squash (Cucurbita maxima) and A. thaliana [86] showed an increase in the number of unsaturated fatty acids present in the plant cell wall, which enhance the cold tolerance to the plants during cold stress. Expression of a plant phosphatase (At PP2CA) in transgenic A. thaliana can accelerate the development of cold acclimation and increase freezing tolerance. It has also shown that transgenic plants expressing a constitutively active kinase NPK1, is more tolerant to chilling and other abiotic stresses [87]. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>This below document has 2 sentences that end with 'improved tolerance to cold stress'. It has approximately 548 words, 24 sentences, and 4 paragraph(s).
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Cold stress is a major environmental factor that limits the agricultural productivity of plants in hilly areas. Plants respond and adapt to this stress to survive under stress conditions at the molecular and cellular levels as well as at the physiological and biochemical levels. However, expression of a variety of genes is induced by different stresses in diverse plants.
Low temperature often affects plant growth and crop productivity, which causes significant crop losses [1]. Plants differ in their tolerance to chilling (0-15 ºC) and freezing (< 0ºC) temperatures. In general, plants from temperate climatic regions are considered to be chilling tolerant with variable degree, and can increase their freezing tolerance by being exposed to chilling, non-freezing temperatures, a process known as cold acclimation [2], which is associated with biochemical and physiological changes [3][4][5] and ultimately showed marked changes in gene expression, biomembrane lipid composition, and small molecule accumulation [6].
*Address correspondence to this author at the Research Associate, Biotechnology Laboratory, Central Institute of Temperate Horticulture, Rangreth, Srinagar, Jammu and Kashmir 190 007, India; Tel: 0194-2305044; Fax: 2305045; Mobile: 09419029020; E-mailBesides, plants of tropical and subtropical origins. are sensitive to chilling stress and lack the mechanism of cold acclimation. Low temperature resistance in plants is a very complex trait, involving many different metabolic pathways and cell compartments [7]. Conventional breeding methods have met with limited success in improving the cold tolerance of important crop plants involving inter-specific or inter-generic hybridization. Besides, in vitro induced variations have also been applied to improve the abiotic stress tolerance of various crop plants but without much success. The conventional breeding approaches are limited by the complexity of stress tolerance traits, low genetic variance of yield components under stress condition and lack of efficient selection criteria. It is important, therefore, to look for alternative strategies to develop cold stress tolerant crops.
Biotechnology offers new strategies that can be used to develop transgenic crop plants with improved tolerance to cold stress. Rapid advance in recombinant DNA technology and development of precise and efficient gene transfer protocols have resulted in efficient transformation and generation of transgenic lines in a number of crop species [8][9][10] (Fig. 1). A number of genes have been isolated and characterized that are responsive to freezing stress. Many studies have suggested that cold regulated gene expression is critical in plants for both chilling tolerance [11] and cold acclimation [12,13]. Advent of molecular tools has made it possible to select directly at the gene label without waiting for the phenotype notype to show up. Transgenic approach is being pursued actively throughout the world to improve traits including tolerance to biotic and abiotic stresses in a number of crops [14]. As compared to other stresses, plant responses to cold stress are complex, so the prospects of improving cold tolerance in crops seem not to be very bright. Despite this, efforts have been made during the last two decades to generate transgenic lines of different crops, which have shown improved tolerance to cold stress. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine
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Engineering Cold Stress Tolerance in Crop Plants Plants respond with changes in their pattern of gene expression and protein products when exposed to low temperatures. Thus ability to adapt has an impact on the distribution and survival of the plant, and on crop yields. Many species of tropical or subtropical origin are injured or killed by non-freezing low temperatures, and exhibit various symptoms of chilling injury such as chlorosis, necrosis, or growth retardation. In contrast, chilling tolerant species are able to grow at such cold temperatures. Conventional breeding methods have met with limited achievers in improving the cold tolerance of important crop plants involving inter-specific or inter-generic hybridization. Recent studies involving full genome profiling/ sequencing, mutational and transgenic plant analysis, have provided a deep insight of the complex transcriptional mechanism that operates under cold stress. The alterations in expression of genes in response to cold temperatures are followed by increases in the levels of hundreds of metabolites, some of which are known to have protective effects against the damaging effects of cold stress. Various low temperature inducible genes have been isolated from plants. Most appear to be involved in tolerance to cold stress and the expression of some of them is regulated by C-repeat binding factor/ dehydration-responsive element binding (CBF/DREB1) transcription factors. Numerous physiological and molecular changes occur during cold acclimation which reveals that the cold resistance is more complex than perceived and involves more than one pathway. The findings summarized in this review have shown potential practical applications for breeding cold tolerance in crop and horticultural plants suitable to temperate geographical locations.
