Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult hom...Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.展开更多
Social network structures can crucially impact complex social processes such as collective behaviour or the transmission of information and diseases. However, currently it is poorly understood how social networks chan...Social network structures can crucially impact complex social processes such as collective behaviour or the transmission of information and diseases. However, currently it is poorly understood how social networks change over time. Previous studies on primates suggest that ＇knockouts＇ （due to death or dispersal） of high-ranking individuals might be important drivers for structural changes in animal social networks. Here we test this hypothesis using long-term data on a natural population of ba- boons, examining the effects of 29 natural knockouts of alpha or beta males on adult female social networks. We investigated whether and how knockouts affected （i） changes in grooming and association rates among adult females, and （2） changes in mean degree and global clustering coefficient in these networks. The only significant effect that we found was a decrease in mean degree in grooming networks in the first month after knockouts, but this decrease was rather small, and grooming networks re- bounded to baseline levels by the second month after knockouts. Taken together our results indicate that the removal of high-ranking males has only limited or no lasting effects on social networks of adult female baboons. This finding calls into question the hypothesis that the removal of high-ranking individuals has a destabilizing effect on social network structures in social animals [Current Zoology 61 （1）： 107-113, 2015].展开更多
Effect of the specific gene knockout on the main metabolism in Escherichia coli was reviewed, and the regulation mechanisms were clarified based on different levels of information such as gene expressions, enzyme acti...Effect of the specific gene knockout on the main metabolism in Escherichia coli was reviewed, and the regulation mechanisms were clarified based on different levels of information such as gene expressions, enzyme activities, intracellular metabolite concentrations, and metabolic fluxes together with fermentation data. The effects of the knockout of such genes as pflA, pta, ppc, pykF, adhE, and ldhA on the metabolic changes were analyzed for the case under anaerobic condition. The effects of the knockout of such genes as pgi, zwf, gnd, ppc pck, pyk, and lpdA on the metabolic changes were also analyzed for the case under aerobic condition. The metabolic regulation analysis was made focusing on the roles of transcription factors.展开更多
Highlights●CRISPR/Cas9 RNP complex-based strategy demonstrates robustness and accuracy in generating gene-edited sheep.●Sheep horn development remains unaffected by partial RXFP2 knockout.●Partial RXFP2 knockout re...Highlights●CRISPR/Cas9 RNP complex-based strategy demonstrates robustness and accuracy in generating gene-edited sheep.●Sheep horn development remains unaffected by partial RXFP2 knockout.●Partial RXFP2 knockout results in unilateral cryptorchidism in sheep.展开更多
Background:C1QL3 is widely expressed in the brain and is specifically produced by a subset of excitatory neurons.However,its function is still not clear.We established C1ql3-deficient rats to investigate the role of C...Background:C1QL3 is widely expressed in the brain and is specifically produced by a subset of excitatory neurons.However,its function is still not clear.We established C1ql3-deficient rats to investigate the role of C1QL3 in the brain.Methods:C1ql3 knockout(KO)rats were generated using CRISPR/Cas9.C1ql3 KO was determined by polymerase chain reaction(PCR),DNA sequencing,and western blot-ting.Microglia morphology and cytokine expression with or without lipopolysaccha-ride(LPS)stimulus were analyzed using immunohistochemistry and real-time PCR.The brain structure changes in KO rats were examined using magnetic resonance imaging.Neuronal architecture alteration was analyzed by performing Golgi staining.Behavior was evaluated using the open field test,Morris water maze test,and Y maze test.Results:C1ql3 KO significantly increased the number of ramified microglia and decreased the number of hypertrophic microglia,whereas C1ql3 KO did not in-fluence the expression of pro-inflammatory factors and anti-inflammatory factors except IL-10.C1ql3 KO brains had more amoeboid microglia types and higher Arg-1 expression compared with the WT rats after LPS stimulation.The brain weights and HPC sizes of C1ql3 KO rats did not differ from WT rats.C1ql3 KO damaged neuronal integrity including neuron dendritic arbors and spine density.C1ql3 KO rats demonstrated an increase in spontaneous activity and an impairment in short working memory.Conclusions:C1ql3 KO not only interrupts the neuronal integrity but also affects the microglial activation,resulting in hyperactive behavior and impaired short memory in rats,which highlights the role of C1QL3 in the regulation of structure and function of both neuronal and microglial cells.展开更多
