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
Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogena...Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.展开更多
Background:Liver injury is one of the most common complications during sepsis.Macrophage migration inhibitory factor(MIF)is an important proinflammatory cytokine.This study explored the role of MIF in the lipopolysacc...Background:Liver injury is one of the most common complications during sepsis.Macrophage migration inhibitory factor(MIF)is an important proinflammatory cytokine.This study explored the role of MIF in the lipopolysaccharide(LPS)-induced liver injury through genetically manipulated mouse strains.Methods:The model of LPS-induced liver injury was established in wild-type and Mif-knockout C57/BL6 mice.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total bilirubin(TBil)were detected,and the expressions of MIF,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were measured.Liver histopathology was conducted to assess liver injury.Moreover,the inhibitions of MIF with(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester(ISO-1)and 4-iodo-6-phenylpyrimidine(4-IPP)were used to evaluate their therapeutic potential of liver injury.Results:Compared with wild-type mice,the liver function indices and inflammation factors presented no significant difference in the Mif-/-mice.After 72 h of the LPS-induced liver injury,serum levels of ALT,AST,and TBil as well as TNF-αand IL-1βwere significantly increased,but the knockout of Mif attenuated liver injury and inflammatory response.In liver tissue,m RNA levels of TNF-α,IL-1βand NF-κB p65 were remarkably elevated in LPS-induced liver injury,while the knockout of Mif reduced these levels.Moreover,in LPS-induced liver injury,the inhibitions of MIF with ISO-1 and 4-IPP alleviated liver injury and slightly attenuated inflammatory response.Importantly,compared to mice with LPS-induced liver injury,Mif knockout or MIF inhibitions significantly prolonged the survival of the mice.Conclusions:In LPS-induced liver injury,the knockout of Mif or MIF inhibitions alleviated liver injury and slightly attenuated inflammatory response,thereby prolonged the survival of the mice.Targeting MIF may be an important strategy to protect the liver from injury during sepsis.展开更多
Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host...Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host’s immune system is compromised.Rhoptry proteins are major virulence factors that enable T.gondii to invade host cells.This study aims to construct a T.gondii rhoptry protein 41(rop41/ROP41)gene knockout strain and preliminarily investigate the biological function of rop41.Methods:Using CRISPR/Cas9 technology,a specific single-guide RNA(sgRNA)for the target gene was designed and linked to a recombinant plasmid.Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes.The recombinant plasmid and the homologous fragments were electroporated into T.gondii,and PCR identification was performed after drug selection and monoclonal screening.Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T.gondii in host cells.Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity.The STRING database was utilized to construct a protein-protein interaction(PPI)network,and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.Results:The T.gondii rop41 gene knockout strain(RHΔku80Δrop41)was successfully constructed and stably inherited.Plaque assays showed that compared with the parental strain,the number of plaques formed by the rop41 gene knockout strain did not significantly decrease,but the reduction in plaque size was statistically significant(P<0.05).After the rop41 gene was knocked out,the invasion ability of T.gondii was reduced,but there was no statistically significant difference in its proliferation ability(P>0.05).The PPI network revealed that ROP41 was associated with other protein kinases and autophagy related proteins.Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction,biosynthesis,metabolism,and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.Conclusion:The T.gondii RHΔku80Δrop41 strain has been successfully constructed.ROP41 primarily affects the ability of T.gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T.gondii and the host.展开更多
The classic method for gene knockout (KO) is based on homologous recombination (HR) and embryonic stem cell technique (Gerlai,1996).Actually,the procedure of homologous replacement is complicated and time consuming,al...The classic method for gene knockout (KO) is based on homologous recombination (HR) and embryonic stem cell technique (Gerlai,1996).Actually,the procedure of homologous replacement is complicated and time consuming,although it has been popular during the past decades.Recent years,genome editing which can cause DNA sequence-specific mutations in the genomes of cellular展开更多
BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no ef...BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system.AIM To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout mice.METHODS We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional(intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin(HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation markers.RESULTS We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted. The intestinal crypt size in inducible conditional Claudin-7 knockout mice was increased compared with control mice(small intestine: 54.1 ± 2.96 vs 38.4 ± 1.63;large intestine: 44.7 ± 1.93 vs 27.4 ± 0.60; P < 0.001).CONCLUSION The knockout of Claudin-7 in vivo causes extensive inflammation, atypical hyperplasia, and adenoma in intestinal tissue as well as animal death in mice.Claudin-7 may act as a tumor suppressor gene in the development of colorectal cancer.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
The CRISPR-Cas system is a powerful genetic engineering tool and can be conveniently used for the generation of diverse gene-knockout models.One CRISPR-Cas system,CRISPR-Cpf1(also known as Cas12a),recognizes the AT-ri...The CRISPR-Cas system is a powerful genetic engineering tool and can be conveniently used for the generation of diverse gene-knockout models.One CRISPR-Cas system,CRISPR-Cpf1(also known as Cas12a),recognizes the AT-rich protospacer-adjacent motif(PAM)present at the 5′end of the target sequence and requires CRISPR RNA(crRNA),but not transactivating crRNA(tracrRNA)for its activity.展开更多
This is a correction to:PingWang,Zunpeng Liu,Xiaoqian Zhang,Jingyi Li,Liang Sun,Zhenyu Ju,Jian Li,Piu Chan,Guang-Hui Liu,Weiqi Zhang,Moshi Song,Jing Qu,CRISPR/Cas9-mediated gene knockout reveals a guard-ian role of NF...This is a correction to:PingWang,Zunpeng Liu,Xiaoqian Zhang,Jingyi Li,Liang Sun,Zhenyu Ju,Jian Li,Piu Chan,Guang-Hui Liu,Weiqi Zhang,Moshi Song,Jing Qu,CRISPR/Cas9-mediated gene knockout reveals a guard-ian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells,Protein&Cell,Volume 9,Issue 11,November 2018,Pages 945-965,https://doi.org/10.1007/s13238-018-0560-5 In the originally published online version of this manuscript,there was an incorrect image in Figure 2,panel G.This should read:This change does not alter the discussions or conclusions of the paper.展开更多
Abstract Objectives To investigate the tissue specificity of reactive oxygen species (ROS) damage to mitochondrial DNA (mtDNA) and to determine whether cochlear mtDNA is a sensitive target for ROS damage. Methods 10...Abstract Objectives To investigate the tissue specificity of reactive oxygen species (ROS) damage to mitochondrial DNA (mtDNA) and to determine whether cochlear mtDNA is a sensitive target for ROS damage. Methods 10 Cu/ZnSOD gene (Cu/Zn superoxide dismutase gene, Sod1) knockout mice and 16 wild-type mice were analyzed by nested polymerase chain reaction (PCR).Results Three deletions were detected in various tissues of Sod1 knockout mice. MtDNA3867bp and mtDNA3726bp deletions were the most visible, and mtDNA4236bp deletion was barely detected in these tissues. There were obvious differences in the ratio of deleted mtDNA/total mtDNA in different tissue. Deleted mtDNA was most abundant in the liver and kidney and less in cochlea, heart and brain. The lowest was in spleen and skin. The ratio in various tissues was 3-20 times in Sod1 knockout mice over wild-type mice. In cochlea, the ratio was about 15. Conclusions Without the protection of Sod1, ROS can lead to mtDNA deletions in various tissues with significant tissue specificity. Cochlear mtDNA is a sensitive target for ROS damage.展开更多
BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remai...BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.展开更多
Objective： TO observe the effect of Huxin Formula （护心方, HXF） on expressions of the chief reverse cholesterol transport （RCT） associated genes, caveolin-1 and scavenger receptor-B I （SR-B I ） in ApoE-gene kno...Objective： TO observe the effect of Huxin Formula （护心方, HXF） on expressions of the chief reverse cholesterol transport （RCT） associated genes, caveolin-1 and scavenger receptor-B I （SR-B I ） in ApoE-gene knockout [ApoE （-/-）] mice. Methods： Thirty ApoE （-/-） mice of 4-6 weeks old were randomly divided into three groups （A-C）. After being fed with high-fat diet for 16 weeks, they were treated with HXF （1 mL/100 g）, pravachol （0.3 mg/100 g）, and saline in equal volume respectively for 16 weeks successively; in addition, a blank group was set up with 10 C57BL/6J mice of 6-week old received 16-week high-fat feeding and saline treatment. Animals were sacrificed at the termination of the experiment, their paraffin sections of aortic tissue were used to measure the size of plaque, expressions of cavolin-1 and SR-B I were detected by immunological histochemical method. Results： As compared with the blank group, levels of caveolin-1 and SR-B I were increased in Groups A and B （P〈0.01）; but the increase in Group A was more significant than that in Group B （P〈0.05）. The plaque/aorta area ratio decreased significantly in Groups A and B, but showed insignificant difference between the two groups. Conclusion： HXF could obviously increase the expressions of RCT associated genes, caveolin-1 and SR-B I, promote the RCT process, so as to reduce the formation of aorta atherosclerotic plaque in ApoE （-/-） mice.展开更多