INTRODUCTION
Abiotic stresses adversely affect growth, productivity and trigger a series of morphological, physiological, biochemical and molecular changes in plants. Cold stress is a major environmental factor that limits the agricultural productivity of plants in hilly areas. Plants respond and adapt to this stress to survive under stress conditions at the molecular and cellular levels as well as at the physiological and biochemical levels. However, expression of a variety of genes is induced by different stresses in diverse plants.
Low temperature often affects plant growth and crop productivity, which causes significant crop losses [1]. Plants differ in their tolerance to chilling (0-15 ºC) and freezing (< 0ºC) temperatures. In general, plants from temperate climatic regions are considered to be chilling tolerant with variable degree, and can increase their freezing tolerance by being exposed to chilling, non-freezing temperatures, a process known as cold acclimation [2], which is associated with biochemical and physiological changes [3][4][5] and ultimately showed marked changes in gene expression, biomembrane lipid composition, and small molecule accumulation [6].
*Address correspondence to this author at the Research Associate, Biotechnology Laboratory, Central Institute of Temperate Horticulture, Rangreth, Srinagar, Jammu and Kashmir 190 007, India; Tel: 0194-2305044; Fax: 2305045; Mobile: 09419029020; E-mailBesides, plants of tropical and subtropical origins. are sensitive to chilling stress and lack the mechanism of cold acclimation. Low temperature resistance in plants is a very complex trait, involving many different metabolic pathways and cell compartments [7]. Conventional breeding methods have met with limited success in improving the cold tolerance of important crop plants involving inter-specific or inter-generic hybridization. Besides, in vitro induced variations have also been applied to improve the abiotic stress tolerance of various crop plants but without much success. We hope the results of these collaborative studies will contribute to the sustainable food production in developing countries and help to prevent the global-scale environmental damage.
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Domain: Biology Environmental Science Engineering Agricultural and Food Sciences Medicine<|endoftext|>
Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. Dorsum with marbled blotches irregularly scattered, without a clear pattern. Some specimens have two dorsolateral stripes, cream, orange, or red, extending from the posterior portion of the eye to the inguinal region. Dorsal surface of head with a bar or an inverted triangle homogeneously dark brown. Dark brown stripe contouring the tympanum from posterior portion of the eye to the arm-trunk junction. Forearms with dark brown transversal stripes or irregular blotches; hand with dorsal surface marbled.
In 70% alcohol preserved specimens, general gray coloration become yellowish or cream over time, evidencing the dark brown blotches and stripes but, at some point, they fade. Heel blotches and dorsolateral stripes become cream or white. Tibial tubercles present or absent in some populations (Fig 4). Specimens from Alcatrazes Island are much larger than specimens from other populations. Snout subelliptical, subovoid, or rounded; loreal region vertical or oblique. Supernumerary tubercles of palmar region distinct or not.
Tadpole. The tadpole was described by Heyer et al. [52] and it is endotrophic, remaining in foam nests throughout the whole development.
Advertisement call. The advertisement call of A. marmorata is composed of single, nonpulsed notes having or not a frequency upsweep. Call duration varies from 21 to 114 ms, the dominant frequency coincides with the first harmonic and ranges from 4027 to 5467 Hz (Tables 4 and 6).
Natural history. Adenomera marmorata occurs mainly along forest edges and is highly abundant in most of its geographical distribution range. Males call on leaf-litter, exposed or not, near the constructed chamber for oviposition. The call activity may occur during all day in rainy days, but mostly from dusk into the first hours of night. On two occasions we observed males calling at dawn from inside collecting bags.
Geographic distribution and type-locality. Adenomera marmorata is distributed in Southeastern Brazil, over the "Serra da Mantiqueira" and northern part of the "Serra do Mar" mountain ranges of the states of Minas Gerais, Rio de Janeiro, and São Paulo, as well as in north-central coastal São Paulo, southern coastal Rio de Janeiro (see map on Fig 1); ranging in elevation from sea level up to more than 1200 m.