Understanding bacterial strategies for coping with heavy metal stress is essential for elucidating their resilience in contaminated environments.However,whether cell wall exfoliation contributes to bacterial tolerance...Understanding bacterial strategies for coping with heavy metal stress is essential for elucidating their resilience in contaminated environments.However,whether cell wall exfoliation contributes to bacterial tolerance under heavy metal stress,such as cadmium(Cd)exposure,remains unclear and requires further investigation.In this study,we reveal a novel self-protective mechanism in Stenotrophomonas sp.H225 isolated from a Cd-contaminated farmland soil,which underwent controlled cell wall exfoliation and regeneration in response to Cd stress up to 200 mg L^(-1).Transmission electron microscopy and energy-dispersive X-ray spectroscopy analyses revealed that the exfoliated cell wall fragments served as extracellular Cd sinks,thereby reducing intracellular Cd accumulation.Fourier-transform infrared spectroscopy and enzyme-linked immunosorbent assay indicated progressive peptidoglycan(PG)degradation,with exfoliated PG concentration in solution increasing from 148 ng mL^(-1) at 0 mg L^(-1) Cd to 240 ng mL^(-1) at 200 mg L^(-1) Cd.This degradation was counteracted by the compensatory upregulation of PG biosynthesis genes,with the enrichment ratio reaching up to 0.83,facilitating cell wall reconstruction.Transcriptomic analysis and gene knockout experiments identified mtgA(encoding a monofunctional transglycosylase)as a key determinant in cell wall repair and Cd resistance.To our knowledge,this is the first mechanistic evidence that bacteria can mitigate heavy metal toxicity through dynamic cell wall remodeling involving exfoliation and regeneration.This finding enhances our understanding of microbial survival strategies under environmental stress and highlights potential targets for engineering metal-tolerant strains for bioremediation applications.展开更多
Objective To investigate the mechanism of in alleviating colonic mucosal inflammation in ten-eleven translocation(TET)protein 2 gene knockout(TET2^(-/-))mice with ulcerative colitis(UC)by regulating DNA methyltransfer...Objective To investigate the mechanism of in alleviating colonic mucosal inflammation in ten-eleven translocation(TET)protein 2 gene knockout(TET2^(-/-))mice with ulcerative colitis(UC)by regulating DNA methyltransferase(DNMT)and DNA hydroxymethylase.Methods Male specific pathogen-free(SPF)grade C57BL/6J wild-type(WT)mice(n=8)and TET2^(-/-)mice(n=20)were used to establish UC models by freely drinking 3%dextran sulfate sodium solution for 7 d.After UC model validation through histopathological examination in two mice from each type,the remaining mice were divided into four groups(n=6 in each group):WT model(WT+UC),TET2^(-/-)model(TET2^(-/-)+UC),TET2^(-/-)mild moxibustion(TET2^(-/-)+MM),and TET2^(-/-)electroacupuncture(TET2^(-/-)+EA)groups.TET2^(-/-)+MM group received mild moxibustion on Tianshu(ST25)and Qihai(CV6)for 10 min daily for 7 d.The TET2^(-/-)+EA group also applied electroacupuncture(1 mA,2/100 Hz)at the same acupoints for 10 min daily for 7 d.The disease activity index(DAI)scores of each group of mice were accessed daily.The colon lengths of mice in groups were measured following intervention.The pathological changes in the colon tissues were observed with hematoxylin and eosin(HE)staining.The concentrations of interleukin(IL)-6,C-C motif chemokine 17(CCL17),and C-X-C motif chemokine ligand 10(CXCL10)in serum were detected by enzyme-linked immunosorbent assay(ELISA).The expression of DNMT proteins(DNMT1,DNMT3A,and DNMT3B)in the colon tissues was detected by immunohistochemistry.The expression of 5-methylcytosine(5-mC),5-hydroxymethylcytosine(5-hmC),histone deacetylase 2(HDAC2),and DNA hydroxymethylase family proteins(TET 1 and TET3)was detected using immunofluorescence,which also determined the co-localization of TET1 and IL-6 protein.Results Compared with WT+UC group,TET2^(-/-)+UC group exhibited significantly higher DAI scores and shorter colon lengths(P<0.01).Both mild moxibustion and electroacupuncture significantly decreased DAI scores and ameliorated colon shortening in TET2^(-/-)mice(P<0.001).Histopathological scores of TET2^(-/-)+UC mice were significantly higher than those of WT+UC group(P<0.001)and were significantly reduced after both mild moxibustion and electroacupuncture interventions(P<0.001).Serum levels of IL-6,CCL17,and CXCL10 were significantly elevated in TET2^(-/-)+UC group compared with WT+UC group(P<0.001).Mild moxibustion significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.001,P<0.001,and P<0.01,respectively),while electroacupuncture also significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.05,P<0.01,and