Objective:To construct the over-expressed lentivirus vector of PRDM5 gene and establish the Neuro-2a cells stably transfected PRDM5,and to provide the basis evidence for exploring the effect of PRDM5 in pathogenesis of ischemic stroke(IS).Methods:The sequence of PRDM5 was searched and designed based on NCBI.The PRDM5 gene was amplified by PCR and ligated with the lentiviral vector GV492 digested by BamH Ⅰ and Age Ⅰ restriction enzymes to form the GV492-PRDM5 over-expression recombinant plasmid.The positive clones with similar length and size to the target gene fragment were screened by PCR and sent to Shenggong Bioengineering(Shanghai)Co.Ltd.for identification.The correctly-sequenced GV492-control plasmid and GV492-PRDM5 over-expression recombinant plasmid were transfected into the HEK293T cells,respectively.After 48 h of transfection,the lentiviruses were collected by centrifugation,and they were GV492-control lentivirus and GV492-PRDMS over-expression lentivirus;the titers of these two lentiviruses were determined by lentiviral titer assay.The Neuro-2a cells were divided into GV492-control group and GV492-PRDM5 group,and then infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus,respectively,with a lentivirus multiplicity of infection(MOI)of 100.The Neuro-2a cells successfully infected with GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were screened with puromycin(10 mng-L-1)after 72 h of infection.The growth status and the expression of green fluorescence protein of Neuro-2a cells in GV492-control group and GV492-PRDM5 group were observed by fluorescence microscope.The expression levels of PRDM5 mRNA and PRDM5 protein in the Neuro-2a cells in two groups were detected by real-time fluorescence quantitative RCR(RT-qPCR)and Western blotting methods.Results:The PCR results showed that the length of the positive transformant of GV492-PRDM5 recombinant plasmid was about 684 bp,and the gene sequence of GV492-PRDM5 over-expression recombinant plasmid was consistent with the designed and synthesized PRDM5 over-expression sequence.The titers of GV492-control lentivirus and GV492-PRDM5 over-expression lentivirus were both 2.5×108TU·mL-1 The Neuro-2a cells in GV492-control group and GV492-PRDM5 group grew well,and the expressions of green fluorescence protein were found under fluorescence microscope.The RT-qPCR results showed that the expression level of PRDM5 mRNA in the Neuro-2a cells in GV492-PRDM5 group was significantly increased compared with GV492-control group(P＜0.01).The Western blotting results showed that the specific bands appeared in the Neuro-2a cells in GV492-control group and GV492-PRDM5 group with a relative molecular weight of 75 000;compared with GV492-control group,the expression level of PRDM5 protein in the Neuro-2a cells in GV492-PRDM5 group was increased(P＜0.01).Conclusion:The over-expression lentivirus vector of PRDM5 gene is successfully constructed,and the stably transfected GV492-PRDM5-Neuro-2a cells are established.

ObjectiveTo determine the prevalence of avian influenza viruses in the external environment of poultry in Anqing City， Anhui Province， and provide scientific evidence for prevention and control of animal-derived influenza in humans. MethodsA total of 28 farmers’ markets/farms in 10 counties （cities， districts） of Anqing City， Anhui Province， were selected as surveillance sites by simple random sampling strategy. Poultry faeces and other related samples were collected for 6 consecutive weeks. Real-time fluorescence quantitative PCR was used to examine the nucleic acids of influenza A virus. Subtypes H5， H7， and H9 of avian influenza virus were further tested in the positive samples. ResultsA total of 426 specimens were collected， among which 113 tested positive with a positive rate of 26.53%. Among the positive specimens， 104 were determined to be subtype H9， accounting for 92.04%. It did not significantly differ in the positive rate between the main and non-main urban areas （χ²<0.01， P>0.05） or among the specimens collected in different weeks （χ²=7.57， P>0.05）. However， it significantly differed in the positive rate among the specimens collected in the third week and other weeks （χ²=6.89， P<0.05）. Furthermore， among the different sampling sites， farms had the highest positive rate of 46.67%. Among the specimens from different sources， the surface-coated specimens from poultry cages had the highest positive rate of 34.78%. ConclusionAvian influenza viruses are prevalent in the external environment of poultry in Anqing City. It warrants strengthening the surveillance and risk assessment to reduce the virus transmission in the external environment and risk of human infection with animal-derived influenza.