Steindachner [17] set the type-locality as "Brasilien" [Brazil]. (DOCX)
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Domain: Biology Medicine
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Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex
The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. For full-length OPG-TASK-1, the bulk of the protein is N-glycosylated ( Figure 5B, low exposure, lanes 6 and 7), in contrast with OPG-TASK-1:N53Q where no modification of the full-length precursor was detectable (cf. Figure 5A, lanes 6 and 7). On this basis, most of the full-length TASK-1 detectable at steady state has TM1 oriented with a native topology. In summary, even under circumstances where aberrant and misfolded precursors are potential targets for ER-associated degradation, we observe patterns of N-glycosylation that strongly support a model where TM1 can assume an inverted topology during TASK-1 biogenesis at the ER, and we conclude that this phenomenon is physiologically relevant to bona fide membrane protein synthesis in vivo. DISCUSSION We have used in vitro translation, cross-linking and Nglycosylation to investigate the biogenesis of two distinct potassium channels at the ER membrane. Our key findings are: (i) the TMs of TASK-1 remain in close proximity to the Sec61 complex for a substantial period during its biogenesis, consistent with their co-ordinated assembly; (ii) TM1 of TASK-1 and Kcv can assume an inverted topology in a cohort of nascent chains during protein biogenesis at the ER; and (iii) the Sec61 translocon can accommodate at least two distinct transmembrane orientations of TASK-TM1 and Kcv-TM1. Taken together, our data are consistent with a model where the first TMs of nascent TASK-1 and Kcv chains do not adopt a stable transmembrane topology immediately upon engaging the ER translocon, but are retained in a Sec61-dependent environment that enables their subsequent reorientation in order to achieve a native topology.
Our data strongly support previous studies [6,22], both of which provide compelling evidence for a pathway involving the 'headfirst' insertion of model signal-anchor sequences followed by their subsequent inversion to achieve their native type II topology (Nterminus cytosolic). Our work underlines the generality of a 'headfirst' membrane insertion pathway at the ER, and shows it operates both in vitro and in cultured mammalian cells. Studies with a synthetic signal-anchor found both its intrinsic hydrophobicity and the 'positive-inside rule' influence its rate of inversion [22], and there is compelling evidence that signal-anchor reorientation is carefully orchestrated event that occurs within an environment created by both the ribosome and the ER translocon [6].
Our cross-linking studies suggest that, for nascent chains of both TASK-1 and Kcv, their TM1 remains associated with the Sec61 complex for a comparatively extensive period of biogenesis, a conclusion that is supported by a delay in TM exit from the ER translocon that was recently observed using an elegant FRET-based approach [14]. Furthermore, for TASK-1, we confirmed TM1 cross-linking to Sec61β from two different conformations/orientations of nascent chain, whereas nascent Kcv chains displayed unusually strong adducts with Sec61β, indicating that a substantial proportion of integration intermediates adopt an inverted topology. N-glycosylation reporters confirmed mixed topologies for both TASK-1 fragments and full-length Kcv. In the case of the shortest TASK-1 fragment studied, we could even detect a fraction of molecules that were triply N-glycosylated. This strongly suggests that at least a subset of TASK-1 chains can assume both a type I and type II transmembrane orientation during their integration into the ER membrane. Overall, the in vitro synthesis of longer TASK-1 fragments typically favoured a native type II topology of TM1 at the expense of the inverted form (cf. Figure 6). Importantly, this effect did not rely on the use of ribosome-tethered precursors that might favour N-terminal translocation (the present study, and [6]), and was also observed following the in vivo expression of TASK-1 derivatives in mammalian cells.
