The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as they transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e. fold change of  -1.5 or  1.5; P< 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g. fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.

Mutation of p53 is a common feature of cancer. Breast cancer is the most common malignancy that develops in women; however, somatic mutation of p53 is rare, suggesting that p53 becomes inactivated by other mechanisms. p53 is expressed as smaller isoforms, some of which inhibit wild-type p53. There are no studies that have examined the relative expression of all isoforms in this disease. We have analysed the relative messenger RNA expression of the p53 isoforms, Δ40, Δ133, β and γ in a panel of 6 breast cancer cell lines, 148 breast cancers specimens and 31 matched normal adjacent tissues by semi-quantitative real-time reverse transcription-PCR and analysed their relationship to clinical features and outcome. We have identified several important clinical associations, particularly with Δ40p53, which was expressed at levels that were ~50-fold higher than the least expressed isoform p53γ. Δ40p53 was significantly upregulated in tumour tissue when compared with the normal breast and was significantly associated with an aggressive breast cancer subtype-triple negative. Additionally, p53β expression was significantly negatively associated with tumour size and positively associated with disease-free survival, where high levels of p53β were protective, particularly in patients with a mutation in p53, suggesting p53β may counteract the damage inflicted by mutant p53. In conclusion, the relative expression of p53 isoforms is related to clinical features of breast cancer and outcome. These results have implications for the stratification of breast cancer based on p53 function and may provide an alternate explanation for deregulated p53 signalling in breast cancer.

BACKGROUND: Subtle histopathologic features such as eosinophilia and increased mast cells have been observed in functional gastrointestinal disorders (FGIDs), including functional dyspepsia (FD) and the irritable bowel syndrome (IBS). The mechanisms that drive recruitment of these cells to the gastrointestinal tract remain unexplained, largely due to the heterogeneity in phenotypes among patients diagnosed with such conditions. We aimed to systematically review the literature and collate the evidence for immune activation in FD and IBS, and where possible, detail the nature of activation. METHODS: Seven literature databases were searched using the keywords: ‘functional gastrointestinal disorder’, FGID, ‘functional dyspepsia’, ‘non-ulcer dyspepsia’, ‘idiopathic dyspepsia’, ‘irritable bowel syndrome’, IBS and ‘immun*’. RESULTS: Fifty-one papers reporting discordant immune features met the selection criteria for this review. Changes in lymphocyte populations, including B and T lymphocyte numbers and activation status were reported in IBS and FD, in conjunction with duodenal eosinophilia in FD and increased colonic mast cells in IBS. Increases in circulating α4+β7+ gut-homing T cells appear to be linked to the pathophysiology of both FD and IBS. Studies in the area are complicated by poor phenotyping of patients into subgroups and the subtle nature of the immune activity involved in FD and IBS. CONCLUSIONS: Alterations in proportions of gut-homing T lymphocytes in both FD and IBS indicate that a loss of mucosal homeostasis may drive the symptoms of FD and IBS. There is indirect evidence that Th17 responses may play a role in FGIDs, however the evidence for a Th2 immune phenotype in FD and IBS is limited. Although immune involvement is evident, large, well-characterised patient cohorts are required to elucidate the immune mechanisms driving the development of FGIDs.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and no targeted treatments. TNBC patients are more likely to develop metastases and relapse than patients with other breast cancer subtypes. We aimed to identify TNBC-specific genes and genes associated with lymph node metastasis, one of the first signs of metastatic spread. A total of 33 TNBCs were used; 17 of which had matched normal adjacent tissues available, and 15 with matched lymph node metastases. Gene expression microarray analysis was used to reveal genes that were differentially expressed between these groups. We identified and validated 66 genes that are significantly altered when comparing tumours to normal adjacent samples. Further, we identified 83 genes that are associated with lymph node metastasis and correlated these with miRNA-expression. Pathway analysis revealed their involvement in DNA repair, recombination and cell death, chromosomal instability and other known cancer-related pathways. Finally, four genes were identified that were specific for TNBC, of which one was associated with overall survival. This study has identified novel genes involved in LN metastases in TNBC and genes that are TNBC specific that may be used as treatment targets or prognostic indicators in the future.

BackgroundBreast cancer is the most common malignancy that develops in women, responsible for the highest cancer-associated death rates. Triple negative breast cancers represent an important subtype that have an aggressive clinical phenotype, are associated with a higher likelihood of metastasis and are not responsive to current targeted therapies. miRNAs have emerged as an attractive candidate for molecular biomarkers and treatment targets in breast cancer, but their role in the progression of triple negative breast cancer remains largely unexplored.MethodsThis study has investigated miRNA expression profiles in 31 primary triple negative breast cancer cases and in 13 matched lymph node metastases compared with 23 matched normal breast tissues to determine miRNAs associated with the initiation of this disease subtype and those associated with its metastasis.Results71 miRNAs were differentially expressed in triple negative breast cancer, the majority of which have previously been associated with breast cancer, including members of the miR-200 family and the miR-17-92 oncogenic cluster, suggesting that the majority of miRNAs involved in the initiation of triple negative breast cancer are not subtype specific. However, the repertoire of miRNAs expressed in lymph node negative and lymph node positive triple negative breast cancers were largely distinct from one another. In particular, miRNA profiles associated with lymph node negative disease tended to be up-regulated, while those associated with lymph node positive disease were down-regulated and largely overlapped with the profiles of their matched lymph node metastases. From this, 27 miRNAs were identified that are associated with metastatic capability in the triple negative breast cancer subtype.ConclusionsThese results provide novel insight into the repertoire of miRNAs that contribute to the initiation of and progression to lymph node metastasis in triple negative breast cancer and have important implications for the treatment of this breast cancer subtype.

