Both glial cells and glia scar greatly affect the development of spinal cord injury and have become hot spots in research on spinal cord injury treatment.The cellular deposition of dense extracellular matrix proteins ...Both glial cells and glia scar greatly affect the development of spinal cord injury and have become hot spots in research on spinal cord injury treatment.The cellular deposition of dense extracellular matrix proteins such as chondroitin sulfate proteoglycans inside and around the glial scar is known to affect axonal growth and be a major obstacle to autogenous repair.These proteins are thus candidate targets for spinal cord injury therapy.Our previous studies demonstrated that 810 nm photo biomodulation inhibited the formation of chondroitin sulfate proteoglycans after spinal cord injury and greatly improved motor function in model animals.However,the specific mechanism and potential targets involved remain to be clarified.In this study,to investigate the therapeutic effect of photo biomodulation,we established a mouse model of spinal cord injury by T9 clamping and irradiated the injury site at a power density of 50 mW/cm~2 for 50 minutes once a day for 7 consecutive days.We found that photobiomodulation greatly restored motor function in mice and down regulated chondroitin sulfate proteoglycan expression in the injured spinal cord.Bioinformatics analysis revealed that photobiomodulation inhibited the expression of proteoglycan-related genes induced by spinal cord injury,and versican,a type of proteoglycan,was one of the most markedly changed molecules.Immunofluorescence staining showed that after spinal cord injury,versican was present in astrocytes in spinal cord tissue.The expression of versican in primary astrocytes cultured in vitro increased after inflammation induction,whereas photobiomodulation inhibited the expression of ve rsican.Furthermore,we found that the increased levels of p-Smad3,p-P38 and p-Erk in inflammatory astrocytes were reduced after photobiomodulation treatment and after delivery of inhibitors including FR 180204,(E)-SIS3,and SB 202190.This suggests that Sma d 3/Sox9 and MAP K/Sox9 pathways may be involved in the effects of photobiomodulation.In summary,our findings show that photobiomodulation modulates the expression of chondroitin sulfate proteoglycans,and versican is one of the key target molecules of photo biomodulation.MAPK/Sox9 and Smad3/Sox9 pathways may play a role in the effects of photo biomodulation on chondroitin sulfate proteoglycan accumulation after spinal cord injury.展开更多
Firmness is one of the most important fruit quality traits in strawberries.The postharvest shelf life of this soft fruit is highly limited by the loss of firmness,where cell wall disassembly plays an important role.Pr...Firmness is one of the most important fruit quality traits in strawberries.The postharvest shelf life of this soft fruit is highly limited by the loss of firmness,where cell wall disassembly plays an important role.Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening.In this study,FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens.Ten independent lines,cv.“Chandler”,were obtained,and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay.The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing.The percentage of edited sequences varied from 47%up to almost 100%,being higher than 95%for seven of the selected lines.Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control,ranging from 33 to 70%increase in firmness.There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness.Minor changes were observed in other fruit quality traits,such as colour,soluble solids,titratable acidity or anthocyanin content.Edited fruits showed a reduced softening rate during postharvest,displayed a reduced transpirational water loss,and were less damaged by Botrytis cinerea inoculation.The analysis of four potential off-target sites revealed no mutation events.In conclusion,editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.展开更多
基金supported by the National Natural Science Foundation of China,Nos.81070996(to ZW),81572151(to XH)Shaanxi Provincial Key R&D Program,Nos.2020ZDLSF02-05(to ZW),2021ZDLSF02-10(to XH)+1 种基金Everest Project of Military Medicine of Air Force Medical University,No.2018RCFC02(to XH)Boosting Project of the First Affiliated Hospital of Air Force Medical University,No.XJZT19Z22(to ZW)。
文摘Both glial cells and glia scar greatly affect the development of spinal cord injury and have become hot spots in research on spinal cord injury treatment.The cellular deposition of dense extracellular matrix proteins such as chondroitin sulfate proteoglycans inside and around the glial scar is known to affect axonal growth and be a major obstacle to autogenous repair.These proteins are thus candidate targets for spinal cord injury therapy.Our previous studies demonstrated that 810 nm photo biomodulation inhibited the formation of chondroitin sulfate proteoglycans after spinal cord injury and greatly improved motor function in model animals.However,the specific mechanism and potential targets involved remain to be clarified.In this study,to investigate the therapeutic effect of photo biomodulation,we established a mouse model of spinal cord injury by T9 clamping and irradiated the injury site at a power density of 50 mW/cm~2 for 50 minutes once a day for 7 consecutive days.We found that photobiomodulation greatly restored motor function in mice and down regulated chondroitin sulfate proteoglycan expression in the injured spinal cord.Bioinformatics analysis revealed that photobiomodulation inhibited the expression of proteoglycan-related genes induced by spinal cord injury,and versican,a type of proteoglycan,was one of the most markedly changed molecules.Immunofluorescence staining showed that after spinal cord injury,versican was present in astrocytes in spinal cord tissue.The expression of versican in primary astrocytes cultured in vitro increased after inflammation induction,whereas photobiomodulation inhibited the expression of ve rsican.Furthermore,we found that the increased levels of p-Smad3,p-P38 and p-Erk in inflammatory astrocytes were reduced after photobiomodulation treatment and after delivery of inhibitors including FR 180204,(E)-SIS3,and SB 202190.This suggests that Sma d 3/Sox9 and MAP K/Sox9 pathways may be involved in the effects of photobiomodulation.In summary,our findings show that photobiomodulation modulates the expression of chondroitin sulfate proteoglycans,and versican is one of the key target molecules of photo biomodulation.MAPK/Sox9 and Smad3/Sox9 pathways may play a role in the effects of photo biomodulation on chondroitin sulfate proteoglycan accumulation after spinal cord injury.
基金supported by the Ministerio de Ciencia,Innovación y Universidades and FEDER EU funds(grant numbers AGL2017-86531-C2-1R and PID2020-118468RB-C21)the University of Malaga(grant number B1-2020_09)+1 种基金CS-R was awarded a PhD Fellowship from the Ministerio de Ciencia,Innovación y Universidades(PRE2018-085509)PhD Program Advanced Biotechnology,University of Málaga.
文摘Firmness is one of the most important fruit quality traits in strawberries.The postharvest shelf life of this soft fruit is highly limited by the loss of firmness,where cell wall disassembly plays an important role.Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening.In this study,FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens.Ten independent lines,cv.“Chandler”,were obtained,and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay.The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing.The percentage of edited sequences varied from 47%up to almost 100%,being higher than 95%for seven of the selected lines.Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control,ranging from 33 to 70%increase in firmness.There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness.Minor changes were observed in other fruit quality traits,such as colour,soluble solids,titratable acidity or anthocyanin content.Edited fruits showed a reduced softening rate during postharvest,displayed a reduced transpirational water loss,and were less damaged by Botrytis cinerea inoculation.The analysis of four potential off-target sites revealed no mutation events.In conclusion,editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.