What is the significance of the increasing fidelity of TM1 topology as more of the TASK-1 polypeptide is synthesized? We favour a model where TM1 of the nascent TASK-1 chain transiently resides in a Sec61-enabled dynamic equilibrium such that the synthesis of additional polypeptide promotes the acquisition of its native orientation ( Figure 6). In this scenario, there is a short window of opportunity during which a signal sequence can reorient within the ER translocon [21], a process that requires an appropriately assembled ribosome/translocon complex [6]. The introduction of artificial stop codons, as used in the present study, will result in the premature release of Nterminal fragments of TASK-1 from both the ribosome and the translocon, presumably resulting in a cohort of precursors where TM1 is locked in an inverted topology. Furthermore, the addition of N-linked glycans can inhibit reorientation within the ER [21], and hence the doubly N-glycosylated OPG tag we used as a reporter for exposure to the ER lumen may have acted as a 'kinetic trap' that stabilizes otherwise transient or reversible topological intermediates (cf. 1 kcal = 4.184 kJ.
Figure S4 Cysteine-dependent adducts with Kcv membrane integration intermediates
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Domain: Biology Medicine<|endoftext|>This below document has 2 sentences that end with 'ERK1/2 inhibition (Fig'. It has approximately 323 words, 23 sentences, and 4 paragraph(s).
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Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions*
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. 5, A and B). Total FOXO1 protein did not change in response to ERK1/2 inhibition (Fig. 5A). Neither AKT and p38 phosphorylation nor total AKT and p38 proteins changed in the trametinib group (Fig. 5C). Trametinib treatment decreased nuclear phosphorylated FOXO1 to total FOXO1 32%, 4 h after trametinib (Fig. 5, D and E). FOXO1 mRNA was also up-regulated at 4 h after ERK1/2 inhibition (Fig. 5F). Further evidence that ERK1/2 inhibition results in increased FOXO1 gene transcription was revealed by increases in catalase and mitochondrial superoxide dismutase 2 (SOD2), two genes regulated by FOXO1 (Fig. 5F) (30,31). We conclude that ERK1/2 regulates the activity of FOXO1 within the nucleus and controls the basal level of PGC-1␣ mRNA and subsequent PGC-1␣ protein levels, as well as downstream MB genes in the kidney.
ERK1/2 Inhibition during AKI Attenuates an Increase in Serum Creatinine and Decreases in PGC-1␣ and NRF1
Expression-To examine the role of ERK1/2 on PGC-1␣ in the renal cortex under injurious conditions, we pretreated mice with trametinib or vehicle for 1 h and then subjected mice to bilateral IR surgery or sham surgery for 18 min to induce AKI; kidneys were collected 3 h later. IR-induced AKI decreases PGC-1␣ mRNA expression, as well as other mitochondrial genes and proteins (32). Trametinib prevented the increase in ERK1/2 phosphorylation following the IR (Fig. 6A). PGC-1␣ mRNA decreased 41% in IR mice after 3 h, and trametinib attenuated this decrease (Fig. 6B). Interestingly, trametinib pretreatment increased NRF1 expression following IR when compared with sham, whereas IR alone resulted in 13% decrease in NRF1 when compared with sham (Fig. 6B). At the protein level, both PGC-1␣ and TFAM proteins increased 1.7-and 2.4-fold, respectively (Fig. was responsible for the funding acquisition.
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Domain: Biology Medicine
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Sources of heterogeneity in human monocyte subsets
Highlights • Variation in monocyte phenotype is explored using the markers CD14, CD16, HLA-DR, CX3CR1 and CCR2.• The CD14++CD16+ monocytes exhibit a spectrum of markers dependent on location within the gate.• Monocyte phenotype varies dependent on genetic background and history of exposure to infection.• Processing technique for PBMC purification does not lead to changes in monocyte subsets.• Processing technique for purification can alter intensity but not pattern of marker expression.
Introduction
Peripheral blood monocytes, which represent around 10% of circulating leukocytes in humans, are recognized as the largest pool of circulating progenitor cells and form a vital part of the immune system [1,2]. Processing was performed in the manner described and cells were suspended at 5 × 10 6 cells/ml. Surface staining was performed straight away in the same manner as for cryopreserved cells as described.
Cryopreserved PBMCs were thawed straight from LN2 in a 37 • C water bath until only a small crystal remained. The contents of the vial were slowly added to complete media (RPMI 1640), supplemented with 10% heat inactivated FCS, 2 mM l-glutamine and 100 U penicillin/streptomycin (all Lonza, Verviers, Belgium). Cells were washed twice with complete media, counted in trypan blue (Sigma-Aldrich, Dorset, UK). Cells were washed in PBS (Lonza, Verviers, Belgium) and resuspended at 5 × 10 5 cells per stain for each staining panel.
