Objective:To investigate the drug resistance status and genomic characterization of Proteus mirabilis (PM) isolated from outpatient cases with diarrhea in Tai′an City and Laizhou City, Shandong Province. Methods:A total of 510 fecal samples were collected from 510 patients with acute diarrhea admitted to 43 sentinel hospitals in Tai′an City and Laizhou City, Shandong Province, between January 2021 and December 2022. The samples were cultured and isolated to identify Proteus spp. by direct inoculation, the drug susceptibility testing was performed by microbroth dilution method, and resistance genes and virulence genes were obtained by whole genome sequencing and bioinformatic analysis, thereby revealing the genetic environment surrounding the blaOXA-1 and blaCTX-M-65 genes. Single nucleotide polymorphism (SNP) analysis and core genome multilocus sequence typing (cgMLST) were conducted on the current strains and 100 PM isolates downloaded from the National Center for Biotechnology Information (NCBI) database via customizable methods utilizing RidomSeqSphere+ software, with the objective of exploring the phylogenetic relationships among the strains. Results:A total of 35 strains of Proteus were isolated from 510 fecal samples, including 31 strains of PM with a detection rate of 6.08% (31/510) and four strains of Proteus vulgaris.The multidrug resistance rate of PM was 100.00% (31/31).The 35 isolates carried a total of 43 resistance genotypes.Thirteen strains of PM carried blaOXA-1, six strains carried both blaOXA-1 and blaCTX-M-65, and 15 PM strains carried at least 15 antibiotic resistance genes (ARGs). The virulence genes included ureA, mrpA, ZapA, hpmA and so on. blaOXA-1 and blaCTX-M-65 genes were surrounded by mobile elements such as Tn3, ISL3 and IS6. cgMLST showed consistency with the SNP clustering results. Isolate 2022LZ41 from Laizhou City clustered with isolates 2022TA018, 2022TA017 and 2022TA019 from Tai′an City, with the number of allelic differences ranging from zero to two, and the Laizhou City isolate 2022LZ40 was highly genetically related to strain CRK0056 (human, USA, 2015). Conclusions:PM isolated from patients with diarrhea is multidrug-resistant, carrying many resistance and virulence genes.The presence of mobile genetic elements can lead to horizontal transfer of resistance genes.

Objective To explore risk factors of poor early prognosis in the treatment of COVID-19 by nematevir and ritonavir tablets Paxlovid and establish the prediction model to provide reference for improving the effect of such patients. Methods 92 inpatients of COVID-19 treated with Paxlovid in three military tertiary hospital in southern Fujian from January 2023 to March 2023 were retrospectively analyzed. The clinical indicators of 92 inpatients were collected for univariate and multivariate analysis by single factor and multiple factors and the independent risk factors of poor early prognosis in Paxlovid were screened out. Logistic model equation was transformed to construct the combined predictors, and ROC curve was used to determine the area under the curve (AUC) and the optimal critical value of the combined predictors. Results Among 92 patients, 31 (33.70%) developed poor early prognosis, including 11 deaths (35.48%), 17 critical cases (54.84%) and 3 severe cases (9.68%). Multi-factor Logistic regression analysis showed that the disease days, lymphocyte count, aspartate aminotransferase(AST), C reactive protein(CRP) and ventilator-assisted ventilation were independent risk factors for poor early prognosis in Paxlovid. A formula for calculating the combined predictors (Y) was established as Y_{combinedpredictors}=7.875X_{disease days}+126.188X_{lymphocyte count}+1.438X_AST+X_CRP+220.500X_{ventilator-assisted ventilation} based on the above independent risk factors, and the ROC curve was drawn. With the maximum area under the ROC curve of the combined predictors being 0.939, the prediction value was best, and the optimal critical value of the ROC curve corresponding to the maximum Youden index (0.756) was 447.920.Theoretical accuracy of the model was 89.10%. Conclusion The disease days, lymphocyte count, AST, CRP and ventilator-assisted ventilation were independent risk factors for poor early prognosis in Paxlovid. Combined predictors could be calculated by the above risk factors before medication. The efficiency should be improved by taking more active treatment, including combining with other anti-COVID-19 drugs when the prediction result exceeds 447.920.