P<0.01,respectively).TET2^(-/-)+UC mice showed increased expression levels of DNMT1,DNMT3A,DNMT3B,and 5-mC(P<0.05,P<0.01 and P<0.001,respectively),with decreased expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001).Mild moxibustion significantly reduced DNMT1,DNMT3B,and 5-mC levels(P<0.05,P<0.01,and P<0.001,respectively),while increasing expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001,P<0.001,P<0.05,and P<0.001,respectively).Electroacupuncture significantly decreased 5-mC and DNMT3B levels(P<0.001 and P<0.01,respectively)and increased 5-hmC and HDAC2 levels(P<0.05 and P<0.001,respectively),but did not significantly affect TET1 and TET3 expression(P>0.05).Compared with TET2^(-/-)+MM group,TET2^(-/-)+EA group showed significantly higher 5-mC expression(P<0.001).TET2^(-/-)+UC group exhibited markedly increased IL-6 expression and higher co-localization of TET1 and IL-6 in mucosal epithelium,whereas minimal IL-6 expression was observed in the other groups.Conclusion Mild moxibustion and electroacupuncture significantly ameliorate colonic inflammation exacerbated by TET2 deficiency in UC mice via epigenetic modulation.Distinct mechanisms exist between the two interventions:mild moxibustion regulates both DNMT and hydroxymethylase,whereas electroacupuncture primarily affects DNMT.展开更多
Magnaporthe oryzae,the causal agent of rice blast,induces significant upregulation of OsPR10b,a pathogenesis-related(PR)pollen allergen(BetV-1)family gene.To investigate its role in immunity,we generated OsPR10b knock...Magnaporthe oryzae,the causal agent of rice blast,induces significant upregulation of OsPR10b,a pathogenesis-related(PR)pollen allergen(BetV-1)family gene.To investigate its role in immunity,we generated OsPR10b knockout mutants in the Zhonghua 11(ZH11)background.OsPR10b was predominantly expressed in rice calli and strongly induced by M.oryzae infection.Knockout mutants(ospr10b-1 and ospr10b-2)exhibited heightened susceptibility to both M.oryzae and Xanthomonas oryzae pv.oryzae(Xoo),demonstrating that OsPR10b positively regulates resistance to blast and bacterial blight.Our findings elucidate OsPR10b’s role in rice immunity and provide genetic resources for disease-resistant breeding.展开更多
The crosstalk between megakaryocytic lineage cells and the skeletal system has just begun to be explored but remains largely elusive.Using conditional gene knockout mouse models,we demonstrated that loss of Beclin 1(B...The crosstalk between megakaryocytic lineage cells and the skeletal system has just begun to be explored but remains largely elusive.Using conditional gene knockout mouse models,we demonstrated that loss of Beclin 1(Becn1),a major regulator of mammalian autophagy,exclusively in the megakaryocytic lineage disrupted autophagy in platelets but did not compromise megakaryopoiesis or the formation and function of platelets.Unexpectedly,conditional Becn1 deletion in male mice led to a remarkable increase in bone mass with improved bone quality,in association with a decrease in sex hormone binding globulin(SHBG)and an increase in free testosterone(FT).In vivo Becn1 overexpression in megakaryocytic lineage-specific cells reduced bone mass and quality,along with an increase in SHBG and a decrease in FT.Transplantation of wild-type bone marrow cells into megakaryocytic lineage Becn1-deficient male mice restored bone mass and normalized SHBG and FT.Furthermore,bilateral orchiectomy of Becn1^(f/f);Pf4-iCre mice,which are crippled with the production of testosterone,resulted in a reduction in bone mass and quality,whereas in vivo overexpression of SHBG,specifically in the liver of Becn1^(f/f);Pf4-iCre mice,decreased FT and reduced bone mass and quality.In addition,metformin treatment,which induces SHBG expression,reduced FT and normalized bone mass in Becn1^(f/f);Pf4-iCre mice.We thus concluded that Becn1 of the megakaryocytic lineage is dispensable locally for platelet hemostasis but limits bone mass by increasing SHBG,which in turn reduces the FT of male mice.Our findings highlight a mechanism by which Becn1 from megakaryocytic lineage cells distally balances bone growth.展开更多
Membrane-initiated estrogen receptorα(mERα)signaling has been shown to affect bone mass in murine models.However,it remains unknown which cell types mediate the mERα-dependent effects on bone.In this study,we gener...Membrane-initiated estrogen receptorα(mERα)signaling has been shown to affect bone mass in murine models.However,it remains unknown which cell types mediate the mERα-dependent effects on bone.In this study,we generated a novel mouse model with a conditional C451A mutation in Esr1,which enables selective knockout of the palmitoylation site essential for the membrane localization of ERα(C451A^(f/f)).First,we used Runx2-Cre mice to generate Runx2-C451A^(f/f)mice with conditional inactivation of mERαsignaling in Runx2-expressing osteoblast lineage cells.No significant changes were observed in body weight,weights of estrogen-responsive organs,or serum concentrations of estradiol between female Runx2-C451A^(f/f)and homozygous C451A^(f/f)littermate controls.High-resolution microcomputed tomography analysis showed a consistent decrease in cortical bone mass in the tibia,femur,and vertebra L5 of Runx2-C451A^(f/f)mice and three-point bending analysis of humerus revealed an impaired mechanical bone strength in Runx2-C451A^(f/f)female mice compared to controls.Additionally,primary osteoblast cultures from mice lacking mERαsignaling showed impaired differentiation compared to controls.展开更多
