ObjectiveTo investigate the knockdown efficiency of the liver-specific knockdown recombinant adeno-associated virus （AAV） thyroid-binding globulin （TBG） （AAV-TBG-shOTUD6B） targeting the ovarian tumor domain 6B （OTUD6B） of the ubiquitin-proteasome system in the liver of C57BL/6J mice and its regulatory effect on liver glucose metabolism. MethodsAn effective siOTUD6B sequence was designed and verified. The DNA sequences of non-specific interference RNA negative control （shNC） and shOTUD6B were inserted into the AAV vector backbone containing the promoter TBG to construct and purify AAV-TBG-shNC and AAV-TBG-shOTUD6B viruses. AAV-TBG-shNC and AAV-TBG-shOTUD6B were injected into mice via the tail vein to target the liver. After 8 weeks of high-fat diet （HFD） feeding， the levels of OTUDB protein and mRNA in the mice liver， as well as blood glucose levels， glucose tolerance， insulin sensitivity， and protein expression levels of phosphorylated protein kinase B （p-AKT） and phosphorylated glycogen synthase kinase-3β （p-GSK-3β） were detected. ResultsCompared with the AAV-TBG-shNC group， the OTUD6B protein level in the liver of the AAV-TBG-shOTUD6B group decreased， the mRNA level of OTUD6B in the liver decreased （P<0.01）， the fasting blood glucose levels of the mice after 4 hours of fasting and 16 hours of fasting both decreased （P<0.05， P<0.01）， the protein level of p-GSK-3β in the liver decreased （P<0.05）， glucose tolerance was enhanced （P<0.05）， insulin sensitivity was enhanced （P<0.01）， and the protein level of p-AKT in the liver increased （P<0.05）. ConclusionThe construction of a recombinant adeno-associated virus for liver OTUD6B knockdown is successfully completed. OTUD6B plays an important role in regulating the homeostasis of liver glucose metabolism. Liver-specific knockdown of OTUD6B can improve the blood glucose levels of mice induced by HFD and enhance insulin sensitivity.

Objective: To analyze differences in the expression levels of lipid metabolism-related proteins in adi- pose tissue exosomes between obese mice and wild-type mice using proteomic techniques . Methods: Wild-type (WT) and obese (ob/ob) model mice of the same age were selected , with 8 mice per group . Adipose tissue from both groups was minced and cultured for 48 hours . Conditioned media was collected , and exosomes were isolated u- sing differential centrifugation . Liquid chromatography-tandem mass spectrometry ( LC-MS/MS) was utilized for proteomic analysis to screen differentially expressed proteins ( DEPs) . Gene ontology ( GO) enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on the DEPs . Heatmaps were generated to visualize DEPs expression patterns , and Western blot was employed to validate DEPs expression levels . Results: Exosomes were successfully extracted from the culture supernatant of adipose tissues from mice in the WT group and the ob/ob group . Mass spectrometry analysis identified a total of 25 629 peptides and 3 376 proteins . Compared with the WT group , there were 699 proteins with high expression and 632 proteins with low expression in the exosomes derived from adipose tissues of ob/ob mice . Both GO and KEGG analyses showed that DEPs were mainly enriched in metabolic pathways . Heatmap analysis visualized the expression patterns of metabolism-related DEPs , and the expression levels of lipid metabolism-related proteins such as acyl-CoA syn- thetase long-chain family member 1 (ACSL1) , apolipoprotein E (ApoE) , and albumin (Alb) changed significant- ly in the obese state (P < 0. 05) . Western blot verification results showed that the expression of ApoE and Alb pro- teins in the adipose tissue-derived exosomes of ob/ob mice decreased ( P < 0. 01) . Conclusion In the obese state , the expression levels of lipid metabolism-related proteins in mouse adipose tissue exosomes are significantly altered . These differentially expressed proteins may thus serve as potential molecular targets for treating obesity and its associated metabolic complications .

BACKGROUND:The second heart field is crucial for the development of the embryonic heart.Abnormal development of the second heart field can result in multiple cardiac malformations.After Cx43 gene knockout,reduced formation and proliferation of cells of the second heart field can be observed,but the specific reason remains unclear. OBJECTIVE:(1)To determine whether β-catenin,Smo and Cx43 were co-expressed in the second heart field and the endoderm,we observed the expression patterns of these proteins.(2)To explore whether Cx43 interacts with the Wnt/β-catenin pathway or the Shh pathway in the development of the second heart field. METHODS:Serial paraffin sections of the mouse embryos at embryonic days 10-12 were selected for immunohistochemical staining,hematoxylin-eosin staining and immunofluorescence staining.The primitive gut of mouse embryos at embryonic day 11 was separated for western blot assay and co-immunoprecipitation. RESULTS AND CONCLUSION:(1)Cx43 and Isl1 were co-expressed in some mesenchymal cells on the ventral side of the foregut and dorsal wall of the pericardial cavity of mouse embryos at embryonic days 10-12;Isl1 positive cells increased while Cx43 positive cells increased.(2)Cx43 and β-catenin were co-expressed in the ventral part of the endoderm at embryonic days 10-12.(3)Cx43 and Smo were co-expressed in the endoderm at embryonic days 10-12.(4)The co-immunoprecipitation results confirmed that there was an interaction between Cx43 and β-catenin,which suggested that Cx43 interacted with β-catenin to participate in the development of the second heart field.

China ; epidemiology ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; classification ; isolation & purification ; metabolism ; Humans ; Shiga Toxin 1 ; biosynthesis ; isolation & purification ; Shiga Toxin 2 ; biosynthesis ; isolation & purification ; Sorbitol