Objective:Evidence on potential cardiovascular benefits of personal-level intervention among the elderly exposed to high levels of particulate matter(PM)remains limited.We aimed to assess improvements in surrogate markers of cardiovascular injury in vulnerable populations at risks by using indoor air filtration units.Methods:We conducted a randomized crossover trial for 2 separate 2-week air filtration interventions in 20 households of patients with stable chronic obstructive pulmonary disease and their partners in the winter of 2013,with concurrent measurements of indoor PM.The changes in biomarkers indicative of cardiac injury,atherosclerosis progression and systemic inflammation following intervention were evaluated using linear mixed-effect models.Results:In the analysis,average levels of indoor PM with aerodynamic diameters＜2.5 μm(PM2.5)decreased significantly by 59.2％(from 59.6 to 24.3 μg/m3,P＜0.001)during the active air filtration.The reduction was accompanied by improvements in levels of high-sensitivity cardiac troponin I by-84.6％(95％confidence interval[CI]:-90.7 to-78.6),growth differentiation factor-15 by-48.1％(95％CI:-31.2 to-25.6),osteoprotegerin by-65.4％(95％CI:-56.5 to-18.7),interleukin-4 by-46.6％(95％CI:-62.3 to-31.0)and myeloperoxidase by-60.3％(95％CI:-83.7 to-3.0),respectively.Conclusion:Indoor air filtration intervention may provide potential cardiovascular benefits in vulnerable popu-lations at risks.

Bacterial ghosts are bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology, which can be used as a new vaccine and delivery vector. In this study, a clinical isolate of avian pathogenic Escherichia coli (APEC) strain DE17 was used to prepare bacterial ghost through three different ways. The results showed that the cleavage efficiency of DE17 bacterial ghost was 99.9% with the lysis plasmid containing the PhiX174 lysis gene E. Scanning electron microscopy showed that transmembrane tunnels were formed in the middle or both ends of the cell envelope of DE17. Furthermore, the DE17 bacterial ghost was prepared with one of cell penetrating peptides (CPPs) named MAP (KLALKLALKALKAALKLA), which will completely inactivate DE17 (OD₆₀₀=0.1) by 10 μmol/L MAP. The cell envelope showed a gully-like structure and obvious transmembrane tunnels were not found through the SEM. However, the DE17 could not be lysed by importing the lysis plasmid (pBV220-MAP), which was used to express MAP. The present study will benefit for research on bacterial ghost preparation methods and provide a reference for biosafety of bacterial ghost vaccines.

Objective To study the effects of thymus transplantation(TT)combined with CD4--DLI on T cell reconstitution after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods BALB/c mice were randomly divided into three groups:hematopoietic stem cell transplantation (HSCT group),hematopoietic stem cell transplantation combined with thymus transplantation(TT group)and hematopoietic stem cell transplanta-tion combined with thymus transplantation plus CD4+ T cell-depleted lymphocyte infusion(CD4--DLI group). On day-1,the mice were treated with the lethal dose of radiotherapy. On day 0,C57BL/6 mice were used as donor for hematopoietic stem cell transplantation. The mice were sacrificed on 5 days,2 weeks,4 weeks and 3 months after transplantation,respectively. The peripheral blood and spleen cells of mice were collected for determinations of T cell surface antigen,T cell receptor,naive T cells and intracellular cytokines. HE staining was used to assess the development of donor thymus. Results TT and CD4--DLI did not impair each other′s effects on T cell reconstitu-tion. TT combined with CD4--DLI increased the number of T cell reconstruction. CD4--DLI promoted the effect of TT on enlargement naive CD4+and CD8+T cell pool. Combination of TT and CD4--DLI enhanced the cytokine pro-duction of T cells. Conclusion TT combined with CD4--DLI had no side effects on TCR repertoire and thymus. Conclusion TT combined with CD4--DLI can enhance the reconstitution of T cell number and function via thymus dependent and thymus independent mechanism.

OBJECTIVE: To construct and obtain ideal protein delivery vectors by researching the delivery efficiency and cytotoxicity to Hela cells using mPEG-PLGA-BSA-FITC-NPs. METHOD: The mPEG-PLGA nanoparticle was obtained through surface modification of PLGA with PEG, and deliver BSA-FITC into Hela cells in vitro. The positive cells were counted by Laser scanning confocal microscopy and the survival rate of Hela cells was calculated by MTT assay at different time points. RESULT: mPEG-PLGA-BSA-FITC-NPs shows the classic nanometer size, and the encapsulation efficiency reached 51. 2%. At the same time, the nanoparticles possess characteristics of slow release. By optimizing the delivery conditions, the highest efficiency of mPEG-PLGA-BSA-FITC-NPs was above 65.2%, and the cellular viability was about 85.7%. CONCLUSION mPEG-PLGA-BSA-FITC-NPs nanoparticles can successfully carry the target protein into cells as safe and effective as novel delivery materials of protein in vitro, and has shown slow release characteristics. The mPEG-PLGA-BSA-FITC-NPs provide ideal delivery vector for future application in clinical treatment of disease using nano-materials.
Drug Carriers ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; HeLa Cells ; Humans ; Nanoparticles ; Particle Size ; Polyesters ; Polyethylene Glycols ; Serum Albumin, Bovine

BACKGROUND:There are myoblasts in human embryonic skeletal muscle. It remains poorly understand whether myoblasts in vitro can form myotube and what are the corresponding markers for identifying myoblasts and myotubes. OBJECTIVE:To investigate whether in vitro cultured myoblasts from human embryonic skeletal muscle can form myotube and whether they can express neural markers. METHODS:Human embryonic muscle-derived myoblasts were cultured in serum-containing medium. When the primary culture was established, cultured cel s were identified with immunocytochemistry for neural markers, such asβ-tubulin markers (desmin, myogenin, smooth muscle actin and myosin). RESULTS AND CONCLUSION:A population of myoblasts could migrate from human embryonic muscle tissues. They could express the markers for skeletal muscle such as desmin and myogenin, and they could express neuron specific enolase, nestin and neurofilament 200. They could form myotubes in vitro, and myotubes expressedβⅢ-tubulin, neurofilament 200 and glial fibril ary acidic protein. The data support the hypothesis that myoblasts from human embryonic muscle express neural markers and muscle markers, and cultured myoblasts and myotubes expressed neuron specific enolase,β-tubulin Ⅲ, nestin, neurofilament 200 and glial fibrillary acidic protein. This indicates that these markers could not be used for cel identification of trans-differentiation study from muscle origin to nervous system.

BACKGROUND:At present,the most frequently agents used for neural induction of bone marrow stromal cells(BMSCs)in vitro are anti-oxidants,such as beta-mercaptoethanol and all trans-retinoids.The majorities of induction from BMSCs are neuron-like cells in these protocols;however,whether it has neuronal function or not should be further studied.OBJECTIVE:TO investigate the differentiated characteristics of inducing human BMSCs into neural cells in serum-free medium.DESIGN:Observational study.SETTING:Chaozhou Central Hospital.MATERIALS:The experiment was carried out in the Chaozhou Central Hospital from April 2004 to December 2005.Adult bone marrows were derived from femoral and tibial bone marrow of three patients with fracture.All patients provided the confirmed consent and were approved by the Ethics Committee of Chaozhou Central Hospital.DMEMIF12 medium(1:1),fetal bovine serum (FBS), glutamine, N2 supplements and B27 Supplements were from GIBCO/BRL Company;recombinant basic fibroblast growth factor(bFGF)and recombinant epidermal growth factor(EGF)from Sigma Company;monoclonal antibody for vimentin(1:100),monoclonal antibody for myelin basic protein(MBP) (1:100),monoclonal antibody for S1 00(1:1 00),monoclonal antibody for neuron specific enolase(NSE)(1:1 00),and monoclonal antibody for neurofilament 200(NF200)(1:1 00)from Beijing Zhongshan Company;monoclonal antibody for glial fibrillary acidic protein (GFAP)(1:200)and polyclonal antibody for nestin(1:100)from Boster Company(Wuhan);mouse monoclonal antibody for beta-tubulin 3(1:1 000)from Sigma Company;SP-9000 kits and quick AEC from Beijing Zhongshan Company; culture dishes and flasks from Coming Company.METHODS:BMSCs from human bone marrow were cultured in serum-containing medium.When the primary culture was established, BMSCs were transferred into serum-free medium containing N2 or B27 supplement with 20 μg/L basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF),and cells were cultured in an incubator containing C02of 0.05 volume fraction at 37℃. Morphological changes of BMSCs in serum-free medium were observed under phase contrast microscope. And two days after culture. Expression of relative markers of BMSCs was detected withimmunocytochemistry.MAIN OUTCOME MEASURES:Morphological changes of BMSCs and expression of relative markers of nerve cells.RESULTS:A population of BMSCs could be isolated from adult human bone marrow,and they were processed to obtain a fibroblast-like population and were expanded as undifferentiated cells in culture for more than 10 passages.indicating their proliferative capacity.They could form spheroid state when they were sub-cultured in serum-free media supplemented with bFGF and EGF.these cells could express the markers for neural stem cells such as vimentin and nestin;they could expressed neuron specific enolase(NSE),beta-tubulin 3,TrkC and neurofilament 200(NF200)when they were plated on dishes with serum-containing medium; some cells exhibited the phenotypes for astrocytes.expressing gilal fibrillary acidic protein(GFAP)and S100 protein.CONCLUSION:The morphology,protein expression and differentiation ability of BMSCs in serum-free medium was similar to those of neural stem cells.The data support the hypothesis that adult bone marrow contains stem cells capable of differentiating into neural cells,the serum-free media make BMSCs overcome their mesenchymal commitment,showing the phenotypes for neural stem cells.

