We studied the function of connexin 43 (Cx43) gene in Xiao Meishan swine bone marrow mesenchymal stem cells (BMSCs) resulting from overexpression. Cx43 eukaryotic expression vector (pEGFP-Cx43) was constructed and transfected into BMSCs by nucleofector, after detecting the transfection efficiency; the expression of Cx43 was verified by RT-PCR, immunofluorescence and western blotting. Furthermore, we detected its cell cycle and apoptosis through flow cytometry. Our results show that pEGFP-Cx43 plasmid was successfully constructed, and green fluorescence in pEGFP-Cx43 transfected BMSCs was highly expressed with 60% transfection efficiency. In transgenic Xiao Meishan swines BMSCs, the expression level of Cx43 mRNA and protein were up-regulated. Meanwhile, the ability of cell proliferation was significantly increased, and the apoptosis rate was significantly reduced. Taken together, Cx43 overexpression could promote the proliferation of Xiao Meishan swine's BMSCs and markedly reduce their apoptosis, which provides evidence for in vivo research.
Animals ; Animals, Genetically Modified ; Apoptosis ; Cell Proliferation ; Connexin 43 ; metabolism ; Flow Cytometry ; Genetic Vectors ; Mesenchymal Stromal Cells ; metabolism ; Plasmids ; Swine ; genetics ; Transfection

The aim of the present study was to investigate the effects and mechanisms of quercetin on plantar incision-induced matrix metalloproteinase (MMP)-9 and MMP-2 activity,microglia activation and the analgesic effect of quercetin on plantar incision surgery-treated mice.Postoperative pain model was mediated by plantar incision surgery and Von Frey Hairs was used to test the mechanical pain threshold.The activity of MMP-9 and MMP-2 in spinal cord was evaluated by gelatin zymography.The marker of microglia ionized calcium binding adapter molecule 1 (IBA-1),phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) was detected by Western blot.Results showed that quercetin (20,40,80 mg/kg,ip) significantly inhibited plantar incisioninduced mechanical allodynia and suppressed the activity of MMP-9 and MMP-2 in the spinal cord.Moreover,quercetin also markedly inhibited plantar incision-induced up-regulation of IBA-1 and p-p38 in spinal cord.In conclusion,quercetin may alleviate postoperative pain by suppressing MMP-9 and MMP-2 activity in microglia.

To obtain the pure and soluble P51 antigen of HIV-1 strain CN54, we transformed the Escherichia. coli strain BL21 codonplus-RIL with recombinant plasmid pTHioHisA51 which carries a gene encoding the Polymerase (Pol) P51 antigen of HIV-1 CN54 formerly, and induced protein expression by IPTG. We purified the recombinant protein with Chelating Sepharose FF-Ni and DEAE-Sepharose FF column chromatography, then renatured the recombinant protein by dialyzation. Purified protein was identified by Western blotting. We labeled and coated antigen P51 in a dual-antigen sandwich system, and tested it with serum samples from HIV-infected individuals. The results showed that P51 was expressed as inclusion body, and represented about 50% of total cellular protein. After purification and renaturation, the purity of P51 was up to 95%. Western blotting and sandwich ELISA demonstrated that recombinant P51 had good anti-HIV antibody specificity and sensitivity. The results suggested that recombinant HIV-1 P51 can be prepared as diagnostic reagent, and provides valuable support for HIV-1 detection and vaccine research.
Escherichia coli ; genetics ; metabolism ; HIV Antibodies ; blood ; immunology ; HIV Infections ; immunology ; virology ; HIV Reverse Transcriptase ; biosynthesis ; genetics ; immunology ; HIV-1 ; classification ; immunology ; Humans ; Protein Renaturation ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sensitivity and Specificity

OBJECTIVEPsoriasis is an autoimmune-related chronic inflammatory skin disease strongly associated with the dysfunction of Th17 cells. Retinoic acid-related orphan nuclear receptor γt (RORγt) plays a critical role in the differentiation and maturation of Th17 cells and in cell-derived immunologic derangement. We conducted this study to investigate potential mechanism by which the derivative of digoxin selectively antagonizes RORγt transcriptional activity.