Intervertebral disc degeneration(IDD)is a progressive and dynamic process in which the senescence-associated secretory phenotype(SASP)of nucleus pulposus cells(NPC)plays a significant role.While impaired chaperone-med...Intervertebral disc degeneration(IDD)is a progressive and dynamic process in which the senescence-associated secretory phenotype(SASP)of nucleus pulposus cells(NPC)plays a significant role.While impaired chaperone-mediated autophagy(CMA)has been associated with inflammation and cellular senescence,its specific involvement in the self-perpetuating feedback loop of NPC senescence remains poorly understood.Through LAMP2A knockout in NPC,we identified a significant upregulation of DYRK1A,a core mediator of premature senescence in Down syndrome.Subsequent validation established DYRK1A as the critical driver of premature senescence in CMA-deficient NPC.Combinatorial transcription factor analysis revealed that under IL1B stimulation or CMA inhibition,elevated DYRK1A promoted FOXC1 phosphorylation and nuclear translocation,initiating transcriptional activation of cell cycle arrest.Intriguingly,CMA impairment concurrently enhanced glutamine metabolic flux in senescent NPC,thereby augmenting their survival fitness.Transcriptomic profiling demonstrated that CMA reactivation in senescent NPC facilitated fate transition from senescence to apoptosis,mediated by decreased glutamine flux via GLUL degradation.Therefore,CMA exerts protective effects against IDD by maintaining equilibrium between premature senescence and senolysis.This study elucidates CMA’s regulatory role in SASP-mediated senescence amplification circuits,providing novel therapeutic insights for IDD and other age-related pathologies.展开更多
[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification...[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.展开更多
Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this ...Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation,as well as expanding the potential pool of usable donor grafts.The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes,increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis.Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury.IRI involves a complex interplay between neutrophils,natural killer T-cells cells,CD4+ T cell subtypes,cytokines,nitric oxide synthases,haem oxygenase-1,survival kinases such as the signal transducer and activator of transcription,Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways.Transgenic animals,particularly genetic knockout models,have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies.Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein.This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.展开更多
Due to rapid urbanization and industrialization, many soils for crop production are contaminated by cadmium(Cd), a heavy metal highly toxic to many organisms. Cereal crops such as rice, wheat, maize, and barley are th...Due to rapid urbanization and industrialization, many soils for crop production are contaminated by cadmium(Cd), a heavy metal highly toxic to many organisms. Cereal crops such as rice, wheat, maize, and barley are the primary dietary source of Cd for humans, and reducing Cd transfer from soil to their grains is therefore an important issue for food safety. During the last decade, great progress has been made in elucidating the molecular mechanisms of Cd transport, particularly in rice. Inter-and intraspecific variations in Cd accumulation have been observed in cereal crops. Transporters for Cd have been identified in rice and other cereal crops using genotypic differences in Cd accumulation and mutant approaches. These transporters belong to different transporter families and are involved in the uptake, vacuolar sequestration, root-to-shoot translocation, and distribution of Cd. Attempts have been made to reduce Cd accumulation in grains by manipulating these transporters through overexpression or knockout of the transporter genes, as well as through marker-assisted selection breeding based on genotypic differences in Cd accumulation in the grains. In this review, we describe recent progress on molecular mechanisms of Cd accumulation in cereal crops and compare different molecular strategies for minimizing Cd accumulation in grains.展开更多
文摘Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.