BACKGROUNDRecent researches have found that NiS can cause the malignant transforming activity and carcinogenicity on human bronchial epithelial cells (16HBE). Its molecular mechanism may be involved in mutation of genes and abnormal expression of transcription factors on 16HBE. And so, this study takes advantage of a model of 16HBE transformed by NiS and screens the differentially expressed genes between 16HBE cells and NiS treated 16HBE cells (NiS-16HBE) using cDNA microarray.

METHODSThe total RNA was extracted from 16HBE cells and NiS-16HBE cells. The cDNA probes were prepared by labeling with Cy3-dCTP and Cy5-dCTP respectively through reverse transcription. The mixed probes were then hybridized to the cDNA microarray chips containing 4000 human genes. The chips were scanned by ScanArray 4000 laser scanner. The acquired fluorescent signals were analyzed by GenPix Pro 3.0 software. Bioinformation function of those differentially expressed genes was analysed.

RESULTSA total of 151 genes exhibited differential expression between 16HBE cells and NiS-16HBE cells. The expression of 70 genes ( 46.36%) was down-regulated and that of 81 genes (53.64%) was up-regulated.

CONCLUSIONSThe regulation of genes including stress response genes, immune related genes, DNA synthesis and repair genes, metabolism genes, pro-oncogenes and tumor suppressor genes may be involved in transforming activity of NiS.

OBJECTIVETo clone differentially expressed cDNA sequences involved in malignant transformation induced by benzo(a)pyrene metabolite dihydroxyepoxy benzo pyrene (BPDE).

METHODThe malignant transformation of human bronchial epithelial cell line 16HBE induced by BPDE in vitro was used as a model for comparing gene expression between the transformed cells and controls. cDNA representational difference analysis (cDNA-RDA) was performed to isolate differentially expressed cDNA fragment in transformed cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA were sequenced and compared with data in GenBank by BLASTN.

RESULTSFive cDNA sequences were found to be novel ones and were registered in dbest database, which assigned accession numbers in GenBank are BG354691, BG354692, BG354693, BG354694 and BG354695, respectively. Eight of the remaining cDNA sequences showed sequence homology to those previously reported such as ribosomal protein S23, MLN137, ACTN4, transforming growth factor and G protein gene.

CONCLUSIONSThese 13 genes may be involved in BPDE-induced malignant transformation, but their biological characteristics and functions are left to further studies.

Benzopyrenes ; metabolism ; pharmacology ; Carcinogens ; pharmacology ; Cell Transformation, Neoplastic ; chemically induced ; genetics ; Cells, Cultured ; Cloning, Molecular ; DNA, Complementary ; analysis ; drug effects ; DNA, Neoplasm ; analysis ; Gene Expression ; drug effects ; Humans