Objective : To investigate the drug resistance and pathogenicity of six clinical isolates of Klebsiella pneu- moniae (Kp) ，and to provide a basis for prevention and treatment of Kp infection． Methods : The six strains from different hospitals were isolated ，cultured ，and identified by species-specific gene khe． Their whole genome se- quences (WGS) were obtained using next-generation sequencing technology (NGS) ．Based on the WGS，the cap- sular serotypes，sequence types (ST) and drug-resistance genes of six strains were identified．The capsular sero- type genes and virulence genes were validated or identified using PCR. Broth microdilution tests were conducted to validate their drug susceptibility，and mice were challenged with Kp aerosols by MicroSprayer aerosolizer to evaluate their pathogenicity. Results : The six strains were all serotype K2 but belonged to four ST types ( ST14 ，ST65， ST700，and ST86) ，and collectively carried six virulence genes and 23 drug-resistance genes．All the six strains were resistant to ampicillin，but only one strain was multidrug-resistant．Four strains exhibited high mucoid charac- teristics．Five strains could cause mortality in mice，which were preliminary identified as high virulence strains. Conclusion For the six Kp clinical isolates from different sources，only one strain named NY 13294 is both multi- drug-resistant and highly virulent，and other four highly virulent strains are resistant to one or two types of antibiot- ics.

Objective To investigate the effect of spleen on hepatic macrophages mediated activation of hepatic stellate cells(HSCs)in mice with liver fibrosis.Methods Eighteen male C57BL/6 mice were randomly divided into three groups.Mice in Group A and Group B were injected intraperitoneally with CCl4 to establish liver fibrosis mouse model,while those in Group C were injected with corn oil as normal control.Four weeks later,mice with liver fibrosis received splenectomy(Spx)or sham operation(Sham),respectively.After continuous injection for 2 weeks,liver homogenates(L-Homo)were prepared and liver cells were isolated from the three groups.Expressions of IL-1β,IL-13,TGF-β,TNF-α,PDGF-β and VEGF in the liver homogenates of the three groups were detected by Luminex multifactor analysis.The expressions of these cytokines in liver macrophages(L-Mψ)and other non-parenchymal cells of Sham and Spx mice were analyzed by Real-time quantitative PCR(RT-qPCR)and flow cytometry.Macrophage cell line RAW264.7 or bone marrow-derived macrophages(BMDMs)were treated with liver homogenates from the Sham and Spx groups.Then the differently treated RAW264.7 cells were analyzed for mRNA expressions of cytokines and glutamine metabolism-related molecules by RT-qPCR,or transwell co-cultured with hepatic stellate cell line JS1.After co-culture,the survival and extracellular matrix expression of JS1 cells were analyzed.For comparison,Student's t test(between two groups)or one-way analysis of variance(among multiple groups)were used.Results Compared with normal control group,the concentrations of IL-1β,IL-13,TGF-β and TNF-α in the L-Homo of model group were significantly increased and showed higher levels in Sham group than in Spx group.Moreover,the hepatic macrophages were indicated as the major source of these cytokines.Consistently,macrophages treated with liver homogenate of Sham mice had increased expressions of IL-1β,TGF-β and TNF-α and glutaminase(GLS).After co-culture with macrophages treated with liver homogenate of Sham group rather than Spx group,JS1 expressed higher expressions of α-SMA and collagens.Conclusion The spleen is involved in regulating the secretion of cytokines by hepatic macrophages and enhancing their ability to activate hepatic stellate cells.

In order to develop a positive quality control products for the detection of porcine repro-ductive and respiratory syndrome virus(PRRSV)nucleic acid by real-time fluorescent quantitative PCR(RT-qPCR),the positive quality control products of PRRSV-1 and PRRSV-2 M genes were prepared using armored RNA technology of MS2 phage.PRRSV-1 and PRRSV-2 M genes were amplified,purified and recovered,and ligated into pET28b vector containing MS2 mature enzyme protein gene and capsid protein.After transformed into BL21(DE3),the gene products were in-duced by IPTG and purified by PEG6000 precipitation method to prepare the armored RNA virus-like particles(AR-PRRSV)containing PRRSV M gene.Following the performance evaluation,as the positive quality control products of PRRSV-1 and PRRSV-2 M genes,AR-PRRSV1M and AR-PRRSV2M were calculated using YY/T 1652-2019 standard.Results showed that it had a good u-niformity,stable storage for the armored virus-like particles at-20,4,25 ℃ for 60 d,and 37 ℃ for 30 d.The prepared armored virus-like particles AR-PRRSV1M and AR-PRRSV2M were deter-mined by digital quantitative PCR(ddPCR)after preliminary quantification by RT-qPCR.The 104 copies/μL of AR-PRRSV1M and AR-PRRSV2M ddPCR fixation was(1.33+0.50)× 104 cop-ies/μL.The above results indicates that the AR-PRRSVM can be used as the quality control of the whole detection process(nucleic acid extraction,reverse transcription and RT-qPCR).

