mTOR pathway plays a critical role in cell proliferation, growth and metabolism. This pathway is composed of two different large protein complexes, mTORC1 and mTORC2, which have their distinct downstream effects. Its inhibitor, rapamycin, has been proved to cause β-cell damage and glucose intolerance. Furthermore, various transgenic mouse models and ex vivo studies have revealed that mTORC1 and mTORC2 are both essential for maintaining normal β cell mass and function, whereas the underlying molecular mechanism and the relevance of the whole mTOR signaling to pathogenesis of type 2 diabetes remain to be explored and further clarified.

Objective To explore the effect of nicotinaide nucleotide transhydrogenase(NNT) mutation on glucose homeostasis in C57BL/6 mice with mix background. Methods We generated wild type NNT homozygous, mutant NNT homozygous and heterozygous by mating the C57BL/6J (with NNT mutation) and 6N (without NNT mutation). At the age of 4 weeks, those mice were randomly assigned to normal control diet(NCD) or high-fat diet(HFD) for 4 weeks. The body weight was measured every week. At the age of 8 weeks, an intraperitoneal glucose tolerance test(IPGTT) and an intraperitoneal insulin tolerance test (ITT) were performed. Results The body weight growth was not affected by NNT mutation during an HFD fed. NNT mutant mice showed significant glucose intolerance. After 4 weeks of high fat diet, the NNT mutant mice showed a decreased insulin sensitivity, while the glucose excursion curve was not elevated in the heterozygous mice. Conclusion NNT mutation had a significant influence on the phenotype of glucose metabolism and insulin resistance of mice, in particular under a metabolic stress. The phenotypes of heterozygous and homozygous mutant ones differed from each other. When using mice with C57BL/6J and C57BL/6N mixed background in research, NNT mutation should be carefully screened in all metabolic studies.

Objective To screen the critical genes related to the development of esophageal squamous cell carcinoma ( ESCC ) by weighted gene co-expression network analysis ( WGCNA ) and to verify by experiments.Methods Gene expression data of ESCC were downloaded from gene expression omnibus (GEO) database based on gene chip platform ( GPL) 570, GPL571, GPL96/97 or GPL14613 platform, respectively. Meanwhile, the obtained differentially expressed genes together with gene expression data of 81 ESCC patients from the cancer genome atlas ( TCGA ) and clinical data were analyzed by WGCNA to set up co-expression networks including mRNA and microRNA ( miRNA ) . The expression of miRNA in ESCC tissues and paracancerous tissues was examined by quantitative real-time polymerase chain reaction ( RT-PCR ) .And the expression of target protein Kruppel like factor 4 ( KLF4 ) and desmocollin 2 ( DSC2 ) were detected by immunohistochemistry .After ESCC cell line ECA-109 cells were transfected with miRNA-92b-3p mimic, cell cycle was tested by flow cytometry ,the cell invasion and migration ability was measured by Transwell chamber assay and scratch-wound assay.The expression of KLF4 and DSC2 was observed by confocal laser scanning microscopy and Western blotting .The target genes were verified by luciferase assay .T-test, rank sum test, chi-square test and Pearson correlation analysis were performed for statistical analysis .Results A total of 4023 differential expression gene ( DEG) and 328 differential expression miRNA ( DEM) were screened and 11 gene modules were set up by WGCNA .Among them, the brown modules were negatively associated with tumor grade and T stage (r=-0.340 and -0.268, P=0.002 and 0.016).Meanwhile, has-miR-92b and the potential target genes KLF4 and DSC2 were all in the brown module .Furthermore, the results of RT-PCR showed the expression of miRNA-92b-3p in ESCC tissues was higher than that in paracancerous tissues (3.052(1.652, 5.371) vs.0.985(0.558, 2.032)), and the difference was statistically significant (Z=-4.021,P<0.01). The results of immunohistochemistry demonstrated that the positive rates of KLF 4 and DSC2 in ESCC tissues were 43.3％(13/30) and 20.0％(6/30), respectively, which were lower than those of paracancerous tissues (70.0％(21/30) and 63.3％(19/30)), and the differences were statistically significant (χ2 =4.344 and 1.589, both P<0.05).After ECA-109 cells were transfected with miRNA-92b-3p mimics, the percentage of cells at G0/G1 phase decreased ((63.71 ±2.83)％vs.(54.62 ±4.00)％) and the percentage of cells at the S phase and G2/M phase increased ((31.81 ±2.88)％vs.(41.20％±2.87)％, and (3.87 ±1.75)％vs. (8.10 ±1.71)％, respectively), and the differences were statistically significant (t =3.215, 4.000 and 2.998;P=0.032, 0.016 and 0.040).The invasion and migration ability of the cells were significantly improved (79.67 ±27.54 vs.280.33 ±46.18, (69.72 ±3.91)％ vs.(84.90 ±5.25)％), and the differences were statistically significant (t=6.465 and 4.019, P=0.003 and 0.016).The results of Western blotting indicated that, compared with control mimic group , the expression of KLF4 and DSC2 was both dramatically downregulated after transfected with miRNA-92b-3p mimics transfected (1.00 ±0.23 vs.0.42 ±0.03, 1.00 ±0.20 vs.0.55 ± 0.21), and differences were statistically significant (t=4.470 and 5.493, P=0.042 and 0.032).The results of luciferase assay demonstrated that miRNA-92b-3p could directly bind KLF4 and DSC2. Conclusion WGCNA is an efficient systemic biological approach by which miRNA-92b-3p is identified as a new cancer-promoting gene .