Triple negative breast cancer (TNBC) is characterised by the lack of receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2). Since it cannot be treated by current endocrine therapies which target these receptors and due to its aggressive nature, it has one of the worst prognoses of all breast cancer subtypes. The only treatments remain chemo- and/or radio-therapy and surgery and because of this, novel biomarkers or treatment targets are urgently required to improve disease outcomes. MicroRNAs represent an attractive candidate for targeted therapies against TNBC, due to their natural ability to act as antisense interactors and regulators of entire gene sets involved in malignancy and their superiority over mRNA profiling to accurately classify disease. Here we review the current knowledge regarding miRNAs as biomarkers in TNBC and their potential use as therapeutic targets in this disease. Further, we review other epigenetic changes and interactions of these changes with microRNAs in this breast cancer subtype, which may lead to the discovery of new treatment targets for TNBC.

Inflammatory bowel disease (IBD) is a progressive disease that includes Crohn's diseases and ulcerative colitis. Over one third of IBD patients have secondary organ pathologies and pulmonary manifestations are common. The platelet activating factor receptor (PAFR) plays a critical role in regulating inflammation and it is expressed on epithelial cells and neutrophils in both colon and lung. Having identified increased expression of PAFR in both the gastrointestinal (GI) tract and lungs of mice with dextran sulfate sodium (DSS) colitis, we hypothesised that PAFR was a mediator of the pulmonary inflammation associated with colitis. We aimed to elucidate the role of PAFR in gut‐lung inflammatory cross‐talk using of the DSS‐induced experimental model of colitis (7 days). DSS mice were treated with PAFR antagonist CV6209 both intranasally and intravenously and lung, colon and blood were assessed for inflammatory cells and mediators by qPCR, immunoblot, immunohistochemistry and flow cytometry. We demonstrated that DSS‐induced colitis resulted in inflammation in mouse bronchoalveolar lavage fluid (BALF) and lungs, as well as increased PAFR protein levels in lungs and colons. Pulmonary neutrophils in DSS animals showed increased PAFR staining. Bacterial 16S mRNA expression were also increased in mouse lungs after 7 days DSS challenge. Both intravenous and intranasal inhibition of PAFR by CV6209 reduced colitis‐induced numbers of neutrophils, but not macrophages, in mouse lungs. Inhibition of PAFR also reduced levels of TNF and IL‐1b proteins and bacterial 16S expression in mouse lungs. Importantly, intravenous administration of CV6209 reduced colitis pathology, TNF and IL‐1b protein levels in mouse colons after 7 days DSS challenge. In the DSS model, increased PAFR protein expression was associated with inflammasome activation, characterized by increased NLRP3 and mature caspase‐1 proteins, however inhibition of PAFR by CV6209 reduced NLRP3 and caspase‐1 levels after DSS challenge in mouse lungs. In vitro, NLRP3, activated caspase‐1 and secreted IL‐1b protein levels were increased in human alveolar epithelial cell culture (A549) after 24h LPS stimulation, and this was inhibited by treatment with CV6209. These data suggest that PAFR regulates colitis‐induced lung inflammation by IL‐1b protein activation via the NLRP3 inflammasome signalling pathway. PAFR may act as an inflammasome‐activating pattern recognition receptor during mucosal inflammation thus is a potential therapeutic target for lung inflammation associated with colitis and bacteraemia. Support or Funding Information This study was supported by a grant from National Health Medical Research Council (NHMRC) Australia This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inflammatory bowel disease (IBD) is associated with several immune-mediated extraintestinal manifestations. More than half of all IBD patients have some form of respiratory pathology, most commonly neutrophil-mediated diseases, such as bronchiectasis and chronic bronchitis. Using murine models of colitis, we aimed to identify the immune mechanisms driving pulmonary manifestations of IBD. We found increased neutrophil numbers in lung tissue associated with the pulmonary vasculature in both trinitrobenzenesulfonic acid- and dextran sulfate sodium-induced models of colitis. Analysis of systemic inflammation identified that neutrophilia was associated with bacteremia and pyrexia in animal models of colitis. We further identified IL-6 as a systemic mediator of neutrophil recruitment from the bone marrow of dextran sulfate sodium animals. Functional inhibition of IL-6 led to reduced systemic and pulmonary neutrophilia, but it did not attenuate established colitis pathology. These data suggest that systemic bacteremia and pyrexia drive IL-6 secretion, which is a critical driver for pulmonary manifestation of IBD. Targeting IL-6 may reduce neutrophil-associated extraintestinal manifestations in IBD patients.