文摘Social network structures can crucially impact complex social processes such as collective behaviour or the transmission of information and diseases. However, currently it is poorly understood how social networks change over time. Previous studies on primates suggest that ＇knockouts＇ （due to death or dispersal） of high-ranking individuals might be important drivers for structural changes in animal social networks. Here we test this hypothesis using long-term data on a natural population of ba- boons, examining the effects of 29 natural knockouts of alpha or beta males on adult female social networks. We investigated whether and how knockouts affected （i） changes in grooming and association rates among adult females, and （2） changes in mean degree and global clustering coefficient in these networks. The only significant effect that we found was a decrease in mean degree in grooming networks in the first month after knockouts, but this decrease was rather small, and grooming networks re- bounded to baseline levels by the second month after knockouts. Taken together our results indicate that the removal of high-ranking males has only limited or no lasting effects on social networks of adult female baboons. This finding calls into question the hypothesis that the removal of high-ranking individuals has a destabilizing effect on social network structures in social animals [Current Zoology 61 （1）： 107-113, 2015].
文摘Effect of the specific gene knockout on the main metabolism in Escherichia coli was reviewed, and the regulation mechanisms were clarified based on different levels of information such as gene expressions, enzyme activities, intracellular metabolite concentrations, and metabolic fluxes together with fermentation data. The effects of the knockout of such genes as pflA, pta, ppc, pykF, adhE, and ldhA on the metabolic changes were analyzed for the case under anaerobic condition. The effects of the knockout of such genes as pgi, zwf, gnd, ppc pck, pyk, and lpdA on the metabolic changes were also analyzed for the case under aerobic condition. The metabolic regulation analysis was made focusing on the roles of transcription factors.
基金supported by the National Key Research and Development Program of China(2022YFD1300200)the National Natural Science Foundation of China(32161143010,32202646,and 32272848)+2 种基金the China Agriculture Research System(CARS-39)the Key Special Project of Ningxia Science and Technology Department,China(2021BEF02024)the local grants,China(NXTS2021-001,2022GD-TSLD-46,NK2022010207,and NXTS2022-001)。
文摘Highlights●CRISPR/Cas9 RNP complex-based strategy demonstrates robustness and accuracy in generating gene-edited sheep.●Sheep horn development remains unaffected by partial RXFP2 knockout.●Partial RXFP2 knockout results in unilateral cryptorchidism in sheep.
基金The present work was supported by the National Natural Science Foundation(31970508)the National Key Research and Development Program of China(2022YFF0710702).
文摘Background:C1QL3 is widely expressed in the brain and is specifically produced by a subset of excitatory neurons.However,its function is still not clear.We established C1ql3-deficient rats to investigate the role of C1QL3 in the brain.Methods:C1ql3 knockout(KO)rats were generated using CRISPR/Cas9.C1ql3 KO was determined by polymerase chain reaction(PCR),DNA sequencing,and western blot-ting.Microglia morphology and cytokine expression with or without lipopolysaccha-ride(LPS)stimulus were analyzed using immunohistochemistry and real-time PCR.The brain structure changes in KO rats were examined using magnetic resonance imaging.Neuronal architecture alteration was analyzed by performing Golgi staining.Behavior was evaluated using the open field test,Morris water maze test,and Y maze test.Results:C1ql3 KO significantly increased the number of ramified microglia and decreased the number of hypertrophic microglia,whereas C1ql3 KO did not in-fluence the expression of pro-inflammatory factors and anti-inflammatory factors except IL-10.C1ql3 KO brains had more amoeboid microglia types and higher Arg-1 expression compared with the WT rats after LPS stimulation.The brain weights and HPC sizes of C1ql3 KO rats did not differ from WT rats.C1ql3 KO damaged neuronal integrity including neuron dendritic arbors and spine density.C1ql3 KO rats demonstrated an increase in spontaneous activity and an impairment in short working memory.Conclusions:C1ql3 KO not only interrupts the neuronal integrity but also affects the microglial activation,resulting in hyperactive behavior and impaired short memory in rats,which highlights the role of C1QL3 in the regulation of structure and function of both neuronal and microglial cells.