Cytoplasmic male sterility is an important way to utilize wheat heterosis. The purpose of thisstudy was to identify cytoplasmic type of three wheat male sterile lines. Amplified fragment length polymorphism (AFLP) marker technique was used to analyze the wheat mitochondrial DNA. We isolated mitochondria by differential centrifugation and density gradient ultracentrifugation. The results show that the extracted mitochondrial DNA was pure. It was suitable for PCR and genetic analysis. We got 4 pairs of specific primers from 64 primers combinations. Primer E1/M7 amplified 3 specific fragments in ms(Kots)-90-110. Primer E4/M2 generated 2 specific fragments in ms(Ven)-90-110. Primer E7/M6 amplified 2 specific fragments in ms(S)-90-110. Primer E6/M4 produced 2 specific fragments in ms(Kots)-90-110. Four specific primers could be used to identify three cytoplasmic types of Aegilops kotschyi, Ae. ventricosa and Triticum spelta. It provided the molecular basis to further study the mechanism of wheat cytoplasmic male sterility.
Amplified Fragment Length Polymorphism Analysis ; methods ; Cytoplasm ; metabolism ; DNA, Mitochondrial ; genetics ; DNA, Plant ; genetics ; Gene Expression Profiling ; Genotype ; Plant Infertility ; genetics ; Triticum ; genetics

Objective:To identify the risk factors for survival prognosis of patients with early-stage hepatocellular carcinoma (HCC) after ultrasound-guided percutaneous microwave ablation (US-PMMA), and to compare the overall survival (OS), cancer specific survival (CSS) and disease-free survival (DFS) between different early-stage HCC patients.Methods:A total of 1 563 patients with early-stage hepatocellular carcinoma (HCC) who underwent MWA in the interventional ultrasound department of the Chiese PLA General Hospital from January 2002 to December 2017 were retrospectively analyzed. Propensity score matching (PSM) balanced the baseline parameters between the elderly group (≥60 years) and the young group (<60 years). Multivariate Cox regression analysis was used to identify the risk factors of OS, CSS and DFS. OS, CSS and DFS probabilities for different patients stratified by respective predictors were calculated with Kaplan-Meier method and compared using the Log-Rank test.Results:All parameters were balanced except for age after PSM.Tumor diameter(95% CI=1.1-1.4, P<0.001), number of tumors(95% CI=1.2-1.9, P<0.001), γ-GT (95% CI=1.0-1.0, P<0.001) and AFP (HR=1.5, 95% CI=1.2-1.8, P<0.001) were shared predictors for OS, CSS and DFS. Age (95% CI=1.2-1.8, P<0.001) and neutrophile to lymphocyte ratio (NLR) (95% CI=1.0-1.0, P=0.043) were another two predictors for both OS and CSS. Albumin predicted OS only, and sex and cirrhosis just predicted DFS. Over the follow-up period (12-156 months), log-rank tests showed that all predictors significantly affected the corresponding OS, CSS or DFS(all P<0.01). Among them, multiple tumors had the greatest impact on OS, CSS and DFS. Compared with patients with single lesion, OS, CSS and DFS in patients with multiple lesions decreased by 9.2%, 2.5% and 4.1% respectively at the 12 years of follow-up, and the median survival time was shortened by 12.3 months, 25.0 months and 11.3 months, respectively (log-rank P=0.049 for OS; P=0.007 for CSS; P<0.001 for DFS). Conclusions:The prognostic benefits from MWA treating early-stage HCC in patients with different survival risk factors are different. Clinically feasible correction of hypoproteinemia and liver disfunction are of great significance to improve the prognosis of early-stage HCC patients after US-PMMA.

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells, indicating a potential candidate method for clinical HER2 positive specimens detection.
Animals ; Breast Neoplasms ; diagnosis ; pathology ; Female ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; MCF-7 Cells ; Receptor, ErbB-2 ; analysis ; Recombinant Fusion Proteins ; genetics ; Sf9 Cells ; Single-Chain Antibodies ; genetics

The main etiology of diabetes mellitus is loss of functional β cell mass, which is responsible for the secretion of the insulin hormone to reduce elevated plasma glucose and to maintain glucose homeostasis. Type 1 diabetes has traditionally been characterized by autoimmune-mediated β-cell death leading to insulin dependence, whereas type 2 diabetes has hallmarks of peripheral insulin resistance, accompanied by β cell dysfunction, and cell death. However, a growing body of evidence suggests that β cell dysfunction and defects of functional maturation in type 2 diabetes involve: (1 ) loss of cell identity, specifically proteins associated with mature cell function and transcription factors like Pdx1,MafA,Nkx6. 1,Glut2,and GK,and (2) de-differentiation,defined by regression to a progenitor or stem cell-like state. Moreover, ectopic expression of genes that are disallowed in β cells is also crucial to maintain the mature phenotype of β cells. In this review, we will combine our preliminary data and summarize the recent literature describing how β cell functional maturation is regulated. We hope that this perspective could shed some lights on possible avenues of new therapeutic intervention for diabetes mellitus.

The outbreak of coronavirus disease 2019 (COVID-19) was declared a global public health emergency on 31 January 2020. Emergency medicine procedures in Emergency Department should be optimized to cope with the current COVID-19 pandemic by providing subspecialty services, reducing the spread of nosocomial infections, and promoting its capabilities to handle emerging diseases. Thus, the Chinese Society of Emergency Medicine and Wuhan Society of Emergency Medicine drafted this consensus together to address concerns of medical staffs who work in Emergency Department. Based on in-depth review of COVID-19 diagnosis and treatment plans, literatures, as well as management approval, this consensus proposes recommendations for improving the rationalization and efficiency of emergency processes, reducing the risk of nosocomial infections, preventing hospital viral transmission, and ensuring patient safety.