BACKGROUND:Studies have found that glucagon-like peptide-1 and its analogues have a significant neuroprotective effect,and some drugs have been applied to the clinical stage Ⅲ study of Alzheimer's disease.However,the mechanism of its neuroprotective effect is still unclear,which needs to be further explored and clarified. OBJECTIVE:To screen out the genes related to the pathogenesis of Alzheimer's disease and the related targets of semaglutide for the treatment of Alzheimer's disease based on bioinformatics and network pharmacology analyses,to identify the potential target genes by comprehensive analysis of the two and to verify them at the cellular level. METHODS:Using DisGeNET database,differentially expressed genes between Alzheimer's disease patients and healthy population were screened out.The chemical structure formula and two-dimensional structure diagram of semaglutide were obtained using PubChem online database.GO/KEGG enrichment analysis was performed using DAVID online database.A protein-protein interaction network was constructed by using the STRING database.The HPA database was used to determine the distribution characteristics of the target proteins in various human tissues.Finally,western blot was used to detect relevant protein expression in HT22 cells after semaglutide intervention. RESULTS AND CONCLUSION:With the dataset in DisGeNET database,3 374 differentially expressed genes between Alzheimer's disease patients and healthy people were obtained,and meanwhile,101 target genes of semaglutide potential drugs were obtained.There were 23 intersection genes between them.Ten key genes were identified based on the protein-protein interaction network,which were silent information regulator 1(SIRT1),CASP9,CCND1,CASP1,KEAP1,DLG4,CASP4,GRB2,GRIA1,and EDNRA.The results of GO gene functional annotation analysis of key genes showed that the positive regulatory activity of cysteine endopeptidase,the positive regulation of proteolysis,and the positive regulation of cysteine endopeptidase involved the cytoplasmic part of the apoptotic activity process;AMPA glutamate receptor complex,inflammatory complex,CARD domain binding,cysteine endopeptidase activity,and cysteine endopeptidase activity were involved in the apoptotic process.The results of KEGG signaling pathway analysis indicated that colorectal cancer,non-small cell carcinoma,and endometrial carcinoma were related to immune infiltration,inflammation and autophagic apoptosis.In addition,according to the association ranking of key genes and their distribution in different tissues of HPA online database,SIRT1 was identified as the most significant differential gene.The expression level of SIRT1 protein was significantly down-regulated in HT22 cells after β-amyloid protein 1-42 treatment,but it could be significantly increased after being treated with semaglutide.To conclude,SIRT1 may be a target gene for semaglutide in the treatment of Alzheimer's disease.

Objective To use bioinformatics analysis to identify the key genes and signaling pathways involved in cardiac dysfunction after acute ischemic stroke.Methods The GSE102558 dataset was downloaded from the Gene Expression Omnibus(GEO)database,and genes with values of P<0.05 and|log2FC|>0.6 were identified as being differentially expressed.The MCODE plugin in Cytoscape software performed a functional module analysis of the protein-protein interaction(PPI)network,while the CytoHubba plugin screened for core genes.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were also performed.A middle cerebral artery occlusion model was constructed to verify core gene expression using real-time PCR.Results Among the screened differential genes,385 were upregulated and 354 were downregulated.The top ten core genes were Col1a1,Col1a2,Col3a1,Fbn1,Postn,Col5a1,Mmp3,Eln,Acta2,and Timp3.The GO enrichment mainly involved the extracellular matrix,the collagen fiber tissue,vascular development,and protease binding.KEGG was mainly enriched in protein digestion and absorption,the relaxin pathway,advanced gly-cation end products-receptor for advanced glycation end products,and platelet activation.Real-time PCR verified that Col3a1and Postn expressions decreased in the heart tissue after acute ischemic stroke.Conclusion Col3a1and Postnexpressions may be closely associ-ated with cardiac dysfunction occurrence and development after acute ischemic stroke.

