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Elevated expression and activity of the epidermal growth factor receptor (EGFR)/protein kinase B (Akt) signaling pathway is associated with development, progression and treatment resistance of head and neck cancer (HNC). Several studies have demonstrated that microRNA-7 (miR-7) regulates EGFR expression and Akt activity in a range of cancer cell types via its specific interaction with the EGFR mRNA 3'-untranslated region (3'-UTR). In the present study, we found that miR-7 regulated EGFR expression and Akt activity in HNC cell lines, and that this was associated with reduced growth in vitro and in vivo of cells (HN5) that were sensitive to the EGFR tyrosine kinase inhibitor (TKI) erlotinib (Tarceva). miR-7 acted synergistically with erlotinib to inhibit growth of erlotinib-resistant FaDu cells, an effect associated with increased inhibition of Akt activity. Microarray analysis of HN5 and FaDu cell lines transfected with miR-7 identified a common set of downregulated miR-7 target genes, providing insight into the tumor suppressor function of miR-7. Furthermore, we identified several target miR-7 mRNAs with a putative role in the sensitization of FaDu cells to erlotinib. Together, these data support the coordinate regulation of Akt signaling by miR-7 in HNC cells and suggest the therapeutic potential of miR-7 alone or in combination with EGFR TKIs in this disease.

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of high-grade gliomas. However, the precise mechanistic role of many miRNAs in this disease remains unclear. Here, we investigate the functional role of miR-331-3p in glioblastoma multiforme (GBM). We found that miR-331-3p expression in GBM cell lines is significantly lower than in normal brain, and that transient overexpression of miR-331-3p inhibits GBM cell line proliferation and clonogenic growth, suggesting a possible tumor suppressor role for miR-331-3p in this system. Bioinformatics analysis identified neuropilin-2 (NRP-2) as a putative target of miR-331-3p. Using transfection studies, we validated NRP-2 mRNA as a target of miR-331-3p in GBM cell lines, and show that NRP-2 expression is regulated by miR-331-3p. RNA interference (RNAi) to inhibit NRP-2 expression in vitro decreased the growth and clonogenic growth of GBM cell lines, providing further support for an oncogenic role for NRP-2 in high-grade gliomas. We also show that miR-331-3p inhibits GBM cell migration, an effect due in part to reduced NRP-2 expression. Finally, we identified a significant inverse correlation between miR-331-3p and NRP-2 expression in The Cancer Genome Atlas GBM cohort of 491 patients. Together, our results suggest that a loss of miR-331-3p expression contributes to GBM development and progression, at least in part via upregulating NRP-2 expression and increasing cell proliferation and clonogenic growth.

Background. Given the apparent high mortality associated with the novel swine-origin influenza A/H1N1 virus (S-OIV) in Mexico, we aimed to study the cytokine profiles induced by S-OIV and the effect of immunomodulators. Methods. We assayed cytokines and their messenger RNA (mRNA) levels in culture supernatants of human macrophages infected with H5N1, S-OIV California/04/2009 (S-OIV-CA), S-OIV Hong Kong/415742 (S-OIV-HK), or seasonal H1N1 with or without celecoxib and mesalazine. Results. Among the 12 cytokines showing detectable levels, levels of 8 proinflammatory cytokines (interleukin [IL] 2R, IL-6, interferon [IFN] α, macrophage inflammatory protein [MIP] α, MIP-1b, IFN-induced protein 10, regulated on activation, normal T cell expressed and secreted [RANTES], and monocyte chemotactic protein [MCP] 1) were higher in cells infected by H5N1 but similar among cells infected with H1N1, S-OIV-CA, or SOIV- HK. The levels of the other 4 cytokines were similar for H5N1, H1N1, S-OIV-CA and S-OIV-HK. Among the 8 cytokines induced by H5N1, 6 were suppressed by celecoxib and mesalazine. The mRNA levels of tumor necrosis factor a, IFN-γ, IL-6, and MCP-1 induced by H5N1 were higher than the levels of other cytokines at 12 and/or 24 h. Conclusions. No major cytokine storm, as seen in H5N1 infection, is associated with S-OIV infection of cell lines. The mainstay of treatment for uncomplicated S-OIV infections should be antiviral agents without immunomodulators. For individual S-OIV-infected patients with severe primary viral pneumonia, severe sepsis, and multiorgan failure, immunomodulators may be considered as an adjunctive therapy in clinical trials.

Pregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear. We showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes. The adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation.

Talaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors. We examined the pathogenic roles of Mp1p and its homologs in a mouse model. All mice died 21 and 30 days after challenge with wild-type T. marneffei PM1 and MP1 complemented mutant respectively. None of the mice died 60 days after challenge with MP1 knockout mutant (P<0.0001). Seventy percent of mice died 60 days after challenge with MP1 knockdown mutant (P<0.0001). All mice died after challenge with MPLP1 to MPLP13 knockdown mutants, suggesting that only Mp1p plays a significant role in virulence. The mean fungal loads of PM1 and MP1 complemented mutant in the liver, lung, kidney and spleen were significantly higher than those of the MP1 knockout mutant. Similarly, the mean load of PM1 in the liver, lung and spleen were significantly higher than that of the MP1 knockdown mutant. Histopathological studies showed an abundance of yeast in the kidney, spleen, liver and lung with more marked hepatic and splenic necrosis in mice challenged with PM1 compared to MP1 knockout and MP1 knockdown mutants. Likewise, a higher abundance of yeast was observed in the liver and spleen of mice challenged with MP1 complemented mutant compared to MP1 knockout mutant. PM1 and MP1 complemented mutant survived significantly better than MP1 knockout mutant in macrophages at 48 hours (P<0.01) post-infection. The mean fungal counts of Pichia pastoris GS115-MP1 in the liver (P<0.001) and spleen (P<0.05) of mice were significantly higher than those of GS115 at 24 hours post-challenge. Mp1p is a key virulence factor of T. marneffei. Mp1p mediates virulence by improving the survival of T. marneffei in macrophages.

Gallbladder cancer (GBC) is a highly aggressive malignancy with limited therapeutic options, particularly in underrepresented populations, including South Africa. Understanding the molecular landscape of GBC may provide novel insights into its pathogenesis and potential therapeutic targets. Molecular changes are known to be associated with GBC, however, there is a paucity of this information especially in African populations. Furthermore, within the tumour microenvironment, different immune cells contribute to GBC progression. We investigated gene expression patterns in GBC tumours and their association with different immune cells in a cohort of South African patients. RNA sequencing was conducted on 2 normal and 8 gallbladder cancer tissues from South African patients to identify differentially expressed genes. Bioinformatics tools were used for pathway analysis, while immune cell quantification was performed using the quanTIseq software and presented as median [IQR]. Verification studies were further carried out using real-time PCR on an independent cohort comprising 7 gallstone samples and 26 gallbladder tumour samples. A total of 65 genes were found to be significantly differentially expressed between the gallbladder tumours and gallstone controls. We also identified 37 upregulated and 28 downregulated genes in this cohort. Among the most upregulated genes, MUC16 was confirmed to be significantly overexpressed in tumours. Normal tissues exhibited a significantly higher proportion of dysregulated genes associated with B cells (17.132 [14.866–18.483], p  < 0.0001) and M1 macrophages (18.943 [1.097–36.790], p  < 0.0001) compared to tumours. In contrast, tumours showed a greater association with dysregulated genes linked to regulatory T cells (Tregs) (14.373 [9.696–20.162]) relative to normal tissues. Pathway analysis further revealed the upregulation of defective GALNT12, defective GALNT3, defective C1GALT1C1 and termination of O-glycan biosynthesis, highlighting key mechanisms potentially involved in tumour progression. The study has shown the dysregulation of key genes in South African gallbladder cancer patients. Specifically, MUC16 was verified to be significantly elevated in tumour samples. Furthermore, the association of these dysregulated genes with key immune cells in this patient group may further highlight their roles in dysfunctional immune processes linked with tumourigenesis.

Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1.

Coronavirus HKU15 is a deltacoronavirus that was discovered in fecal samples of pigs in Hong Kong in 2012. Over the past three years, Coronavirus HKU15 has been widely detected in pigs in East/Southeast Asia and North America and has been associated with fatal outbreaks. In all such epidemiological studies, the virus was generally only detected in fecal/intestinal samples. In this molecular epidemiology study, we detected Coronavirus HKU15 in 9.6% of the nasopharyngeal samples obtained from 249 pigs in Hong Kong. Samples that tested positive were mostly collected during winter. Complete genome sequencing of the Coronavirus HKU15 in two nasopharyngeal samples revealed quasispecies in one of the samples. Two of the polymorphic sites involved indels, but the other two involved transition substitutions. Phylogenetic analysis showed that the two nasopharyngeal strains in the present study were most closely related to the strains PDCoV/CHJXNI2/2015 from Jiangxi, China, and CH/Sichuan/S27/2012 from Sichuan, China. The outbreak strains in the United States possessed highly similar genome sequences and were clustered monophyletically, whereas the Asian strains were more diverse and paraphyletic. The detection of Coronavirus HKU15 in respiratory tracts of pigs implies that in addition to enteric infections, Coronavirus HKU15 may be able to cause respiratory infections in pigs and that in addition to fecal-oral transmission, the virus could possibly spread through the respiratory route. The presence of the virus in respiratory samples provides an alternative clinical sample to confirm the diagnosis of Coronavirus HKU15 infection. Quasispecies were unprecedentedly observed in the 5′-untranslated region of coronavirus genomes. Emerging Microbes & Infections (2017) 6, e53; doi:10.1038/emi.2017.37; published online 21 June 2017

IntroductionThe current COVID-19 pandemic has forced hospices to look for more ways to support people remotely, including psychological support. Emotional disclosure-based interventions hold potential as a way of providing support remotely. However, evidence of their efficacy in people with terminal illness is mixed. Reviews have highlighted this may be due to interventions not being tailored to the unique needs of this population. In response to this, we are developing Let It Out (LIO), an online, self-guided emotional disclosure-based intervention tailored for people living with terminal illness.AimsThe primary objective of the study is to optimise the design of the LIO intervention. Secondary objectives include assessing its acceptability and feasibility; exploring potential impact on well-being; identifying potential adverse effects; and informing choice of outcome measures for potential future evaluation.Methods and analysisA single arm, mixed-methods, multisite, longitudinal study. Up to 40 people living with a terminal illness under the care of hospices in England and Scotland will receive the online LIO intervention. LIO consists of 3, self-guided expression sessions over 2 weeks. The primary outcome measures are (1) a structured feedback form completed by participants after the final expression session; and (2) semi-structured interviews and focus groups with ≤15 patient participants, ≤30 hospice staff and ≤15 informal carers. These quantitative and qualitative data will be triangulated via process evaluation to inform optimisation of the intervention design. Secondary outcome measures include validated measures of physical and psychological health collected at baseline and after the final expression session (immediately, 1, 4 and 8 weeks after); and data on recruitment, retention and fidelity.Ethics and disseminationThe study is approved by the University College London Research Ethics Committee (reference: 15281/002). The findings will be shared through peer-reviewed scientific journals and conferences, and traditional, online and social media platforms.