In this study, we established the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) bacmid system for the construction of a Bac-to-Bac expression system and the generation of virus mutants. The CopyRight pSMART BAC cloning vector harboring the chloramphenicol resistance gene was introduced into the AnpeNPV genome to produce the AnpeNPV bacmid that could be propagated in Escherichia coli with stable replication. The enhanced green fluorescent protein (EGFP) was successfully expressed in both Tn-Hi5 cells and A. pernyi pupae using the AnpeNPV Bac-to-Bac expression system. To generate the AnpeNPV mutants, we developed the AnpeNPV bacmid/λ Red recombination system that facilitated the deletion of viral genes from the AnpeNPV genome. The genes cathepsin and chitinase were deleted and a derivative AnpeNPV Bac-to-Bac expression system was constructed. Furthermore, we demonstrated that the novel expression system could be used to express human epidermal growth factor in A. pernyi pupae. Taken together, the AnpeNPV bacmid system provides a powerful tool to create the AnpeNPV Bac-to-Bac expression system for protein expression in A. pernyi pupae. Further, it helps to knock-out genes from the AnpeNPV genome with λ Red recombination system for identification of the role of viral genes involved in regulating gene expression, DNA replication, virion structure, and infectivity during the AnpeNPV infection process.
The multidrug-resistant rate of Klebsiella pneumoniae has risen rapidly worldwide. To better understand the multidrug resistance situation and molecular characterization of Klebsiella pneumoniae, a total of 153 Klebsiella pneumoniae isolates were collected, and drug susceptibility test was performed to detect its susceptibility patterns to 13 kinds of antibiotics. Phenotypic tests for carbapenemases ESBLs and AmpC enzyme-producing strains were performed to detect the resistance phenotype of the isolates. Then PCR amplification and sequencing analysis were performed for the drug resistance determinants. The results showed that 63 strains harbored blaCTX-M gene, and 14 strains harbored blaDHA gene. Moreover, there were 5 strains carrying blaKPC gene, among which 4 strains carried blaCTX-M, blaDHA and blaKPC genes, and these 4 strains were also resistant to imipenem. Our data indicated that drug-resistant Klebsiella pneumoniae were highly prevalent in the hospital. Thus it is warranted that surveillance of epidemiology of those resistant isolates should be a cause for concern, and appropriate drugs should be chosen.
Abstract Background The gut microbiome and its metabolites can impact brain health and are altered in Parkinson’s disease (PD) patients. It has been recently demonstrated that PD patients have reduced fecal levels of the potent epigenetic modulator butyrate and its bacterial producers. Here, we investigate whether the changes in the gut microbiome and associated metabolites are linked to PD symptoms and epigenetic markers in leucocytes and neurons. Methods Stool, whole blood samples, and clinical data were collected from 55 PD patients and 55 controls. We performed DNA methylation analysis on whole blood samples and analyzed the results in relation to fecal short-chain fatty acid concentrations and microbiota composition. In another cohort, prefrontal cortex neurons were isolated from control and PD brains. We identified the genome-wide DNA methylation by targeted bisulfite sequencing. Results We show that lower fecal butyrate and reduced Roseburia, Romboutsia , and Prevotella counts are linked to depressive symptoms in PD patients. Genes containing butyrate-associated methylation sites include PD risk genes and significantly overlap with sites epigenetically altered in PD blood leucocytes, predominantly neutrophils, and in brain neurons, relative to controls. Moreover, butyrate-associated methylated-DNA (mDNA) regions in PD overlap with those altered in gastrointestinal, autoimmune, and psychiatric diseases. Conclusions Decreased levels of bacterially produced butyrate are linked to epigenetic changes in leucocytes and neurons from PD patients, and to the severity of their depressive symptoms. PD shares common butyrate-dependent epigenetic changes with certain gastrointestinal and psychiatric disorders, which could be relevant for their epidemiological linkage.
Circular RNAs (circRNAs) play vital roles in the development and progression of various diseases. CircRNA coiled-coil domain containing 66 (circ-CCDC66) has been reported to be involved in several cancers, but its biological function and underlying mechanism in papillary thyroid carcinoma (PTC) remain unclear. We detected the relative expression level of circ-CCDC66 in PTC specimens and cell lines using real-time reverse transcription PCR. In addition, EdU assay, transwell assay, and xenograft analysis were performed to measure the effect of circ-CCDC66 on the proliferative, migratory, and invasive capacities of PTC cells. We also investigated the potential mechanism of circ-CCDC66 by bioinformatics analysis, RNA immunoprecipitation, and dual-luciferase reporter assay. We observed that circ-CCDC66 expression was upregulated in PTC specimens and cell lines and was correlated with poor clinical characteristics of PTC patients. Moreover, in vitro experiments demonstrated that knockdown of circ-CCDC66 markedly suppressed the proliferative, migratory, and invasive capacities of PTC cells. Mechanistically, miR-129-5p was a target gene of circ-CCDC66 and was downregulated in PTC tissues. LARP1, a downstream target of miR-129-5p, was upregulated in PTC tissues. In addition, we confirmed that inhibition of circ-CCDC66 could repress xenograft tumor growth. Circ-CCDC66 promoted PTC proliferation, migration, invasion, and tumor growth by sponging miR-129-5p and promoting LARP1 expression.