In response to the challenges of small scale and huge labor cost in biomedical feature modeling in current skin disease assisted diagnosis,as well as the inability to accurately describe the time series of patient disease features,a fusion text classification algorithm is used to integrate commonly used text classification models(TextLSTM,TextCNN,and RCNN)to obtain a model based on transfer learning and neural networks(TLNN model).By extracting the medical features of image sensors and quantizing them,the pretreatment reduces the number of foci and eliminates the feature information with large deviations,thus improving the accuracy of decision data.TLNN model achieves an accuracy of 72.36%on ISIC2018 and PH2 datasets,which is higher than those of the other 3 text classification models.The diagnostic accuracy of TLNN model is close to doctor's diagnosis(92%vs 94%),but the effective diagnostic efficiency is significantly higher than doctor's diagnosis(1.17 min/case vs 4.57 min/case),and the overall efficiency is improved by 290%.The results demonstrate that the fusion text classification algorithm model can obtain accurate diagnosis in less time than the traditional manual diagnosis.TLNN model can be applied to disease diagnosis,and assist doctors in medical decision-making,thereby providing patients with high-quality and convenient intelligent diagnosis and treatment services.

OBJECTIVES: This study aimed to analyze the bacteria in dental caries and establish an optimized dental-ca-ries diagnosis model based on 16S ribosomal RNA (rRNA) data of oral flora. METHODS: We searched the public databa-ses of microbiomes including NCBI, MG-RAST, EMBL-EBI, and QIITA and collected data involved in the relevant research on human oral microbiomes worldwide. The samples in the caries dataset (1 703) were compared with healthy ones (20 540) by using the microbial search engine (MSE) to obtain the microbiome novelty score (MNS) and construct a caries diagnosis model based on this index. Nonparametric multivariate ANOVA was used to analyze and compare the impact of different host factors on the oral flora MNS, and the model was optimized by controlling related factors. Finally, the effect of the model was evaluated by receiver operating characteristic (ROC) curve analysis. RESULTS: 1) The oral microbiota distribution obviously differed among people with various oral-health statuses, and the species richness and species diversity index decreased. 2) ROC curve was used to evaluate the caries data set, and the area under ROC curve was AUC=0.67. 3) Among the five hosts' factors including caries status, country, age, decayed missing filled tooth (DMFT) indices, and sampling site displayed the strongest effect on MNS of samples (P=0.001). 4) The AUC of the model was 0.87, 0.74, 0.74, and 0.75 in high caries, medium caries, low caries samples in Chinese children, and mixed dental plaque samples after controlling host factors, respectively. CONCLUSIONS The model based on the analysis of 16S rRNA data of oral flora had good diagnostic efficiency.
Humans ; Child ; Bacteria/genetics* ; Dental Caries/microbiology* ; Dental Caries Susceptibility ; Microbiota/genetics* ; RNA, Ribosomal, 16S

OBJECTIVE: To assess the influence of rs2910164 G/C single nucleotide polymorphism (SNP) of the miR-146a gene on its expression and susceptibility to gastric cancer. METHODS: Fifty three gastric cancer patients and six gastric cancer cell lines were selected for determining the miR-146a expression by Taqman quantitative PCR. A model was constructed to assess the influence of miR-146a overexpression on the growth of AGS gastric cancer cells. A case-control study involving 417 gastric cancer patients and 420 cancer-free individuals was then conducted, and the allelic and genotypic frequencies of the rs2910164 G/C SNP were compared. The genotypes of all subjects were determined by using a Taqman allelic discrimination assay. A Taqman assay was also used to quantify mature and pri-miR-146a transcripts among 65 gastric cancer patients with known genotypes. RESULTS: The expression of miR-146a was down-regulated among the 53 gastric cancer patients and six gastric cancer cell lines. Over-expression of miR-146a has suppressed the growth of gastric cancer by inhibiting the G1/S-phase transition of AGS cells. The case-control study showed that subjects with GC/CC genotypes had significantly lower risk for gastric cancer compared with those with GG genotype. In addition, miR-146a G/C SNP has significantly increased the level of mature miR-146a in those with GC/CC genotype compared with GG genotype. CONCLUSION Down-regulation of miR-146a may play an important role in the pathogenesis of gastric cancer. The rs2910164 polymorphism of the miR-146a gene may reduce the risk of gastric cancer by influencing the processing of mature miR-146a.
Case-Control Studies ; Genotype ; Humans ; MicroRNAs/genetics* ; Polymorphism, Single Nucleotide ; Stomach Neoplasms/genetics*

HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited cell proliferation of multiple cancer cell lines and macrophages, and the anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.

This article summarizes the attribute conditions to the combination products designation from 2009 to 2018 in China,analyzes the common problems of combination products attribution definition.It is hoped to be helpful for researchers and manufacturers of combination products.
China ; Equipment and Supplies ; Terminology as Topic

Objective:To explore the clinical efficacy, especially the comprehensive improvement of blood glucose and lipid of probucol and metformin in the treatment of type 2 diabetes mellitus with hyperlipidemia. Methods:Totally 105 patients with type 2 di-abetes mellitus complicated with hyperlipidemia were randomly divided into the control group1 (35 cases), the control group 2 (34 ca-ses) and the observation group (36 cases). The control group 1 was treated with diet control, exercise and metformin, the control group 2 was treated with rosuvastatin calclum tablets based on the group 1, and the observation group was treated with probucol based on the group 1. The three groups were continuously treated for 12 weeks. The improvement of the fasting blood-glucose (FPG), 2h postprandial blood glucose (PBG), hemoglobin A1C (HbA1C), fasting insulin levels (Fins), insulin resistance indices (HOMA-IR) and TC, TG and HDL-C, and the adverse reactions among the three groups were compared. Results:The total effective rate in the ob-servation group was higher than that in the control group 1 and 2 (P<0. 05). After the treatment, all the indices of blood glucose and lipid in the three groups were significantly improved (P<0. 05), and those of blood glucose in the observation were better than those in the control group 1 and 2(P<0. 05), those of blood lipid in the observation group and the control group 2 were better than those in the control group 1 (P<0. 05), and the level of HDL-C in the observation group was much higher than that in the control group 2 (P<0. 05). The adverse drug reactions in the three groups were mild without statistical significance (P>0. 05). Conclusion:Probucol as one of lipid-lowing drugs with antioxidant action combined with metformin can improve blood lipid and lower blood glucose at the same time, which is worthy of promoted application in clinics.

Objective The relationship was analyzed between clinic and the expression intensity of HBsAg and HBcAg with in the hepatic tissue from the serum HBeAg negative group and the positive group.Methods A total of 317 liver biopsy specimens were divided into the HBeAg negative group and the positive group,and the relationship was analyzed between the expression inten sity of HBsAg and HBcAg within the hepatic tissue and their age,gender,ALT level,serum HBV-DNA load,hepatic inflammatory activity grading and fibrosis staging in the two groups.Results Age,ALT level,hepatic inflammatory activity grading and fibrosis of the serum HBeAg negative patients were greater than those of the serum HBeAg positive patients,while their serum HBV-DNA load and the expression intensity of HBcAg within the hepatic tissue were lower than those of the serum HBeAg positive patients (P＜0.05).The expression intensity of HBsAg within the hepatic tissue between the serum HBeAg patients and the serum HBeAg positive patients was not significantly different,and it was not correlated with age,ALT level,hepatic inflammatory grading and fi brosis staging (P＞0.05).After the serum HBeAg turned negative,the expression intensity of HBcAg within the hepatic tissue was decreased (P=0.00,t=12 349.0),and it became positively correlated with the serum HBV DNA load(P=0.007,r=0.251) and its negative correlation with the hepatic inflammatory activity and fibrosis was weakened.Conclusion After the serum HBeAg turned negative,other antigenic components of HBV may still maintain the adequately active immune status within the hepatic tis sue of organisms.After the serum HBeAg turned negative,the expression intensity of HBcAg within the hepatic tissue was de creased and became positively correlated with the serum HBV DNA,while its negative correlation with the hepatic inflammatory activity grade and fibrosis stagings was weakened.