Fresh and thawed cells for staining were incubated with 10% FCS at 4 • C for 10 minutes prior to staining and stained with Alexa488-conjugated anti-CD14 (clone M5E2), PE-Cy7-conjugated HLA-DR (clone L243; all from BD Biosciences, San Jose, CA), Pacific Blue-conjugated CD16 (clone CB16; eBiosciences, San Diego, CA), Alexa647-conjugated CX3CR1 (clone 2A9-1; BioLegend, San Diego, CA), PerCP-conjugated CCR2 (clone 48607; RnD Systems, Minneapolis, MN) or the relevant isotype control for 30 min at 4 • C. Unbound antibodies were washed off and cells were resuspended in PBS prior to acquisition of at least 50,000 live events on a BD FACS LSR II (BD Biosciences, San Jose, CA). Compensation was performed prior to acquisition of each experiment using BD FacsComp beads (BD Biosciences, San Jose, CA). Analysis was performed using FlowJo software (TreeStar, USA) and Mean fluorescence index (MFI) was calculated for each marker with the relevant isotype control subtracted.
Monocyte discrimination
To ensure that only CD14+ cells representing monocytes were analyzed, a gating strategy was employed to gate only HLA-DR, CD14 expressing cells. Briefly a live gate, to include all leukocytes, was drawn based on forward scatter (FSC) and side scatter (SSC).
HLA-DR positive cells were gated to exclude any CD16+ natural killer (NK) cells and other non-MHC expressing cells [12], and true monocytes were gated based on expression of CD14 and CD16 surface markers.
Determination of exposure to P. falciparum and S. haematobium
In order to determine if any arising differences between the Caucasian and African participants were due to undetected schistosome or Plasmodium parasite infection (current or previous) or parasite-unrelated mechanisms such as genetic differences, serological assays were conducted to determine parasite exposure history. Enzyme linked immunosorbent assay (ELISA) was used to measure antigen-specific antibodies to malaria schizont (IgG and IgM) and schistosome adult worm (IgG4, IgM, IgE) in the serum. Lyophilized soluble S. haematobium adult worms (SWAP) was obtained from the Theodor Bilharz Institute (Giza, Egypt) and reconstituted as recommended by the manufacturer. Schizont extract was a kind gift from David Cavanagh (University of Edinburgh, UK). ELISAs were performed as reported elsewhere [27,28], and all ELISAs were performed in duplicate on the same day with positive and negative controls on each plate.
Statistical analysis
All statistical analyses were conducted using the statistical package SPSS version 19. Parametric tests were used when assumptions of parametric tests were met, otherwise non-parametric tests were used [29]. When using parametric tests data were transformed using appropriate transformations: surface marker expression (measured as MFI) was log transformed (log 10 (x + 1)), proportions of subsets were arcsine square root transformed, and antibody responses were square root transformed. In parametric models age was taken as a continuous variable, sex (male/female) and donor ethnicity (African/Caucasian) were categorical.
To test the hypothesis that the whole monocyte population is composed of a continuum of 'subsets' consisting of distinct phenotypic profiles, differences in expression of surface markers were analyzed using a one way analysis of variance (ANOVA) with subset as a grouping variable and post hoc tests used to determine significant differences between adjacent subsets. Differences in the proportion of each subset were analyzed using an arcsine square root transformation and a one way ANOVA using type I sequential sums of squares in a similar manner as discussed. Importantly our study indicates that conformity across research groups in gating of these subsets is necessary in order to compare studies.
