G0/G1 arrest and apoptosis induced by SARS-CoV 3b protein in transfected cells

Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), cause of the life-threatening atypical pneumonia, infects many organs, such as lung, liver and immune organ, and induces parenchyma cells apoptosis and necrosis. The genome of SARS-CoV, not closely related to any of the previously characterized coronavirus, encodes replicase and four major structural proteins and a number of non-structural proteins. Published studies suggest that some non-structural proteins may play important roles in the replication, virulence and pathogenesis of viruses. Among the potential SARS-CoV non-structural proteins, 3b protein (ORF4) is predicted encoding 154 amino acids, lacking significant similarities to any known proteins. Till now, there is no report about the function of 3b protein. In this study, 3b gene was linked with the EGFP tag at the C- terminus. Through cell cycle analysis, it was found that over-expression of 3b-EGFP protein in Vero, 293 and COS-7 cells could induce cell cycle arrest at G0/G1 phase, and that especially in COS-7 cells, expression of 3b-EGFP was able to induce the increase of sub-G1 phase from 24 h after transfection, which was most obvious at 48 h. The apoptosis induction of 3b fusion protein in COS-7 cells was further confirmed by double cell labeling with 7-AAD and Annexin V, the function of 3b protein inducing cell G0/G1 arrest and apoptosis may provide a new insight for further study on the mechanism of SARS pathogenesis

Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions

Plant-Wide Target Metabolomics Provides a Novel Interpretation of the Changes in Chemical Components during <i>Dendrobium officinale</i> Traditional Processing

The traditional processing of Dendrobium officinale (DO) is performed in five necessary processing steps: processing fresh strips, drying at 85 °C, curling, molding, and drying at 35 °C (Fengdou). The antioxidant activity of DO is increased after it is processed into Fengdou. To comprehensively analyze the changes in the functional components, a plant-wide target metabolomics approach was employed. In total, 739 differential chemical components were identified in five processing treatments, mainly highlighting differences in the levels of phenolic acids, flavonoids, lipids, and amino acids and their derivatives, and the glycosylation of aglycone resulted in the upregulation of flavonoid glycoside levels. Temperature is a key factor in DO processing during production. In addition, the enrichment of specific differential chemical components was found mainly in five different metabolic pathways: glucosinolate biosynthesis, linoleic acid metabolism, flavonoid biosynthesis, phenylpropanoid biosynthesis, and ubiquinone and other terpene quinone biosynthesis. A correlation analysis clarified that total phenols and flavonoids show a significant positive correlation with antioxidant capacity. This study provides new insights into the influence of the processing processes on DO quality, which may provide guidance for the high-quality production of DO

Predictable regulation of survival by intratumoral microbe-immune crosstalk in patients with lung adenocarcinoma

Intratumoral microbiota can regulate the tumor immune microenvironment (TIME) and mediate tumor prognosis by promoting inflammatory response or inhibiting anti-tumor effects. Recent studies have elucidated the potential role of local tumor microbiota in the development and progression of lung adenocarcinoma (LUAD). However, whether intratumoral microbes are involved in the TIME that mediates the prognosis of LUAD remains unknown. Here, we obtained the matched tumor microbiome and host transcriptome and survival data of 478 patients with LUAD in The Cancer Genome Atlas (TCGA). Machine learning models based on immune cell marker genes can predict 1- to 5-year survival with relative accuracy. Patients were stratified into high- and low-survival-risk groups based on immune cell marker genes, with significant differences in intratumoral microbial communities. Specifically, patients in the high-risk group had significantly higher alpha diversity (p < 0.05) and were characterized by an enrichment of lung cancer-related genera such as Streptococcus. However, network analysis highlighted a more active pattern of dominant bacteria and immune cell crosstalk in TIME in the low-risk group compared to the high-risk group. Our study demonstrated that intratumoral microbiota-immune crosstalk was strongly associated with prognosis in LUAD patients, which would provide new targets for the development of precise therapeutic strategies

Chemical structure of Tg.

<p>Chemical structure of Tg.</p

Involvment of [Ca²⁺]_i increase and CaM activity in Tg-induced uterine contractions and MLC20 phosphorylation.

<p>(A) Confocal fluorescence images of myometrial smooth muscle cells loaded with Fluo-4 AM before and after 2.5 µM Tg stimulation. The fluorescence images were observed at the excitation wavelength of 510 nm and 12 s after Tg stimulation. (B) Time course of the change of the fluorescence in a Fluo-4 AM-loaded myometrial smooth muscle cells in response to Tg (2.5 µM). The arrow indicates the time at which Tg was added. (<b>C</b>) Inhibitory effects of cumulative doses of W-7 on contractile responses after stimulation with Tg in rat myometrial strips (n = 3). Contractions were measured as the area under the curve (AUC) and expressed as a percentage of the response to 5 µM Tg. (<b>D–E</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of W-7 (500 nM), a common antagonist of calmodulin (D). Signal intensities for MLC20 and pMLC20 from three different blots were used for the quantitative analyses (E). Data are expressed as means ± SEM. ** <i>P</i><0.01 compared to control.</p

MLCK is involved in Tg-induced MLC20 phosphorylation and uterine contractions.

<p>(<b>A</b>) Representative recording of the inhibitory effects of cumulative doses of MLCK inhibitor ML-7 on rat myometrial contractions induced by 5 µM Tg, while the control strips were added with vehicle at the same time after stimulation with Tg. (<b>B–C</b>) Representative antibody reaction blots for the relative levels of MLC20 and pMLC20 in protein samples from Tg (400 nM) -treated or untreated rat myometrial cells in the presence or absence of ML-7 (500 nM), a specific inhibitor of MLCK (B). Signal intensities for MLC20 and pMLC20 from three different blots were used for the quantitative analyses (C). Data are expressed as means ± SEM. ** <i>P</i><0.01 compared to control.</p