METHODUsing molecular docking in combination with molecular electrostatic potential (MEP), we detected the interaction between the derivative of digoxin (Dhd) and ROR transcription factor (RORα,RORβ and RORγt), and the results were further confirmed by bioluminescent assay.

RESULTMolecular docking demonstrated that Dhd could exclusively inhibit the conformation of RORγt; bioluminescent assay further indicated that RORγt was selectively antagonized by Dhd in a dose- and time-dependent manner.

CONCLUSIONDhd can selectively suppress RORγt transcriptional activity.

Digoxin ; analogs & derivatives ; pharmacology ; Humans ; Models, Chemical ; Molecular Docking Simulation ; Nuclear Receptor Subfamily 1, Group F, Member 1 ; antagonists & inhibitors ; genetics ; Transcription, Genetic

Background/Aims: Epstein-Barr virus-associated gastric cancers (EBVaGCs) have unique molecular and clinicopathological characteristics. The cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway is recently recognized as the critical innate immunity against pathogens and tumors. STING is also a master regulator in the cancer-immunity cycle and targeting STING could synergize with existing immune-checkpoint therapies. However, the role of STING in GC, especially in EBVaGC, and its correlation with programmed death-ligand 1 (PD-L1) remain largely unclear. Methods: We collected 78 cases of EBVaGCs and 210 cases of EBV-negative GC (EBVnGC) from a total of 1,443 cases of GC analyzed by EBV-encoded small RNA in situ hybridization. We investigated STING and PD-L1 expression and their concomitant prognostic value in EBVaGCs and EBVnGCs using tissue microarray and immunohistochemistry. The effects of STING and PD-L1 expression on the overall survival of patients with EBVaGC or EBVnGC were assessed by univariate and multivariate analysis. Results: We found that both STING and PD-L1 exhibited significantly higher expression in the EBVaGCs than that in the EBVnGCs. The expression of STING was positively correlated with that of PD-L1 in EBVaGCs. Simultaneous negative expression of STING and PD-L1, and positive expression of STING were independent prognostic risk factors for EBVaGC and EBVnGC, respectively. Conclusions This is the first prognostic retrospective study of STING and PD-L1 expression and the prognosis among EBVaGC and EBVnGC. The expression and prognostic value of STING and PD-L1 are different in the two types of GCs. STING and PD-L1 are promising prognostic biomarkers and therapeutic targets for EBVaGC and EBVnGC.

Objetive Psoriasis is an autoimmune-related chronic inflammatory skin disease strongly associated with the dysfunction of Th17 cells. Retinoic acid-related orphan nuclear receptorγt (RORγt) plays a critical role in the differentiation and maturation of Th17 cells and in cell-derived immunologic derangement. We conducted this study to investigate potential mechanism by which the derivative of digoxin selectively antagonizes RORγt transcriptional activity. Method Using molecular docking in combination with molecular electrostatic potential (MEP), we detected the interaction between the derivative of digoxin (Dhd) and ROR transcription factor (RORα,RORβ and RORγt), and the results were further confirmed by bioluminescent assay. Result Molecular docking demonstrated that Dhd could exclusively inhibit the conformation of RORγt;bioluminescent assay further indicated that RORγt was selectively antagonized by Dhd in a dose- and time-dependent manner. Conclusion Dhd can selectively suppress RORγt transcriptional activity.