p38 regulated/activated protein kinase(PRAK)plays a key role in cell senescence and tumor suppression.The aim of this study was to investigate if PRAK had effect on cell proliferation.The growth of PRAK+/+and PRAK^(–...p38 regulated/activated protein kinase(PRAK)plays a key role in cell senescence and tumor suppression.The aim of this study was to investigate if PRAK had effect on cell proliferation.The growth of PRAK+/+and PRAK^(–/–)mouse embryonicfibroblast(MEF)cells was measured by methylthiazoletetrazolium(MTT)colorimetric assay,and the proportion of the cell number in different phases of the cell cycle was analyzed byflow cytometry.The growth curves showed that the growth rate was notably decreased,and cell double time was elongated in PRAK^(–/–)cells;moreover,the number of PRAK^(–/–)cells was decreased by 44.5%compared with that of PRAK+/+cells cultured for 96 h,suggesting that G2/M transition is inhibited in PRAK^(–/–)cells.Meanwhile,G1/S transition was also inhibited in PRAK^(–/–)cells,observed withflow cytometry analysis.The ratios of G0/G1,G2/M,and S phases of PRAK+/+cells were 44.9%,12.2%,and 42.9%,respec-tively,while those of PRAK^(–/–)cells were 55.3%,7.3%,and 37.4%,respectively.There were 23.1%increase and 12.7%decrease of the number of PRAK^(–/–)cells in G1 and S phases in comparison with that of PRAK+/+cells,respectively.Taken together,PRAK gene knockout in MEF cells leads to cell cycle arrest and proliferation inhibition.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
Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first ...Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first commercial hybrid maize was introduced in the 1930s（Duvick,2001）.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.
基金Supported by the National Natural Science Foundation of China (30970089,20876181,20831006)the Natural Science Foundation of Guangdong Province (9351027501000003)
文摘Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.
基金supported by grants from the National Natural Science Foundation of China(81971881)Medical Science and Technology Project of Henan Provincial Health Commission(SB201901045)。
文摘Background:Liver injury is one of the most common complications during sepsis.Macrophage migration inhibitory factor(MIF)is an important proinflammatory cytokine.This study explored the role of MIF in the lipopolysaccharide(LPS)-induced liver injury through genetically manipulated mouse strains.Methods:The model of LPS-induced liver injury was established in wild-type and Mif-knockout C57/BL6 mice.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total bilirubin(TBil)were detected,and the expressions of MIF,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were measured.Liver histopathology was conducted to assess liver injury.Moreover,the inhibitions of MIF with(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester(ISO-1)and 4-iodo-6-phenylpyrimidine(4-IPP)were used to evaluate their therapeutic potential of liver injury.Results:Compared with wild-type mice,the liver function indices and inflammation factors presented no significant difference in the Mif-/-mice.After 72 h of the LPS-induced liver injury,serum levels of ALT,AST,and TBil as well as TNF-αand IL-1βwere significantly increased,but the knockout of Mif attenuated liver injury and inflammatory response.In liver tissue,m RNA levels of TNF-α,IL-1βand NF-κB p65 were remarkably elevated in LPS-induced liver injury,while the knockout of Mif reduced these levels.Moreover,in LPS-induced liver injury,the inhibitions of MIF with ISO-1 and 4-IPP alleviated liver injury and slightly attenuated inflammatory response.Importantly,compared to mice with LPS-induced liver injury,Mif knockout or MIF inhibitions significantly prolonged the survival of the mice.Conclusions:In LPS-induced liver injury,the knockout of Mif or MIF inhibitions alleviated liver injury and slightly attenuated inflammatory response,thereby prolonged the survival of the mice.Targeting MIF may be an important strategy to protect the liver from injury during sepsis.
基金supported by the National Natural Science Foundation of China(32170510)the Innovation Training Program of Central South University(20240026020055),China.
文摘Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host’s immune system is compromised.Rhoptry proteins are major virulence factors that enable T.gondii to invade host cells.This study aims to construct a T.gondii rhoptry protein 41(rop41/ROP41)gene knockout strain and preliminarily investigate the biological function of rop41.Methods:Using CRISPR/Cas9 technology,a specific single-guide RNA(sgRNA)for the target gene was designed and linked to a recombinant plasmid.Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes.The recombinant plasmid and the homologous fragments were electroporated into T.gondii,and PCR identification was performed after drug selection and monoclonal screening.Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T.gondii in host cells.Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity.The STRING database was utilized to construct a protein-protein interaction(PPI)network,and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.Results:The T.gondii rop41 gene knockout strain(RHΔku80Δrop41)was successfully constructed and stably inherited.Plaque assays showed that compared with the parental strain,the number of plaques formed by the rop41 gene knockout strain did not significantly decrease,but the reduction in plaque size was statistically significant(P<0.05).After the rop41 gene was knocked out,the invasion ability of T.gondii was reduced,but there was no statistically significant difference in its proliferation ability(P>0.05).The PPI network revealed that ROP41 was associated with other protein kinases and autophagy related proteins.Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction,biosynthesis,metabolism,and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.Conclusion:The T.gondii RHΔku80Δrop41 strain has been successfully constructed.ROP41 primarily affects the ability of T.gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T.gondii and the host.
基金supported by the National Key Research and Development Plan of China(2017YFD0501602)the Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five Plan(IDHT20170516)
文摘The classic method for gene knockout (KO) is based on homologous recombination (HR) and embryonic stem cell technique (Gerlai,1996).Actually,the procedure of homologous replacement is complicated and time consuming,although it has been popular during the past decades.Recent years,genome editing which can cause DNA sequence-specific mutations in the genomes of cellular
基金the National Natural Science Foundation of China,No.81372585 and No.81772557Beijing Health System High Level Training Plan of Health Technical Personnel,No.2014-3-048
文摘BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system.AIM To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout mice.METHODS We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional(intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin(HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation markers.RESULTS We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted. The intestinal crypt size in inducible conditional Claudin-7 knockout mice was increased compared with control mice(small intestine: 54.1 ± 2.96 vs 38.4 ± 1.63;large intestine: 44.7 ± 1.93 vs 27.4 ± 0.60; P < 0.001).CONCLUSION The knockout of Claudin-7 in vivo causes extensive inflammation, atypical hyperplasia, and adenoma in intestinal tissue as well as animal death in mice.Claudin-7 may act as a tumor suppressor gene in the development of colorectal cancer.
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 National Research Foundation of Korea(NRF)grants funded by the Ministry of Science,ICT,and Future Planning[2017M3A9C4065958 and 2018R1A2B6002192]by Korea Drug Development Fund(KDDF)funded by Ministry of Science and ICT,Ministry of Trade,Industry,and Energy,and Ministry of Health and Welfare[RS-2023-00283544]by a grant from the Asan Institute for Life Sciences,Asan Medical Center,Seoul,Republic of Korea[2024IP0045].
文摘The CRISPR-Cas system is a powerful genetic engineering tool and can be conveniently used for the generation of diverse gene-knockout models.One CRISPR-Cas system,CRISPR-Cpf1(also known as Cas12a),recognizes the AT-rich protospacer-adjacent motif(PAM)present at the 5′end of the target sequence and requires CRISPR RNA(crRNA),but not transactivating crRNA(tracrRNA)for its activity.
文摘This is a correction to:PingWang,Zunpeng Liu,Xiaoqian Zhang,Jingyi Li,Liang Sun,Zhenyu Ju,Jian Li,Piu Chan,Guang-Hui Liu,Weiqi Zhang,Moshi Song,Jing Qu,CRISPR/Cas9-mediated gene knockout reveals a guard-ian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells,Protein&Cell,Volume 9,Issue 11,November 2018,Pages 945-965,https://doi.org/10.1007/s13238-018-0560-5 In the originally published online version of this manuscript,there was an incorrect image in Figure 2,panel G.This should read:This change does not alter the discussions or conclusions of the paper.