Antibodies, Neutralizing ; Antibodies, Viral/isolation & purification* ; B-Lymphocytes/virology* ; COVID-19/virology* ; Humans ; Immunity/genetics* ; RNA, Viral/immunology* ; SARS-CoV-2/pathogenicity* ; Single-Cell Analysis

Objective To understand the epidemiological characteristics of patients with advanced schistosomiasis in Hunan Province,so as to provide the evidence for formulating the advanced schistosomiasis prevention strategies and measures. Meth-ods The data of advanced schistosomiasis patients were collected and analyzed retrospectively with the cross section research method and description method in Hunan Province,2012. Results There were 5 722 advanced schistosomiasis patients in Hu-nan Province,and among them,4 112 patients were male(71.86%),and 1 610 were female(28.14%). Totally 5 311 patients came from the schistosomiasis endemic areas(92.82%)and 411 patients from non-schistosomiasis endemic areas(7.18%). The prevalence rate of advanced schistosomiasis was 8.46/10 000. The mean age of advanced schistosomiasis patients was 60.30 ± 11.63 years,and the youngest was 17 years old and the oldest 92 years old. In the age composition of advanced schistosomiasis pa-tients,the greatest number of cases was in the 60-70 years age group (32.72%). There were 3 595 cases of ascites type (62.83%),2107 cases of splenomegaly type(36.82%),11 cases of dwarf type(0.16%),and 11 cases of colon proliferation type (0.35%). Conclusion The prevalence rate of advanced schistosomiasis is relatively stable in Hunan Province,and the age of the patients showed an old aging trend. The salvation of advanced schistosomiasis patients in non-endemic areas should be strength-ened.

Objective To explore the application value of liquid chromotography tandem mass spectrometry (LC-MS/MS) in screening of inherited metabolic disease risk children ,and to provide a reference for the scientific prevention and control .Methods 246 cases of patients with inherited metabolic children blood samples were collected through the blood spots ,LC-MS/MS was used to examine blood samples from high risk cases of inherited metabolic who came from general hospital of Ningxia medical university . Results 30 cases were confirmed with inherited metabolic disease by the LC-MS/MS ,the positive rate was 12 .2% ,including 9 ca-ses of methylmalonic academia ,4 cases of isovaleric acidemia ,6 cases of phenylketonuria ,2 cases of propionic acidemia ,2 cases of citrullinemia ,1 cases of very long-chain acyl-CoA dehydrogenase ,1 cases of mitochondrial trifunctional protein deficiency ,1 cases of maple syrup urine disease ,3 cases of homocystinuria ,2 cases of argininesuccinic aciduria .Conclusion Liquid chromatography tan-dem mass spectrometry(L-MS/MS) technology is highly efficient and high reliability method in screening inherited metabolic dis-ease .

Objective To explore the effects of astrocytes in cervical spinal cord posterior horn on allodynia in rat models of migraine.Methods Fifty-four male SD rats were randomly divided into 9 groups (each group n =6): blank group,sham surgery group,new inflammatory soup 3 d group (IS 3 d group),new inflammatory soup 7 d group(IS 7 d group),saline group,fluorocitrate (FCA)prevention group,FCA prevention control group,FCA treatment group,FCA treatment control group.Following the IS stimulation in the rat dual matter,Von-Frey hair was used to monitor the pain threshold of periorbital skin.Immunofluorescence technique was used to observe the expression of specific marker of astrocyte,glial fibrilary acidic protein (GFAP),and neuron activation marker,C-fos.Results ①Von-Frey hair study showed the thresholds of IS 3 d group and IS 7 d group were significantly decreased compared with the control group(3 d:5.3 ±0.3 vs 6.4 ±0.3,F =40.15;7 d:3.0 ±0.3 vs 6.3 ±0.2,F =382.5,both P ＜0.01).The pain thresholds of FCA prevention group and FCA treatment group both began to significantly increase at the day 4 (FCA prevention:5.5 ± 5.1 vs 5.1 ± 0.2,F =16.00 ;FCA treatment:4.3 ± 0.2 vs 3.0 ± 0.2,F =138.0,both P ＜ 0.01).②GFAP immunofluorescence showed that the mean optical density (A) values of IS 3 d group and IS 7 d group were significantly increased compared with the control group(3 d:24.5 ±4.4 vs 14.8 ± 2.5,F =32.54;7 d:38.9 ± 7.1 vs 14.6 ± 1.8,F =63.56,both P ＜ 0.01).()C-fosimmunofluorescence showed that mean A value of IS 3 d group was significantly increased compared with the control group (20.4 ± 2.3 vs 8.4 ± 1.1,F =129.0,P ＜ 0.01).The difference was not significant between the IS 7 d group and the control group.Conclusions Activated astrocytes contribute to the facial allodynia induced by chronic dural inflammation.Its inhibitor FCA could both prevent and treat the allodynia behaviour.All of these suggest that astrocytes may not only contribute to the initiation of mechanical allodynia but also the maintenance.

In situ hybridization and immunocytochemical techniques were employed to examine the expression of matrix metalloproteinases-1 (MMP-1) and to identify the pattern of its distribution in rat pancreas. The results indicated that the signal of MMP-1 mRNA and MMP-1 positive immunoreaction were detected in some fiberoblasts around interlobular ducts and exocrine cell in margin acinus of some lobules, but the signal of MMP-1 mRNA and MMP-1 positive immunoreaction could not be detected in most of other acinus and islets of pancreas. It is concluded that the expression of MMP-1 in above cells of rat might play an important role in acinar proliferation and differentiation of rat pancreatic tissues.
Animals ; Cell Division ; Immunohistochemistry ; In Situ Hybridization ; Male ; Matrix Metalloproteinase 1 ; analysis ; biosynthesis ; genetics ; Pancreas ; enzymology ; RNA, Messenger ; analysis ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley