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Abstract: Neuregulins are growth factors that signal via the ErbB3 and ErbB4 receptors. Here we show using immunohistochemistry that they are often expressed in the nucleus of a range of tumour types including soft tissue and breast. The Neuregulin 1 type I-β3 (NRG1-β3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-β3 tagged with photoactivatable GFP and demonstrated that this re-localised from nucleoli to spliceosomes over 90min. Tyrosine kinase activity was not required for retaining the NRG1-β3 within the nucleus. Mutation of the lysines 14 and 16 or 15 and 16 together prevented nucleolar uptake while four positively charged residues were identified which were required for spliceosome uptake. Molecular modelling suggests that three of these may form a binding site. We showed using a kinome array that NRG1-β3 and a mutant exclusively localising to spliceosomes increased phosphorylation and/or expression of the HER4 and HER2 receptors. Using a transcriptomic analysis the same two constructs induced expression of several messenger RNAs and we confirmed the increased expression at the protein level of the most highly induced, Heat Shock Protein 70B′. These results suggest that Neuregulin activates receptor signalling in spliceosomes leading to altered gene expression. [ABSTRACT FROM AUTHOR]

Abstract: In Trypanosoma brucei the GPI-anchored variant surface glycoprotein (VSG) represents ∼90% of cell surface protein and a major proportion of endoplasmic reticulum (ER) biosynthetic output. We identified four trypanosomatid-specific genes encoding candidate ER-resident proteins; all were required for normal proliferation. For Tb11.01.2640 and Tb11.01.8120, an increase in VSG abundance was found on silencing, while the protein products localized to the ER; we designated these ERAP32 and ERAP18 for ER-associated protein of 32kDa and 18kDa. Silencing ERAP32 or ERAP18 did not alter expression levels of ISG65 or ISG75, the major surface trans-membrane domain proteins. Surface biotinylation or immunoflorescence did not identify intracellular VSG accumulation, while FACS and fluorescence microscopy indicated that the cells were not increased in size, arguing for increased VSG density on the cell surface. Therefore, ERAP32 and ERAP18 are trypanosome-specific ER-localized proteins with a major role in VSG protein export and, contrary to current paradigms, VSG is not saturated on the cell surface. [Copyright &y& Elsevier]

MicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood. A microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression. The results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49 cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49 cells in vivo. Our results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC. • The expression of miR-3960 in BC patients were positively correlated with survival. • DEXI knockdown inhibits MB49 cell proliferation. • Transfection of miR-3960 inhibited BC organoid growth. • Inhibition of DEXI suggested as a promising therapeutic strategy against bladder BC. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignant disease with a poor prognosis. We previously found that p62 presented a marked nuclear-cytoplasmic translocation in ESCC cells as compared that in normal esophageal epithelial cells, but its effects on ESCC cells remain unclear. This study aims to clarify the impacts of different cellular localization of p62 on the function of ESCC cells and the underlying molecular mechanisms. We here demonstrated that cytoplasmic p62 enhances the migration and invasion abilities of esophageal cancer cells, whereas nuclear p62 has no effect. We further explored the interaction protein of p62 by using GST pull-down experiment and identified EPLIN as a potential protein interacting with p62. In addition, reducing EPLIN expression significantly inhibited the migration and invasion of ESCC cells, which were rescued when EPLIN expression was restored after the p62 knockdown. At a molecular level, p62 in cytoplasm positively regulated the expression of EPLIN via enhancing its protein stability. Data from the TCGA and GEO database displayed a significant up-regulation of EPLIN mRNA expression in ESCC tissues compared with corresponding paired esophageal epithelial samples. Our findings present evidence that the nuclear-cytoplasmic translocation of p62 protein contributes to an aggressive malignancy phenotype, providing candidate molecular biomarkers and potential molecular targets for the diagnosis and treatment of ESCC. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the biological function of hpsh4590 in Helicobacter pylori . After Hpsh4590 was expressed using a prokaryotic expression system, the cytotoxic effects and IL-8 production of Hpsh4590 were analyzed by co-culturing with GES-1 cells. Meanwhile, the antibody of rHpsh4590, produced by immunizing rabbit, was used for localization and protein interaction studies. Hpsh4590 fusion protein was expressed successfully in Escherichia coli Rosetta (DE3), and the polyclonal antibody was produced at high titers. The MTT assay showed that the inhibition ratio of GES-1 cells cultured with 0.1 μg/mL rHpsh4590 (3.02% ± 0.02%) was significantly lower than that of 20 μg/mL rHpsh4590 (57.57% ± 0.03%, p < 0.01 ), while DAPI staining showed the cytotoxic effects of rHpsh4590 for GES-1 cells. The up-regulation of cleaved caspase-3 and cleaved PARP was observed after GES-1 cells co-cultured with rHpsh4590 by Western blot. Co-culturing of GES-1 cells with rHpsh0459 (20 μg/mL) led to significant production of IL-8 at 12 h(1097.74 ± 212.37 pg/mL) and 24 h (1379.55 ± 209.58 pg/mL) then at 6 h(134.68 ± 14.64 pg/mL, p < 0.01). These observations suggest that the cytotoxicity of Hpsh4590 occurred in a concentration dependent manner, which is related with IL-8 secretion from gastric mucosal epithelial cells. Hpsh4590 was found localized in the membrane and the periplasm of H. pylori , interacted with zinc finger protein and methionine ABC transporter ATP-binding protein, and potentially regulates DNA uptake or transfer. [ABSTRACT FROM AUTHOR]

Abstract: CD4+ T cells are the main targets of HIV-1 and play a central role during the progression of AIDS, but the mechanism has not been clearly elucidated. In the present study, blood samples were collected from HIV-1-infected Chinese individuals, including typical progressors (TPs) and long-term nonprogressors (LTNPs). More HIV-1 productively infected CD4+ T cells were obtained through co-cultures and the infected CD4+ T cells were discriminated from bystander cells by intracellular p24 staining. The activation level and dynamics of cytokine expression of CD4+ T cells were analyzed. After stimulating the freshly isolated PBMCs with PHA, the frequencies of CD69+CD4+ T cells/CD25+CD4+ T cells were higher in TP than in LTNP group and were positively correlated with viral load and negatively correlated with CD4+ T cell counts. The activation level of CD4+ T cells in the co-cultured PBMCs was higher in TP than in LTNP group, and HIV-1 productively infected CD4+ T cells were more activated than bystander CD4+ T cells. The expression of Th1 cytokines (IL-2 and IFN-γ) and the frequency of Th1 cells in co-cultured PBMCs were lower in TP than in LTNP group. HIV-1 productively infected CD4+ T cells expressed higher level of Th1/Th2 cytokines than bystander cells. More productive HIV-1 infection occurred in Th1 than in Th2 cells, followed by Th0 cells. The present results suggest that the excessive activation level of CD4+ T cells and the preferential replication of HIV-1 in Th1 cells that lead to the shift of Th1 to Th2 are likely crucial to AIDS progression in HIV-1-infected Chinese individuals. [Copyright &y& Elsevier]

Abstract: To develop a real-time thallium flux assay for high-throughput screening (HTS) of human KCNQ4 (Kv7.4) potassium channel openers, we used CHO-K1 cells stably expressing human KCNQ4 channel protein and a thallium-sensitive dye based on the permeability of thallium through potassium channels. The electrophysiological and pharmacological properties of the cell line expressing the KCNQ4 protein were found to be in agreement with that reported elsewhere. The EC50 values of the positive control compound (retigabine) determined by the thallium and 86rubidium flux assays were comparable to and consistent with those documented in the literature. Signal-to-background (S/B) ratio and Z factor of the thallium influx assay system were assessed to be 8.82 and 0.63, respectively. In a large-scale screening of 98,960 synthetic and natural compounds using the thallium influx assay, 76 compounds displayed consistent KCNQ4 activation, and of these 6 compounds demonstrated EC50 values of less than 20μmol/L and 2 demonstrated EC50 values of less than 1μmol/L. Taken together, the fluorescence-based thallium flux assay is a highly efficient, automatable, and robust tool to screen potential KCNQ4 openers. This approach may also be expanded to identify and evaluate potential modulators of other potassium channels. [Copyright &y& Elsevier]

Abstract: Transcription factors (TFs) are proteins that bind to specific promoter regions of their target genes and regulate gene transcription. Many of these factors have been found to influence flowering. Lycoris longituba exhibits a great deal of diversity in flower color and flower form, making it a suitable model for the study of floral development. We have identified 338 putative TFs from more than thirty thousand ESTs sequenced from the floral tissue of L. longituba, and validated them using real-time RT-PCR. Fifty-one of the TFs were recognized as being potentially flower-specific, and the expression patterns of some of them during six flowering phases have been elucidated. Homolog annotation and phylogenetic analysis revealed that some TFs that belong to several TF families, such as MADS, MYB-related, NAC, and ABI3-VP1, were suggested to play important roles in the flowering process. Our dataset may be used to identify priority target TF genes for further study. [Copyright &y& Elsevier]