OLA1 promotes colorectal cancer tumorigenesis by activation of HIF1α/CA9 axis.

BACKGROUND: Obg-like ATPase 1 (OLA1) is a highly conserved GTPase, which was over expressed in a variety of malignant tumors, but its role in colorectal cancer (CRC) was poorly studied. PATIENTS AND METHODS: Three public CRC gene databases were applied for OLA1 mRNA expression detection. The clinical data of 111 CRC patients were retrospectively collected from the Second Affiliated Hospital of Zhejiang University (SAHZU) for OLA1 protein expression and Kaplan-Meier Survival analysis. OLA1 stably knocked out CRC cell lines were conducted by CRISPR-Cas9 for experiments in vitro and in vivo. RESULTS: OLA1 was highly expressed in 84% CRC compared to matched surrounding tissues. Patients with OLA1 high expression had a significantly lower 5-year survival rate (47%) than those with OLA1 low expression (75%). OLA1 high expression was an independent factor of poor prognosis in CRC patients. OLA1-KO CRC cell lines showed lower ability of growth and tumorigenesis in vitro and in vivo. By mRNA sequence analysis, we found 113 differential express genes in OLA1-KO cell lines, of which 63 were hypoxic related. HIF1α was a key molecule in hypoxic regulation. Further molecular mechanisms showed HIF1α /CA9 mRNA and/or protein levels were heavily downregulated in OLA1-KO cell lines, which could explain the impaired tumorigenesis. According to previous studies, HIF1α was a downstream gene of GSK3ß, we verified GSK3ß was over-activated in OLA1-KO cell lines. CONCLUSION: OLA1 was a new gene that was associated with carcinogenesis and poor outcomes in CRC by activation of HIF1α/CA9 axis, which may be interpreted by GSK3ß.

Anchoring proteins to Escherichia coli cell membranes using hydrophobic anchors derived from a Bacillus subtilis integral membrane protein.

Anchored periplasmic expression (APEx) technology aims to express and localize proteins or peptides in the Escherichia coli periplasm. Some reports have suggested that transmembrane segments of integral membrane proteins can be used as membrane anchors in the APEx system. In this study, a series of hydrophobic anchors derived from the first putative transmembrane helix of a Bacillus subtilis integral membrane protein, MrpF, and its truncated forms were investigated for anchored periplasmic expression of alkaline phosphatase (PhoA) in E. coli. Anchoring efficiency of hydrophobic anchors was evaluated by monitoring the expression and activity of anchored PhoA. The length of hydrophobic anchors was found to be critical for anchoring proteins to cell membranes. This study may open new avenues for applying transmembrane segments derived from native membrane proteins as membrane anchors in the APEx system.

Unique characteristics of G719X and S768I compound double mutations of epidermal growth factor receptor (EGFR) gene in lung cancer of coal-producing areas of East Yunnan in Southwestern China.

BACKGROUND: The principal objective of this project was to investigate the Epidermal Growth Factor Receptor (EGFR) gene mutation characteristics of lung cancer patients, which can provide a molecular basis for explaining the clinicopathological features, epidemiology and use of targeted therapy in lung cancer patients in the coal-producing areas of East Yunnan. METHODOLOGY: We collected 864 pathologically confirmed lung cancer patients' specimens in First People's Hospital of Qujing City of Yunnan Province from September 2016 to September 2021. We thereafter employed Next Generation Sequencing (NGS) technology to detect all exons present in the EGFR gene. RESULTS: The overall mutation frequency of the EGFR gene was 47.22%. The frequency of EGFR gene mutations in the tissue, plasma, and cytology samples were found to be 53.40%, 23.33%, and 62.50%, respectively. Univariate analysis indicated that the coal-producing areas and Fuyuan county origin were significantly associated with relatively low EGFR gene mutation frequency. Female, non-smoking history, adenocarcinoma, non-brain metastasis, and tissue specimens were found to be related to high EGFR gene mutation frequency. Multivariate logistic regression analysis suggested the lung cancer patients in the central area of Qujing City, stage Ia, non-coal-producing areas, non-Fuyuan origin, and non-Xuanwei origin were more likely to develop EGFR gene mutations. The most common mutations were L858R point mutation (33.09%) and exon 19 deletion (19-del) (21.32%). Interestingly, the mutation frequency of G719X (p = 0.001) and G719X + S768I (p = 0.000) in the coal-producing areas were noted to be more significant than those in non-coal-producing regions. CONCLUSION: This findings of this study might be important in establishing the correlation between routine using NGS for EGFR gene mutation diagnosis and clinical practice in the lung cancer patients.