基金partially supported by the National Natural Science Foundation of China (Nos. 42377004 and 41991334)the Fundamental Research Funds for the Central Universities (No. 226-2025-0004)+1 种基金the China Agriculture Research System (No. CARS-01)the opportunity granted by the China Scholarship Council (No. 202406320448)
文摘Understanding bacterial strategies for coping with heavy metal stress is essential for elucidating their resilience in contaminated environments.However,whether cell wall exfoliation contributes to bacterial tolerance under heavy metal stress,such as cadmium(Cd)exposure,remains unclear and requires further investigation.In this study,we reveal a novel self-protective mechanism in Stenotrophomonas sp.H225 isolated from a Cd-contaminated farmland soil,which underwent controlled cell wall exfoliation and regeneration in response to Cd stress up to 200 mg L^(-1).Transmission electron microscopy and energy-dispersive X-ray spectroscopy analyses revealed that the exfoliated cell wall fragments served as extracellular Cd sinks,thereby reducing intracellular Cd accumulation.Fourier-transform infrared spectroscopy and enzyme-linked immunosorbent assay indicated progressive peptidoglycan(PG)degradation,with exfoliated PG concentration in solution increasing from 148 ng mL^(-1) at 0 mg L^(-1) Cd to 240 ng mL^(-1) at 200 mg L^(-1) Cd.This degradation was counteracted by the compensatory upregulation of PG biosynthesis genes,with the enrichment ratio reaching up to 0.83,facilitating cell wall reconstruction.Transcriptomic analysis and gene knockout experiments identified mtgA(encoding a monofunctional transglycosylase)as a key determinant in cell wall repair and Cd resistance.To our knowledge,this is the first mechanistic evidence that bacteria can mitigate heavy metal toxicity through dynamic cell wall remodeling involving exfoliation and regeneration.This finding enhances our understanding of microbial survival strategies under environmental stress and highlights potential targets for engineering metal-tolerant strains for bioremediation applications.
基金National Natural Science Foundation of China(82274641,81873372,and 82105012).
文摘Objective To investigate the mechanism of in alleviating colonic mucosal inflammation in ten-eleven translocation(TET)protein 2 gene knockout(TET2^(-/-))mice with ulcerative colitis(UC)by regulating DNA methyltransferase(DNMT)and DNA hydroxymethylase.Methods Male specific pathogen-free(SPF)grade C57BL/6J wild-type(WT)mice(n=8)and TET2^(-/-)mice(n=20)were used to establish UC models by freely drinking 3%dextran sulfate sodium solution for 7 d.After UC model validation through histopathological examination in two mice from each type,the remaining mice were divided into four groups(n=6 in each group):WT model(WT+UC),TET2^(-/-)model(TET2^(-/-)+UC),TET2^(-/-)mild moxibustion(TET2^(-/-)+MM),and TET2^(-/-)electroacupuncture(TET2^(-/-)+EA)groups.TET2^(-/-)+MM group received mild moxibustion on Tianshu(ST25)and Qihai(CV6)for 10 min daily for 7 d.The TET2^(-/-)+EA group also applied electroacupuncture(1 mA,2/100 Hz)at the same acupoints for 10 min daily for 7 d.The disease activity index(DAI)scores of each group of mice were accessed daily.The colon lengths of mice in groups were measured following intervention.The pathological changes in the colon tissues were observed with hematoxylin and eosin(HE)staining.The concentrations of interleukin(IL)-6,C-C motif chemokine 17(CCL17),and C-X-C motif chemokine ligand 10(CXCL10)in serum were detected by enzyme-linked immunosorbent assay(ELISA).The expression of DNMT proteins(DNMT1,DNMT3A,and DNMT3B)in the colon tissues was detected by immunohistochemistry.The expression of 5-methylcytosine(5-mC),5-hydroxymethylcytosine(5-hmC),histone deacetylase 2(HDAC2),and DNA hydroxymethylase family proteins(TET 1 and TET3)was detected using immunofluorescence,which also determined the co-localization of TET1 and IL-6 protein.Results Compared with WT+UC group,TET2^(-/-)+UC group exhibited significantly higher DAI scores and shorter colon lengths(P<0.01).Both mild moxibustion and electroacupuncture significantly decreased DAI scores and ameliorated colon shortening in TET2^(-/-)mice(P<0.001).Histopathological scores of TET2^(-/-)+UC mice were significantly higher than those of WT+UC group(P<0.001)and were significantly reduced after both mild moxibustion and electroacupuncture interventions(P<0.001).Serum levels of IL-6,CCL17,and CXCL10 were significantly elevated in TET2^(-/-)+UC group compared with WT+UC group(P<0.001).Mild moxibustion significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.001,P<0.001,and P<0.01,respectively),while electroacupuncture also significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.05,P<0.01,and P<0.01,respectively).TET2^(-/-)+UC mice showed increased expression levels of DNMT1,DNMT3A,DNMT3B,and 5-mC(P<0.05,P<0.01 and P<0.001,respectively),with decreased expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001).Mild moxibustion significantly reduced DNMT1,DNMT3B,and 5-mC levels(P<0.05,P<0.01,and P<0.001,respectively),while increasing expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001,P<0.001,P<0.05,and P<0.001,respectively).Electroacupuncture significantly decreased 5-mC and DNMT3B levels(P<0.001 and P<0.01,respectively)and increased 5-hmC and HDAC2 levels(P<0.05 and P<0.001,respectively),but did not significantly affect TET1 and TET3 expression(P>0.05).Compared with TET2^(-/-)+MM group,TET2^(-/-)+EA group showed significantly higher 5-mC expression(P<0.001).TET2^(-/-)+UC group exhibited markedly increased IL-6 expression and higher co-localization of TET1 and IL-6 in mucosal epithelium,whereas minimal IL-6 expression was observed in the other groups.Conclusion Mild moxibustion and electroacupuncture significantly ameliorate colonic inflammation exacerbated by TET2 deficiency in UC mice via epigenetic modulation.Distinct mechanisms exist between the two interventions:mild moxibustion regulates both DNMT and hydroxymethylase,whereas electroacupuncture primarily affects DNMT.