Objective:To establish a rapid quantitative PCR (qPCR) technique for Mycobacterium marinum skin infections, and to analyze its clinical diagnostic efficiency. Methods:DNA was extracted from Mycobacterium marinum colonies and serially diluted (10 -1 to 10 -8). Twelve pairs of previously reported primers and probes, as well as 6 pairs of newly designed primers and probes in this study, were used for qPCR amplification to identify the most sensitive primers and probes for the detection of Mycobacterium marinum. Skin lesion tissues were collected from 72 patients with confirmed Mycobacterium marinum infections (experimental group) and 68 with other mycobacterial infections (control group) at Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Venereology, Shandong First Medical University & Shandong Academy of Medical Sciences in 2021. These skin tissues were subjected to qPCR amplification, interferon-gamma release assay (IGRA), acid-fast staining, and tissue culture to evaluate the diagnostic efficacy. Results:The newly designed primers and probes targeting the mycobacterial enhanced infection locus 2 (Mel2) demonstrated the highest sensitivity, with a detection limit of 0.86 copies/μl (cycle threshold value = 37) ; the qPCR amplification with the Mel2 primers/probes did not yield positive results when used for the detection of other mycobacteria (including Mycobacterium leprae and Staphylococcus spp) . Among the 72 patients in the experimental group, 44 were positive for qPCR with a sensitivity of 61.1% (95% CI: 49.6% - 71.5%), and 47 were positive for culture with a sensitivity of 65.2% (95% CI: 53.8% - 75.3%) ; all the 68 controls were negative for both qPCR and culture, with their specificities both being 100%. Among 65 patients subjected to IGRA, 31 were positive with a sensitivity of 47.7% (95% CI: 36.0% - 59.6%), while 16 out of 25 controls were negative for IGRA with a specificity of 64.0% (95% CI: 44.5% - 79.8%). Among 58 patients subjected to acid-fast staining, 37 were positive with a sensitivity of 63.8% (95% CI: 50.9% - 74.9%), and 52 out of 66 controls were negative for acid-fast staining with a specificity of 78.8% (95% CI: 67.5% - 86.9%). The combination of qPCR and culture resulted in a sensitivity of 93% and a specificity of 100% for the detection of Mycobacterium marinum. Conclusion:In this study, a highly sensitive qPCR assay was developed for the detection of Mycobacterium marinum, and its combination with culture could further improve the detection sensitivity.

Objective:To express and purify VP7 protein of group A rotavirus (RVA) G1P[8]. The VP7 polyclonal antibody was prepared and its function was evaluated.Methods:The G1 VP7 protein was expressed by baculovirus expression system and purified by affinity chromatography. Polyclonal antibody against G1 VP7 was obtained by immunizing rabbits with G1 VP7 protein. The function of the G1 VP7 polyclonal antibody was verified by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay.Results:The soluble G1 VP7 protein of human RVA G1P[8] was obtained using the baculovirus expression system and the VP7 protein was mainly in trimer state. The G1 VP7 polyclonal antibody was prepared and displayed relatively high binding titer to G1 VP7 protein by ELISA. The VP7 polyclonal antibodies could recognize multiple G-type RVAs by WB and ELISA. Immunofluorescence assay further demonstrated that G1 VP7 polyclonal antibody can bind to different RVAs, including Wa (genotype G1P[8]), DS-1(genotype G2P[4]), SA11 (genotype G3P[2]), and human G9P[8] RV strains. In addition, double sandwich ELISA showed that VP7 polyclonal antibody could be used to detect rotavirus in clinical samples.Conclusions:The soluble G1 VP7 protein was successfully expressed and VP7 antibody was obtained. The G1 VP7 polyclonal antibody could bind to a variety of G-type rotaviruses, which lays a foundation for the establishment of detection method of different G type rotaviruses.

Objective:To understand the epidemiological characteristics of group A rotavirus (RVA) infection in hospitalized children under 5 years of age with diarrhea in 2019, and to provide reference for the surveillance of RVA.Methods:Stool samples and clinical information of hospitalized children under 5 years of age with diarrhea were collected from sentinel hospitals in 20 provinces in 2019. RVA nucleic acid detection and genotyping were performed according to the rotavirus detection method in the National Viral Diarrhea Surveillance Program.Results:A total of 5 395 viral diarrhea samples were collected, 5 038 were tested, and 1 247 diarrhea samples showed RVA positive results (1 247/5 038, 24.75%). The positive rate of RVA in Fujian province was the lowest (30/319, 9.40%), and the positive rate of RVA was the highest in Henan province (182/338, 53.85%). The positive rate of RVA in male and female children was 25.24%(762/3 019)and 24.02%(485/ 2 019), respectively. There was no significant gender distribution of RVA infection ( χ2 = 0.96, P=0.326). Children aged 12 to 17 months were mainly susceptible to RVA (342/1 033, 33.11%), and the positive rate of RVA in children aged 48 to 59 months was lower (35/227, 15.42%). RVA infection showed significant age distribution characteristics ( χ2 = 86.78, P<0.001). RVA infection had significant difference between urban and rural areas ( χ2 = 20.92, P<0.001) and seasonal characteristics ( χ2 =411.42, P<0.001). RVA genotyping showed that G9P[8] type (994/1 122, 88.59%) was the dominant epidemic strain. Conclusions:In 2019, the main genotype of RVA infection in hospitalized children under 5 years of age with diarrhea was G9P[8], and RVA infection had significant age, region and season characteristics.