Improving the sensitivity of single-strand conformation polymorphism (SSCP) to study the variability of PLMVd.

Single-strand conformation polymorphism (SSCP) was used to characterize viroids. Eight cDNA clones, which showed identical profiles in preliminary existing SSCP analysis but had different sequences, were chosen to develop a sensitive SSCP technique for identifying the variability of Peach latent mosaic viroid (PLMVd). Polyacrylamide Gel Electrophoresis (PAGE) conditions were optimized to improve the sensitivity of the existing SSCP, and a modified SSCP protocol was developed. The results indicated that the modified SSCP protocol provided an overall sensitivity in identifying the variability of these clones, and showed higher resolution than the existing one and its improved versions. As shown by sequence analyses of cDNA clones of PLMVd and the modified SSCP profiles, there is no close correlation between the number of base changes and variation of the modified SSCP band patterns. The potential use of the modified SSCP analysis is discussed as a tool for viroids characterization.

Heavy Metal Resistances and Chromium Removal of a Novel Cr(VI)-Reducing Pseudomonad Strain Isolated from Circulating Cooling Water of Iron and Steel Plant.

Three bacterial isolates, GT2, GT3, and GT7, were isolated from the sludge and water of a circulating cooling system of iron and steel plant by screening on Cr(VI)-containing plates. Three isolates were characterized as the members of the genus Pseudomonas on the basis of phenotypic characteristics and 16S rRNA sequence analysis. All isolates were capable of resisting multiple antibiotics and heavy metals. GT7 was most resistant to Cr(VI), with a minimum inhibitory concentration (MIC) of 6.5 mmol L-1. GT7 displayed varied rates of Cr(VI) reduction in M2 broth, which was dependent on pH, initial Cr(VI) concentration, and inoculating dose. Total chromium analysis revealed that GT7 could remove a part of chromium from the media, and the maximum rate of chromium removal was up to 40.8 %. The Cr(VI) reductase activity of GT7 was mainly associated with the soluble fraction of cell-free extracts and reached optimum at pH 6.0∼8.0. The reductase activity was apparently enhanced by external electron donors and Cu(II), whereas it was seriously inhibited by Hg(II), Cd(II), and Zn(II). The reductase showed a K m of 74 µmol L-1 of Cr(VI) and a V max of 0.86 µmol of Cr(VI) min-1 mg-1 of protein. The results suggested that GT7 could be a promising candidate for in situ bioremediation of Cr(VI).

Respiratory toxicity of cyanobacterial aphantoxins from Aphanizomenon flos-aquae DC-1 in the zebrafish gill.

Aphantoxins from Aphanizomenon flos-aquae are frequently identified in eutrophic waterbodies worldwide. These toxins severely endanger environmental safety and human health due to the production of paralytic shellfish poisons (PSPs). Although the molecular mechanisms of aphantoxin neurotoxicity have been studied, many questions remain to be resolved such as in vivo alterations in branchial histology and neurotransmitter inactivation induced by these neurotoxins. Aphantoxins extracted from a naturally isolated strain of A. flos-aquae DC-1 were determined by high performance liquid chromatography. The basic components of the isolated aphantoxins identified were gonyautoxin 1 (GTX1), gonyautoxin 5 (GTX5), and neosaxitoxin (neoSTX), which comprised 34.04, 21.28, and 12.77% of the total, respectively. Zebrafish (Danio rerio) was administrated 5.3 or 7.61mg STX equivalents (eq)/kg (low and high doses, respectively) of the A. flos-aquae DC-1 aphantoxins by intraperitoneal injection. Histological alterations and changes in neurotransmitter inactivation in the gills of zebrafish were investigated for 24h following exposure. Aphantoxin exposure significantly increased the activities of gill alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and resulted in histological alterations in the gills during the first 12h of exposure, indicating the induction of functional and structural damage. Gill acetylcholinesterase (AChE) and monoamine oxidase (MAO) activities were inhibited significantly, suggesting an alteration of neurotransmitter inactivation in zebrafish gills. The observed alterations in gill structure and function followed a time- and dose-dependent pattern. The results demonstrate that aphantoxins or PSPs lead to structural damage and altered function in the gills of zebrafish, including changes in histological structure and increases in the activities of AST and ALT. The inhibition of the activities of AChE and MAO suggest that aphantoxins or PSPs could induce respiratory toxicity in the zebrafish gill. Furthermore, these parameters may be used as bioindicators for investigating aphantoxin exposure and cyanobacterial blooms in nature.