An electrochemiluminescence (ECL) sensor provides a sensitive and convenient method for early diagnosis of diseases; however, it is still a challenge to develop simple and sensitive sensing platforms based on efficient ECL signals and luminophore groups. Porphyrin-based metal-organic frameworks (MOFs) show great potential in ECL sensing; however, the mechanism and structure-activity relationship, as well as application, are rarely reported. Herein, hydrothermal reactions obtained porphyrin Zr-MOFs (PCN-222) with different specific surface areas, pore sizes, structures, and surface charge states by tuning the reaction time were developed, which served both as the ECL luminophore, coreaction promoter for S2O82-, and a connection in the ECL immunoassay. By progressively controlling the condition of the hydrothermal reaction, PCN-222 with large surface area-abundant micropores can be obtained, which has good conductivity and positively charged surfaces, obtaining excellent ECL performance. The ECL performance and the enhancement mechanism were investigated in detail. Using PCN-222-6h with the best ECL intensity as the immobilization matrix for the aptamer, a highly sensitive and selective assay for thrombin was developed. The decrease of the ECL signal was logarithmically linear with the concentration of thrombin in the range from 50 fg mL-1 to 100 pg mL-1 with a low detection limit of 2.48 fg/mL. This proposed strategy provides a brand new approach for tuning of the structures of MOFs as effective ECL signal probes, thus providing wider possibilities for effective ECL immunoassays in the detection of other biomarkers in diagnosis of diseases.
Abstract An open challenge in human genetics is to better understand the link between genotype variation and the various molecular, cellular, anatomical and physiological systems that it can affect. To address this challenge, we performed genotype-phenotype-systems analysis for accuracy in nine cognitive tasks from the Philadelphia Neurodevelopmental Cohort (3,319 individuals aged 8-21 years). We report a region of genome-wide significance within the 3’ end of the FBLN1 gene (p=4.6×10 −8 ), associated with nonverbal reasoning, a heritable form of complex reasoning ability. Integration of published brain-specific ‘omic maps reveals that FBLN1 shows greatest expression in the fetal brain, is a marker of neural progenitor cells, is differentially expressed in schizophrenia and increases genetic risk for bipolar disorder. These findings suggest that nonverbal reasoning and FBLN1 variation warrant further investigation in studies of neurodevelopmental disorders and psychosis. Using genotype-pathway analysis, we identify pathways related to development and to autonomic nervous system dysfunction associated with working memory accuracy. Top-ranking pathway genes include those genetically associated with multiple diseases with working memory deficits, such as schizophrenia and Parkinson’s disease, and that are also markers for specific brain cell types. Our findings identify novel molecular players involved in specific cognitive tasks and link variants to genes, pathways, cell types, diseases and drugs. This work advances the “molecules-to-behaviour” view of cognition, and provides a framework for using systems-level organization of data for other biomedical domains.
Abstract Increasing evidences suggest that insufficient radiofrequency ablation (IRFA) can paradoxically promote tumor invasion and metastatic processes, whereas the effects of moderate hyperthermia on cancer progression are not well illustrated. Our study found that IRFA can increase the in vitro migration, invasion, and epithelial–mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells via induction of Snail, a master regulator of EMT events. Among measured miRNAs, IRFA can decrease the expression of miR-148a-5p in HCC cells. Whereas overexpression of miR-148a-5p can reverse IRFA-induced migration of HCC cells and upregulation of Snail, mechanistically overexpression of miR-148a-5p can directly target and decrease the expression of protein kinase ATM (ataxia telangiectasia mutated), which can increase protein stability of Snail. Collectively, our data suggest that IRFA can regulate the miR-148a-5p/ATM/Snail axis to trigger migration of HCC cells.
Abstract Background: Glutamine is one of the primary nutrients utilized by cancer cells for energy production and biosynthesis. Interfering with glutamine metabolism may impose anti-tumor effects. In this study, we aimed to investigated the anti-tumorigenic effects of GLS1 inhibition in endometrial cancer cells in vitro and in vivo . Methods: In this study, we assessed the relationship between glutaminase-1 enzyme (GLS1) expression and prognosis in endometrial cancer by bioinformatics analyses. Overall survival (OS) and progression-free survival (PFS) analyses were performed using the Kaplan-Meier method. The effects of compound 968 on cell proliferation, cell cycle, apoptosis, cellular stress, invasion, and AKT/mTOR/S6 pathway inhibition in human endometrial cancer cell lines were assessed. The in vivo therapeutic potential of compound 968 in endometrial cancer was evaluated using tumor xenografts. Results: We found that GLS1 expression is elevated during endometrial cancer progression and is associated with poor prognosis. The GLS1-targeting compound 968 was able to reduce cancer cell proliferation, induce cell cycle arrest at the G1 phase, inhibit cell invasion, as well as promote cellular stress and cancer cell apoptosis. Compound 968 treatment significantly increased the sensitivity of cells to paclitaxel. Moreover, the treatment of endometrial cancer cells with compound 968 resulted in AKT/mTOR/S6 signaling pathway inhibition. In xenograft mouse models of endometrial cancer, compound 968 significantly suppressed tumor growth. Conclusion: We conclude that compound 968 is a promising anti-tumorigenic agent and that combination with paclitaxel may be a valuable strategy for the treatment of endometrial cancer.
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