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Domain: Biology Medicine<|endoftext|>14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
To further explore the potential signaling pathways associated with mammalian Eag, we set forth to identify novel binding partners of rEag1 channels in the brain. Figure S1 Phosophorylation-dependent interaction of Herg with 14-3-3e. Herg was co-expressed with an empty vector or myc-tagged 14-3-3e in HEK293T cells. 24 hours after transfection, cells were left untreated, or were treated with 1 mM okadaic acid or staurosporine for 60 min. (Left panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or antiphosphorylated Akt (pAkt) antibody. b-actin was run as a loading control. (Right panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or anti-Herg antibody. In the presence of the phosphatase inhibitor okadaic acid, enhanced immunopreciation efficiency was observed for Herg. By contrast, pretreatment with the protein kinase-inhibitor staurosporine led to a small but conspicuous decrease in 14-3-3e interaction. (TIF)
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Domain: Biology Medicine<|endoftext|>Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. V5 , indicated below also as gH st , we followed a similar strategy; the annealing oligonucleotides encoding the Factor Xa protease cleavage site, followed by V5 and One-strep-tag epitopes, were 5Ј-GGA GAC GCA TGC TAA TCG AAG GGC GAG GTA AGC CTA TCC CTA ACC CTC TCC TAG GCC TCG ATT CTA CGA GCG CTT GGA GCC ACC CGC AGT TCG AGA AAG G- 3Ј and 5Ј-GGT AGT AGA TCT CAT TTT TCG AAC TGC GGG TGG CTC CAC GAT CCA CCT CCC GAT CCA CCT CCG GAA CCT CCA CCT TTC TCG AAC TGC GGG TGG CTC CAA G-3Ј. They were ligated to the SphI/BglII-digested gH. The aa sequence of gH st. V5 cytoplasmic tail became ILKVLRT-SVPFFWRRMLIEGRGLPIPNPLLGLDSTSAWSHPQFELGG-GSGGGSGGGSWSHPQFEL. To generate gL 5E1. Her , we followed essentially a similar strategy and inserted the NheI restriction site by site-directed mutagenesis, in place of the stop codon, by means of oligonucleotides 5Ј- CCC CAC TCC CGG CGC CTG CTA GCA TGA ATT CAC GGA AAC CCG TCC GGG TTC GGG- 3Ј and 5Ј- CCC GAA CCC GGA CGG GTT TCC GTG AAT TCA TGC TAG CAG GCG CCG GGA GTG GGG-3Ј. The annealing oligonucleotides encoding the trombin-5E1-His epitopes were 5Ј-CTA TAG CTA GCC CTG GTT CCG CGT GGA TCC TCC AGA CCA GGA AGC ACT ACA CCC TCT GGG AAC TCT GCA AGA TAT GGG AA- 3Ј and 5Ј-GGA AGA TCT TCA ATG GTG ATG GTG ATG ATG CGG AGT TAT ACT TCT AGG TGT GTT ATT CCC ATA TCT TGC AGA GTT CCC-3Ј and were ligated to NheI/BglII digested gL. The aa sequence of the gL C terminus was changed from SRRL to SRRLLALVPRGSSRPGSTTPSGNSARYG-NNTPRSITPHHHHHH. To generate gL V5. Her , the annealing oligonucleotides encoding the V5 and Her epitopes were 5Ј-ATG CTC GCT AGC TGG TAA GCC TAT CCC TAA CCC TCT CCT CGG TCT CGA TTC TAC GC- 3Ј and 5Ј-TTA GCG AGA TCT CAA TGG TGA TGA TGG TGA TGA TGA ACG GTA CGC GTA GAA TCG AGA C-3Ј and were ligated to NheI/BglII-digested gL. The aa sequence of gL C terminus was changed from SRRL to SRRLLAGLPIPNPLLGLDSTRTVHH-HHHHH. gC V5 was generated as follows. The gC ORF was PCR-amplified from HSV-5(F) DNA with primers encoding the V5 epitope 5Ј-AGA TCT AGG CCT ATG GCC CCG GGG CGG GTG GGC CTT GCC G TG GTC CTG TGG AGC CTG- 3Ј and 5Ј-GCA CGG GGC GGC CGC TTA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG GAT AGG CTT ACC GGC TAG CCG ATG ACG CTG CCG CGA CTG TGA TGT GCG G-3Ј. The StuI-NotI-digested gC amplimer was ligated to MTS vector. For all plasmids, the ORF was sequenced.
Genetic Engineering of HSV1(BAC)-gD st -HSV1(BAC)-gD st was generated by bacterial artificial chromosome (BAC) "galK recombineering," employing the Escherichia coli strain SW102 and galK (galactokinase) positive/negative selectable marker, kindly provided by Dr. N. G. Copeland) (45). pYEbac102 HSV-BAC was provided by Mireia Hudson and carries the BAC sequences inserted between UL3 and UL4 genes. The galK cassette was recombined in HSV-1(BAC) to replace the gD stop codon. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine
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