MiRNA-15a mediates cell cycle arrest and potentiates apoptosis in breast cancer cells by targeting synuclein-γ.

BACKGROUND: Recent studies have indicated that microRNA-15a (miR-15a) is dysregulated in breast cancer (BC). We aimed to evaluate the expression of miR-15a in BC tissues and corresponding para-carcinoma tissues. We also focused on effects of miR-15a on cellular behavior of MDA-MB-231 and expression of its target gene synuclein-γ (SNCG). MATERIALS AND METHODS: The expression levels of miR-15a were analysed in BC formalin fixed paraffin embedded (FFPE) tissues by microarray and quantitative real-time PCR. CCK-8 assays, cell cycle and apoptosis assays were used to explore the potential functions of miR-15a in MDA-MB-231 human BC cells. A luciferase reporter assay confirmed direct targets. RESULTS: Downregulation of miR-15a was detected in most primary BCs. Ectopic expression of miR-15a promoted proliferation and suppressed apoptosis in vivo. Further studies indicated that miR-15a may directly interact with the 3'-untranslated region (3'-UTR) of SNCG mRNA, downregulating its mRNA and protein expression levels. SNCG expression was negatively correlated with miR-15a expression. CONCLUSIONS: MiR-15a has a critical role in mediating cell cycle arrest and promoting cell apoptosis of BC, probably by directly targeting SNCG. Thus, it may be involved in development and progression of BC.

Direct and rapid quantum dots labelling of Escherichia coli cells.

Here, we supplied a direct and rapid quantum dots labelling method of bacterial cells in food, water and environmental contaminations. Outer layers of Escherichia coli cells prevent cells from direct interactions with molecules and objects such as quantum dots. Permeabilization treatment of E. coli cells may facilitate macromolecules penetrate cell walls and improve internal bacterial quantum dots (QDs) labelling. In this work, we investigated direct internal QDs labelling of E. coli cells permeabilized using three methods including chloroform-SDS treatment, lysozyme-EDTA treatment and osmotic shock treatment. Effects of permeabilization were analysed by scanning electronic microscopy and measuring activity of alkaline phosphatase (PhoA) released from periplasm. Internal bacterial QDs labelling was monitored by flow cytometry and fluorescent microscopy. After chloroform-SDS or lysozyme-EDTA treatment, cells could be directly labelled with QDs. No interaction was observed between osmotic shock treated cells and QDs. The mechanism of cell permeabilization explaining different labelling efficiency has been established. The QDs labelling approach presented in this work provides a simple, rapid and sensitive detection method for multiple purpose bacterial analysis in combination with biological techniques.

Characterization of Citrus tristeza virus strains from southern China based on analysis of restriction patterns and sequences of their coat protein genes.

Citrus tristeza virus (CTV) isolates collected from southern China were characterized by biological indexing on citrus indicators, restriction fragment length polymorphism (RFLP), and bi-directional reverse transcription-polymerase chain reaction (BD-PCR) analysis of their coat protein (CP) genes. Of the 30 isolates, only two isolates, N3 and N4, did not induce visible symptoms. Twenty-eight other isolates induced stem pitting and vein clearing, plant stunting, and leaf yellowing symptoms on Mexican lime, Duncan grapefruit, and sour orange seedlings. In BD-PCR analysis, a 392-bp fragment specific for the mild strains was amplified from isolates N3 and N4, and a 320-bp fragment specific for the severe strains was produced from the other 28 isolates. The RFLP analysis for RT-PCR products of the CP gene with restriction enzyme HinfI identified seven groups representing groups I-VI and a new group, which was not involved in the seven groups defined by Gillings (J Virol Methods 44:305-317, 1993). The sequences of the CP genes from 12 Chinese CTV isolates showed a high divergence, with 91.5-99.7% identities at the nucleotide level and 94.2-99.6% identities at the amino acid level. Our results suggest that the composition of CTV populations from China has a high genetic diversity in the CP gene.