Objetive Psoriasis is an autoimmune-related chronic inflammatory skin disease strongly associated with the dysfunction of Th17 cells. Retinoic acid-related orphan nuclear receptorγt (RORγt) plays a critical role in the differentiation and maturation of Th17 cells and in cell-derived immunologic derangement. We conducted this study to investigate potential mechanism by which the derivative of digoxin selectively antagonizes RORγt transcriptional activity. Method Using molecular docking in combination with molecular electrostatic potential (MEP), we detected the interaction between the derivative of digoxin (Dhd) and ROR transcription factor (RORα,RORβ and RORγt), and the results were further confirmed by bioluminescent assay. Result Molecular docking demonstrated that Dhd could exclusively inhibit the conformation of RORγt;bioluminescent assay further indicated that RORγt was selectively antagonized by Dhd in a dose- and time-dependent manner. Conclusion Dhd can selectively suppress RORγt transcriptional activity.

Objective To establish a dynamic model of lipopolysaccharide-induced acute lung injury in mice based on the NLRP3/Caspase-1/gasdermin D(GSDMD)pyroptosis pathway,and observe the result ing lung injury at different time points.We aimed to identify the optimal time for modelling according to the injury at different time points and the expression of pyroptosis pathway-related proteins,to lay the foundation for animal models for subsequent experiments.Methods Fifty-four 6～8 weeks old male SPF BALB/c mice were divided randomly into nine groups,including Con group and model groups at 1,3,6,12,18,24,48,and 72 h.Body weight and lung tissue were detected by general and pathological observations and semi-quantitative scoring,including lung index,lung water content,and wet and dry weight ratio.The white blood cell count and concentrations of tumor necrosis factor-α,interleukin(IL)-6,IL-1β,IL-18,and BCA protein were detected in bronchoalveolar lavage fluid(BALF).The classic pyroptosis pathway-related proteins NLRP3,pro-Caspase 1,Caspase 1,and GSDMD were detected by Western Blot.Results Body weight decreased in all experimental groups,with the most significant weight loss in the 24 and 48 h groups.Gross observation and pathological examination of lung tissue showed that the most severe lung injury occurred at 24～72 h,with significant differences between each group and the control group.The lung index,lung water content,and wet/dry weight ratio were also significantly increased at 24～72 h.White blood cells in BALF started to increase from 6 h after model initiation,48 h can reach a peak,72 h all keep increasing.IL-18 in BALF began to increase at 24 h and continued to increase at 72 h.The inflammatory factors tumor necrosis factor-α,IL-1β,IL-6 were highest at 6 h and significantly reduced at 48 h.Protein concentrations in BALF were significantly increased within 24,48,and 72 h compared with those in the control group.The pyroptosis pathway proteins NLRP3,pro-Caspase-1,Caspase-1,and GSDMD were significantly enhanced in each time series,and channel protein expression was significantly enhanced at 24～72 h compared with that in the Con group.Conclusions Comprehensive analysis of experimental indicators,inflammatory factors,and pathway proteins at different times showed that the mechanism of pyroptosis was closely related to the occurrence and progression of acute lung injury.Expression of the pyroptosis pathway was most obvious and lung injury was most serious at 24～48 h.This study provides a model reference and experimental basis for subsequent studies of the specific mechanism and intervention targets of acute lung injury.

The co-existence of different neurodegenerative diseases in the same case has received increasing attention and reports. A rare comorbidity of amyotrophic lateral sclerosis (ALS) and progressive supranuclear palsy (PSP) in a sporadic patient was reported. The patient presented with pseudobulbar palsy, freezing gait, and developed muscle weakness and fasciculation. Brain magnetic resonance imaging, positron emission computed tomography and whole exomesequencing showed no evident abnormality. The patient had no response towards the treatment of levodopa, and received supportive treatment and gastrostomy. He is in stable condition by now. Totally 23 cases of ALS with PSP were reviewed, and found that the clinical manifestations were related to the main distribution of pathological inclusion bodies and the location of neuron loss, and the deposition of the same pathological protein in multiple systems may lead to the coexistence of different neurodegenerative diseases. Traditional treatment is generally ineffective, thus support treatment is pivotal. The genetic background and pathogenesis of this comorbidity should be studied in the future.