基金NationalOutstandingYouthSciencesFoundation (No 3972 5 0 2 6)andPostdoctoralSciencesFoundationofChina (No 2 0 0 0 2 3)
文摘Abstract Objectives To investigate the tissue specificity of reactive oxygen species (ROS) damage to mitochondrial DNA (mtDNA) and to determine whether cochlear mtDNA is a sensitive target for ROS damage. Methods 10 Cu/ZnSOD gene (Cu/Zn superoxide dismutase gene, Sod1) knockout mice and 16 wild-type mice were analyzed by nested polymerase chain reaction (PCR).Results Three deletions were detected in various tissues of Sod1 knockout mice. MtDNA3867bp and mtDNA3726bp deletions were the most visible, and mtDNA4236bp deletion was barely detected in these tissues. There were obvious differences in the ratio of deleted mtDNA/total mtDNA in different tissue. Deleted mtDNA was most abundant in the liver and kidney and less in cochlea, heart and brain. The lowest was in spleen and skin. The ratio in various tissues was 3-20 times in Sod1 knockout mice over wild-type mice. In cochlea, the ratio was about 15. Conclusions Without the protection of Sod1, ROS can lead to mtDNA deletions in various tissues with significant tissue specificity. Cochlear mtDNA is a sensitive target for ROS damage.
基金the Natural Science Foundation of Shandong Province,No. Y2008C54
文摘BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.
基金Supported by the National Natural Science Foundation of China （No. 81102584）Guangdong Provincial Administration of Traditional Chinese Medicine （No. 2009386）
文摘Objective： TO observe the effect of Huxin Formula （护心方, HXF） on expressions of the chief reverse cholesterol transport （RCT） associated genes, caveolin-1 and scavenger receptor-B I （SR-B I ） in ApoE-gene knockout [ApoE （-/-）] mice. Methods： Thirty ApoE （-/-） mice of 4-6 weeks old were randomly divided into three groups （A-C）. After being fed with high-fat diet for 16 weeks, they were treated with HXF （1 mL/100 g）, pravachol （0.3 mg/100 g）, and saline in equal volume respectively for 16 weeks successively; in addition, a blank group was set up with 10 C57BL/6J mice of 6-week old received 16-week high-fat feeding and saline treatment. Animals were sacrificed at the termination of the experiment, their paraffin sections of aortic tissue were used to measure the size of plaque, expressions of cavolin-1 and SR-B I were detected by immunological histochemical method. Results： As compared with the blank group, levels of caveolin-1 and SR-B I were increased in Groups A and B （P〈0.01）; but the increase in Group A was more significant than that in Group B （P〈0.05）. The plaque/aorta area ratio decreased significantly in Groups A and B, but showed insignificant difference between the two groups. Conclusion： HXF could obviously increase the expressions of RCT associated genes, caveolin-1 and SR-B I, promote the RCT process, so as to reduce the formation of aorta atherosclerotic plaque in ApoE （-/-） mice.
基金supported by grants from the National Key Basic Research Program(973 Program)of China(No.2002CB513005)Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT,No.IRT0731)+4 种基金National Natural Science Foundation of China(No.30670828No.30572151,No.30700291)Joint Fund of NSFC with the Guangdong Provincial Government(No.U0632004)Project of Science and Technology Program of Guangdong Province(No.A1090202)Project of Science and Technology Program of Guangzhou City(No.2007J1-C0301).
文摘p38 regulated/activated protein kinase(PRAK)plays a key role in cell senescence and tumor suppression.The aim of this study was to investigate if PRAK had effect on cell proliferation.The growth of PRAK+/+and PRAK^(–/–)mouse embryonicfibroblast(MEF)cells was measured by methylthiazoletetrazolium(MTT)colorimetric assay,and the proportion of the cell number in different phases of the cell cycle was analyzed byflow cytometry.The growth curves showed that the growth rate was notably decreased,and cell double time was elongated in PRAK^(–/–)cells;moreover,the number of PRAK^(–/–)cells was decreased by 44.5%compared with that of PRAK+/+cells cultured for 96 h,suggesting that G2/M transition is inhibited in PRAK^(–/–)cells.Meanwhile,G1/S transition was also inhibited in PRAK^(–/–)cells,observed withflow cytometry analysis.The ratios of G0/G1,G2/M,and S phases of PRAK+/+cells were 44.9%,12.2%,and 42.9%,respec-tively,while those of PRAK^(–/–)cells were 55.3%,7.3%,and 37.4%,respectively.There were 23.1%increase and 12.7%decrease of the number of PRAK^(–/–)cells in G1 and S phases in comparison with that of PRAK+/+cells,respectively.Taken together,PRAK gene knockout in MEF cells leads to cell cycle arrest and proliferation inhibition.
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supported by grants from the National Natural Science Foundation of China (Nos. 31501376 and 31570369)the National Key Research and Development Program of China (No. 2016YFD0101804)the National Transgenic Science and Technology Program (No. 2016ZX08010002)
文摘Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first commercial hybrid maize was introduced in the 1930s（Duvick,2001）.
