Heterosubtypic immunity to influenza A virus infection requires a properly diversified antibody repertoire

Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT(-/-)) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (D(H)) with an altered D(H) segment (namely, Delta D-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT(-/-) mice compared to those in Delta D-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.     

Structure-activity relationship (SAR) investigations of substituted imidazole analogs as TRPV1 antagonists

A novel series of 4,5-biarylimidazoles as TRPV1 antagonists were designed based on the previously reported 4,6-disubstituted benzimidazole series. The analogs were evaluated for their ability to block capsaicin- or acid-induced calcium influx in TRPV1-expressing CHO cells. These studies led to the identification of a highly potent and orally bioavailable TRPV1 antagonist, imidazole 33.     

ERbeta ligands. Part 6: 6H-Chromeno[4,3-b]quinolines as a new series of estrogen receptor beta-selective ligands

A new class of estrogen receptor beta (ERbeta) ligands based on the 6H-chromeno[4,3-b]quinoline scaffold has been prepared. Several C7-substituted analogues displayed high affinity and modest selectivity for ERbeta.     

Isolation of New Delhi metallo‐β‐lactamase 1‐producing Vibrio cholerae non‐O1, non‐O139 strain carrying ctxA,st and hly genes in southern Vietnam

Vibrio cholerae non‐O1, non‐O139 (VC_NAG) organisms are universally present in the aquatic environment and regarded as non‐pathogenic bacteria. However, considering that they do occasionally induce gastroenteritis, a study of their virulence and antibiotic resistance genes is important. The presence of enteropathogenic genes, including ctxA, VC_NAG‐specific heat‐stable toxin gene (st), hemolysin (hly), and zona occludens toxin (zot) was determined by PCR in 100 VC_NAG strains isolated in southern Vietnam in 2010–2013 from 94 environmental and six human origins. These 100 VC_NAG strains were also tested phenotypically and genotypically for the presence of the New Delhi metallo‐β‐lactamase (NDM‐1). Of the 100 VC_NAG strains tested, six were positive for ctxA; five from the environment and one of human origin. The st gene was detected in 17 isolates, 15 and two of which were of environmental and human origins, respectively. Gene hly was detected in 19 VC_NAG strains examined, two of which were isolated from humans and 17 from environments. The zot gene was not detected in any of the strains tested. Three VC_NAG strains of environmental origin were confirmed to produce NDM‐1 and the bla_NDM‐1 gene was detected in those strains by PCR. Of note, one of the three NDM‐1‐producing VC_NAG strains was confirmed to carry ctxA, st and hly genes concurrently. This is the first report of isolation of NDM‐1‐producing VC_NAG strains in Vietnam.     

RUN and FYVE domain–containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17–positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.     

The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) represents an anomalous subset of breast cancer with a greatly reduced (30%) 5-year survival rate. The enhanced mortality and morbidity of TNBC arises from the high metastatic rate, which requires the acquisition of AnR, a process whereby anchorage-dependent cells become resistant to cell death induced by detachment. In this study TNBC cell lines were selected for AnR, and these cell lines demonstrated dramatic enhancement in the formation of lung metastases as compared with parental cells. Genetic analysis of the AnR subclones versus parental cells via next generation sequencing and analysis of global alternative RNA splicing identified that the mRNA splicing of cytoplasmic polyadenylation element binding 2 (CPEB2), a translational regulator, was altered in AnR TNBC cells. Specifically, increased inclusion of exon 4 into the mature mRNA to produce the CPEB2B isoform was observed in AnR cell lines. Molecular manipulations of CPEB2 splice variants demonstrated a key role for this RNA splicing event in the resistance of cells to anoikis. Specifically, down-regulation of the CPEB2B isoform using siRNA re-sensitized the AnR cell lines to detachment-induced cell death. The ectopic expression of CPEB2B in parental TNBC cell lines induced AnR and dramatically increased metastatic potential. Importantly, alterations in the alternative splicing of CPEB2 were also observed in human TNBC and additional subtypes of human breast cancer tumors linked to a high metastatic rate. Our findings demonstrate that the regulation of CPEB2 mRNA splicing is a key mechanism in AnR and a driving force in TNBC metastasis.     

Long ncRNA expression associates with tissue-specific enhancers

Long non-coding RNAs (ncRNA) have recently been demonstrated to be expressed from a subset of enhancers and to be required for the distant regulation of gene expression. Several approaches to predict enhancers have been developed based on various chromatin marks and occupancy of enhancer-binding proteins. Despite the rapid advances in the field, no consensus how to define tissue specific enhancers yet exists. Here, we identify 2,695 long ncRNAs annotated by ENCODE (corresponding to 28% of all ENCODE annotated long ncRNAs) that overlap tissue-specific enhancers. We use a recently developed algorithm to predict tissue-specific enhancers, PreSTIGE, that is based on the H3K4me1 mark and tissue specific expression of mRNAs. The expression of the long ncRNAs overlapping enhancers is significantly higher when the enhancer is predicted as active in a specific cell line, suggesting a general interdependency of active enhancers and expression of long ncRNAs. This dependency is not identified using previous enhancer prediction algorithms that do not account for expression of their downstream targets. The predicted enhancers that overlap annotated long ncRNAs generally have a lower ratio of H3K4me1 to H3K4me3, suggesting that enhancers expressing long ncRNAs might be associated with specific epigenetic marks. In conclusion, we demonstrate the tissue-specific predictive power of PreSTIGE and provide evidence for thousands of long ncRNAs that are expressed from active tissue-specific enhancers, suggesting a particularly important functional relationship between long ncRNAs and enhancer activity in determining tissue-specific gene expression.