基金supported by the Special Fund for Agro-scientific Research in the Public Interest of Fujian Province,China(Grant No.2023R1021006)the National Natural Science Foundation of China(Grant No.32402387)+1 种基金the extended research project of the National Natural Science Foundation of China(Grant No.GJYS202511)the 5511 Collaborative Engineering Project,China(Grant No.XTCXGC2021001).
文摘Magnaporthe oryzae,the causal agent of rice blast,induces significant upregulation of OsPR10b,a pathogenesis-related(PR)pollen allergen(BetV-1)family gene.To investigate its role in immunity,we generated OsPR10b knockout mutants in the Zhonghua 11(ZH11)background.OsPR10b was predominantly expressed in rice calli and strongly induced by M.oryzae infection.Knockout mutants(ospr10b-1 and ospr10b-2)exhibited heightened susceptibility to both M.oryzae and Xanthomonas oryzae pv.oryzae(Xoo),demonstrating that OsPR10b positively regulates resistance to blast and bacterial blight.Our findings elucidate OsPR10b’s role in rice immunity and provide genetic resources for disease-resistant breeding.
基金supported in part by grants from the National Natural Science Foundation of China(No.81673093,No.82170227,No.91649113,No.82470165,No.82000121,No.31771640)the Jiangsu Science and Technology Department(No.SBK20200191)+1 种基金the State Key Laboratory of Radiation Medicine and Protection of Soochow University(No.GZC00201)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘The crosstalk between megakaryocytic lineage cells and the skeletal system has just begun to be explored but remains largely elusive.Using conditional gene knockout mouse models,we demonstrated that loss of Beclin 1(Becn1),a major regulator of mammalian autophagy,exclusively in the megakaryocytic lineage disrupted autophagy in platelets but did not compromise megakaryopoiesis or the formation and function of platelets.Unexpectedly,conditional Becn1 deletion in male mice led to a remarkable increase in bone mass with improved bone quality,in association with a decrease in sex hormone binding globulin(SHBG)and an increase in free testosterone(FT).In vivo Becn1 overexpression in megakaryocytic lineage-specific cells reduced bone mass and quality,along with an increase in SHBG and a decrease in FT.Transplantation of wild-type bone marrow cells into megakaryocytic lineage Becn1-deficient male mice restored bone mass and normalized SHBG and FT.Furthermore,bilateral orchiectomy of Becn1^(f/f);Pf4-iCre mice,which are crippled with the production of testosterone,resulted in a reduction in bone mass and quality,whereas in vivo overexpression of SHBG,specifically in the liver of Becn1^(f/f);Pf4-iCre mice,decreased FT and reduced bone mass and quality.In addition,metformin treatment,which induces SHBG expression,reduced FT and normalized bone mass in Becn1^(f/f);Pf4-iCre mice.We thus concluded that Becn1 of the megakaryocytic lineage is dispensable locally for platelet hemostasis but limits bone mass by increasing SHBG,which in turn reduces the FT of male mice.Our findings highlight a mechanism by which Becn1 from megakaryocytic lineage cells distally balances bone growth.
基金supported by the Swedish Research Council(2017-01286,2020-01840)the Swedish state under the agreement between the Swedish government and the county councils(ALF-agreement)(ALFGBG721581)+2 种基金the Gustaf V 80-years fund(FAI-2018-0466)the IngaBritt and Arne Lundberg Foundation(LU2017-0076)the Novo Nordisk Foundation(26844).