Objective:To explore the pathogenic spectrum and epidemiological characteristics of respiratory viruses in children with acute lower respiratory tract infection in Changsha, and provide scientific basis for disease prevention and treatment.Methods:A total of 1 092 respiratory tract specimens of children were collected and 12 respiratory viruses were detected by real-time quantitative transcription polymerase chain reaction.Results:Among the samples from 1 092 cases, those from 437 cases (40%) were positive for respiratory syncytial virus (RSV), 337 cases (30.9%) were positive for parainfluenza virus-3 (PIV-3), 263 cases (24.1%) were positive for human bocavirus (HBOV) and 228 cases (20.8%) were positive for adenovirus (ADV). The detection rates of boys and girls were 82.26% and 83.42%. The infection rate of RSV was higher in the group ≤ 6 months of age, the infection rates of PIV-3 and HBOV ≤2 years old were higher, and the infection rate of ADV was higher in the group between 6 months to 5 years old. The detection rates of virus infection of spring, summer, autumn and winter were 90.48%, 83.50%, 62.26% and 82.80%, respectively, there were significant differences among them.Conclusions:The main viruses in children with acute lower respiratory tract infection in Changsha were RSV and PIV-3. Mixed infections were common. Children under 2 years of age were more likely to get acute lower respiratory infections. Viruses had seasonal trends and peaked in winter and spring.

Objective:To study the binding characteristics of horse-derived P[12] rotavirus GST-VP8*-Horse P[12]E403 protein to oligosaccharides and saliva receptors, provides an important scientific basis for the cross-species transmission and the mechanism of interaction between the bodies.Methods:The E. coli expression system was used to express and purify the horse-derived P[12] rotavirus GST-VP8*-Horse P[12]E403 protein. The receptor binding characteristics of this genotype were analyzed by saliva and oligosaccharide binding experiments. Results:Horse-derived GST-VP8*-Horse P[12]E403 protein binds well with mucin core 2 sugar, but does not bind to other oligosaccharides such as A, B, Lewis, and HBGAs in saliva.Conclusions:The potential receptor of VP8*-Horse P[12]E403 protein may be mucin core 2, and it did not bind to human saliva.

Objective To evaluate the clinical efficacy and adverse reactions of S-1 chemotherapy plus high-low oxygen radiotherapy synchronously in treatment of patients with locally advanced pancreatic cancer. Methods Sixty-four patients with locally advanced pancreatic cancer in Rizhao People's Hospital and the Affiliated Hospital of Qingdao University from January 2013 to October 2015 were randomly divided into study group and control group by using envelope method, in which the study group was treated with oral administration of S-1 plus high-low oxygen radiation synchronously and the control group with intravenous gemcitabine chemotherapy. The efficiency, disease control rate, clinical benefit rate, distant metastasis rate, survival rate and adverse reactions of the two groups were compared. Results The effective rates of the study group and the control group were 70.4 %(19/27)and 32.1 %(9/28),the difference was statistically significant (χ2=8.04,P<0.01), and the disease control rates were 88.9 % (24/27) and 67.9 % (19/28) (χ2= 3.56, P >0.05). The clinical benefit rates of the study group and the control group were 77.8 % (21/27) and 57.1 % (16/28) (χ2=2.66,P >0.05), and the distant metastasis rates were 63.0 %(17/27) and 71.4 %(20/28) (χ2=0.45, P > 0.05). The 1-year survival rates of the study group and the control group were 63.0 % (17/27) and 32.1 % (9/28), and the 2-year survival rates were 37.0 % (10/27) and 10.7 % (3/28), the differences were statistically significant (χ2= 5.24, P < 0.05; χ2= 5.28, P< 0.05). While there were no significant difference in the incidence of serious adverse reactions between the two groups (P> 0.05). Conclusion The treatment of locally advanced pancreatic cancer with S-1 plus high-low oxygen radiotherapy synchronously is better than that with intravenous gemcitabine chemotherapy in terms of effective rate, 1-year survival rate and 2-year survival rate,with no increase of adverse reactions.

Objective: To explore the receptor binding specificity of VP8^* protein of porcine P[19] rotaviruses (RVs) with oligosaccharides. Methods: The porcine P[19] VP8^* protein was expressed and purified. The receptor binding specificity of P[19] VP8^* was analyzed by oligosaccharide binding and saliva binding assay. Results: The P[19] VP8^* protein showed significant binding to mucin cores, especially mucin core 2. Conclusions Mucin core 2 may be a potential receptor for the porcine P[19] RV, which provides certain basis for the study of virus infection mechanism and RV surveillance.

Objective: The VP8^* core protein of rotavirus P[6] genotype LL4260 was purified by prokaryotic expression, which is important for further study of protein structure and function. Methods: The P[6] genotype LL4260 strain was obtained by PCR.The recombinant plasmid pET30 a-LL4260VP8^*core was inserted into pET30 a vector and transformed into BL21 (DE3) competent cells with the correct recombinant plasmid. The expressed protein is purified by affinity chromatography and molecular sieve chromatography. Results: The pET30 a-LL4260VP8^* core region protein is soluble in the supernatant and proteins of approximately 22 kDa are identified by electrophoresis using purified proteins. Conclusions In this study, LL4260 containing pET30 a-LL4260VP8^* core plasmid was successfully constructed and LL4260 strain VP8^* protein was expressed.