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
OBJECTIVE To observe the effects of serum metabolites by using ~1H-NMR-based metabonomic approach to explore the possible mechanisms of total flavonoids in Ocimum BasilicumLinn(OBL) on atherosclerosis in apolipomtein ...OBJECTIVE To observe the effects of serum metabolites by using ~1H-NMR-based metabonomic approach to explore the possible mechanisms of total flavonoids in Ocimum BasilicumLinn(OBL) on atherosclerosis in apolipomtein E knockout(ApoE-/-) mice.METHODS Six-week-old male apoE knockout mice were divided into four groups(n=10) and fed with high fet diet:model,Simv.astatin,OBL-H,OBL-M and OBL-L groups.The homogeneous male mice of C57 BL/6 J were used as the normol group and fed with normal chow diet.After 14 weeks,~1H-NMR technology was used to ex.plore the variability of serum metabolites by the method of PLS-DA and OPLS-DA.RESULTS Com.pared with normal group,Model group showed a significant increase in the serum levels of VLDL,LDL,β-hydroxyisobutyrate,lactate,myo-inositol and showed a significant decrease in the serum levels of al.anine,glutamine,proline,carnitine,methylamine,citrate,creatine,choline,taurine,pyruvate,β-glu.cose,α-glucose,glycine,lysine.Combined with model group OBL-H,OBL-M,OBL-L groups showed the effects of regulating the levels of different metabolites of the glucose,lipid and amino acid metabo.lism.CONCLUSION The anti-atheros-clerotic activity of total flavonoids in Ocimum BasilicumLinn may be related not only to regulation of lipid metabolism,but also glycometabolism and amino acid metabolism.展开更多
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 National Natural Science Foundation of China (81560586) and Xinjiang Natural Science Foundation (2016D01C161)
文摘OBJECTIVE To observe the effects of serum metabolites by using ~1H-NMR-based metabonomic approach to explore the possible mechanisms of total flavonoids in Ocimum BasilicumLinn(OBL) on atherosclerosis in apolipomtein E knockout(ApoE-/-) mice.METHODS Six-week-old male apoE knockout mice were divided into four groups(n=10) and fed with high fet diet:model,Simv.astatin,OBL-H,OBL-M and OBL-L groups.The homogeneous male mice of C57 BL/6 J were used as the normol group and fed with normal chow diet.After 14 weeks,~1H-NMR technology was used to ex.plore the variability of serum metabolites by the method of PLS-DA and OPLS-DA.RESULTS Com.pared with normal group,Model group showed a significant increase in the serum levels of VLDL,LDL,β-hydroxyisobutyrate,lactate,myo-inositol and showed a significant decrease in the serum levels of al.anine,glutamine,proline,carnitine,methylamine,citrate,creatine,choline,taurine,pyruvate,β-glu.cose,α-glucose,glycine,lysine.Combined with model group OBL-H,OBL-M,OBL-L groups showed the effects of regulating the levels of different metabolites of the glucose,lipid and amino acid metabo.lism.CONCLUSION The anti-atheros-clerotic activity of total flavonoids in Ocimum BasilicumLinn may be related not only to regulation of lipid metabolism,but also glycometabolism and amino acid metabolism.