文摘Membrane-initiated estrogen receptorα(mERα)signaling has been shown to affect bone mass in murine models.However,it remains unknown which cell types mediate the mERα-dependent effects on bone.In this study,we generated a novel mouse model with a conditional C451A mutation in Esr1,which enables selective knockout of the palmitoylation site essential for the membrane localization of ERα(C451A^(f/f)).First,we used Runx2-Cre mice to generate Runx2-C451A^(f/f)mice with conditional inactivation of mERαsignaling in Runx2-expressing osteoblast lineage cells.No significant changes were observed in body weight,weights of estrogen-responsive organs,or serum concentrations of estradiol between female Runx2-C451A^(f/f)and homozygous C451A^(f/f)littermate controls.High-resolution microcomputed tomography analysis showed a consistent decrease in cortical bone mass in the tibia,femur,and vertebra L5 of Runx2-C451A^(f/f)mice and three-point bending analysis of humerus revealed an impaired mechanical bone strength in Runx2-C451A^(f/f)female mice compared to controls.Additionally,primary osteoblast cultures from mice lacking mERαsignaling showed impaired differentiation compared to controls.
基金supported by the National Natural Science Foundation of China (NSFC) (No.82172497)
文摘Intervertebral disc degeneration(IDD)is a progressive and dynamic process in which the senescence-associated secretory phenotype(SASP)of nucleus pulposus cells(NPC)plays a significant role.While impaired chaperone-mediated autophagy(CMA)has been associated with inflammation and cellular senescence,its specific involvement in the self-perpetuating feedback loop of NPC senescence remains poorly understood.Through LAMP2A knockout in NPC,we identified a significant upregulation of DYRK1A,a core mediator of premature senescence in Down syndrome.Subsequent validation established DYRK1A as the critical driver of premature senescence in CMA-deficient NPC.Combinatorial transcription factor analysis revealed that under IL1B stimulation or CMA inhibition,elevated DYRK1A promoted FOXC1 phosphorylation and nuclear translocation,initiating transcriptional activation of cell cycle arrest.Intriguingly,CMA impairment concurrently enhanced glutamine metabolic flux in senescent NPC,thereby augmenting their survival fitness.Transcriptomic profiling demonstrated that CMA reactivation in senescent NPC facilitated fate transition from senescence to apoptosis,mediated by decreased glutamine flux via GLUL degradation.Therefore,CMA exerts protective effects against IDD by maintaining equilibrium between premature senescence and senolysis.This study elucidates CMA’s regulatory role in SASP-mediated senescence amplification circuits,providing novel therapeutic insights for IDD and other age-related pathologies.
基金Supported by the National Natural Science Fundation Item of China(30970578,31070651)"Excellent Talent Support Plan in NewCentury"of Ministry of Education(NECT-08-0731)~~
文摘[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.
文摘Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation,as well as expanding the potential pool of usable donor grafts.The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes,increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis.Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury.IRI involves a complex interplay between neutrophils,natural killer T-cells cells,CD4+ T cell subtypes,cytokines,nitric oxide synthases,haem oxygenase-1,survival kinases such as the signal transducer and activator of transcription,Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways.Transgenic animals,particularly genetic knockout models,have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies.Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein.This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.
基金supported by the Grant-in-Aid for Specially Promoted Research, Japan (JSPS KAKENHI Grant No. 16H06296 to J. F. Ma.)the grants from the Scientific Research Development Foundation of Zhejiang A&F University for the Talents, China (No. 2019FR002)the Major Special Science and Technology Project of Zhejiang Province, China (No. 2016C02G2101016)。
文摘Due to rapid urbanization and industrialization, many soils for crop production are contaminated by cadmium(Cd), a heavy metal highly toxic to many organisms. Cereal crops such as rice, wheat, maize, and barley are the primary dietary source of Cd for humans, and reducing Cd transfer from soil to their grains is therefore an important issue for food safety. During the last decade, great progress has been made in elucidating the molecular mechanisms of Cd transport, particularly in rice. Inter-and intraspecific variations in Cd accumulation have been observed in cereal crops. Transporters for Cd have been identified in rice and other cereal crops using genotypic differences in Cd accumulation and mutant approaches. These transporters belong to different transporter families and are involved in the uptake, vacuolar sequestration, root-to-shoot translocation, and distribution of Cd. Attempts have been made to reduce Cd accumulation in grains by manipulating these transporters through overexpression or knockout of the transporter genes, as well as through marker-assisted selection breeding based on genotypic differences in Cd accumulation in the grains. In this review, we describe recent progress on molecular mechanisms of Cd accumulation in cereal crops and compare different molecular strategies for minimizing Cd accumulation in grains.
