BackgroundHigh-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.MethodsSeven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.ResultsNo significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.ConclusionsUsing the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.

Elevated expression of COX-2 and increased levels of PGE 2 are found in numerous cancers and are associated with tumour development and progression. Although epidemiological, clinical and preclinical studies have shown that the inhibition of PGE 2 synthesis through the use of either non-steroidal anti-inflammatory drugs (NSAIDs) or specific COX-2 inhibitors (COXibs) has the potential to prevent and treat malignant disease, toxicities due to inhibition of COX-2 have limited their use. Thus, there is an urgent need for the development of strategies whereby COX-2 activity may be reduced without inducing any side effects. The biological effects of PGE 2 are mediated by signalling through four distinct E-type prostanoid (EP) receptors -EP 1 , EP 2 , EP 3 and EP 4 . In recent years, extensive effort has gone into elucidating the function of PGE 2 and the EP receptors in health and disease, with the goal of creating selective inhibitors as a means of therapy. In this review, we focus on PGE 2 , and in particular on the role of the individual EP receptors and their signalling pathways in neoplastic disease. As knowledge concerning the role of the EP receptors in cancer grows, so does the potential for exploiting the EP receptors as therapeutic targets for the treatment of cancer and metastatic disease. AbbreviationsCOXibs, specific COX-2 inhibitors; EMT, epithelial-mesenchymal transition; NSAIDs, non-steroidal anti-inflammatory drugs; YB-1, Y-box binding protein 1 BJP IntroductionInflammation has been established in recent years as playing a major role in cancer, with cancer-promoting inflammation an enabling characteristic underlying many, if not all, of the six hallmarks of cancer (Hanahan and Weinberg, 2011). In some cancers, the inflammatory conditions precede the development of malignancy, for example, ulcerative colitis is a major risk factor for colon cancer (Gupta et al., 2007). Alternatively, oncogenic mutations can drive tumour-promoting inflammation in tumours that are epidemiologically unrelated to overt inflammatory conditions (Del Prete et al., 2011). One key inflammatory mediator deregulated in many cancers is the COX enzyme, COX-2 (Janakiram and Rao, 2014). COX-2 expression has been shown in many cancers to be inversely associated with patient survival (Gallo et al., 2002;Peng et al., 2013;Sicking et al., 2014), with epidemiological studies suggesting that regular aspirin use decreases colorectal cancer incidence and mortality through the inhibition of COX-2 (Chan et al., 2009). Thus, drugs that target COX-2 may have chemopreventative or chemotherapeutic functions. Although drugs that target the COX enzymes have entered the clinic, albeit for different diseases, inhibition of COX-2 using either non-steroidal anti-inflammatory drugs (NSAIDs) or specific COX-2 inhibitors (COXibs) is associated with various side effects including gastric ulceration and myocardial infarction (Ranger, 2014). Such toxicities have limited their clinical applications. Dysregulation of COX-2 leads to elevated levels of its princ...

TLR signaling is a crucial component of the innate immune response to infection. MicroRNAs (miRNAs) have been shown to be upregulated during TLR signaling. Specifically, microRNA-146a (miR-146a) plays a key role in endotoxin tolerance by downregulating interleukin-1 receptor-associated kinase 1 (IRAK-1). The aim of this study was to assess the role of miR-146a in the TLR2 signaling and development of bacterial lipoprotein (BLP) self-tolerance and cross-tolerance to bacteria. Expression of miR-146a increased in a dose- and time-dependent manner in BLP-stimulated human THP-1 promonocytic cells. In BLP-tolerised cells miR-146a was even further upregulated in response to BLP re-stimulation (p<0.001). Re-stimulation of BLP-tolerised cells with heat-killed gram-negative Salmonella typhimurium (S. typhimurium), but not gram-positive Staphylococcus aureus (S. aureus), led to significant overexpression of miR-146a (p<0.05). Transfection of naive cells with a miR-146a mimic substantially suppressed TNF-α production (p<0.05). Furthermore, overexpression of miR-146a resulted in strong reduction in IRAK-1 and phosphorylated IκBα expression in naive and S. typhimurium-stimulated THP-1 cells. Collectively, miR-146a is upregulated in response to BLP and bacterial stimulation in both naive and BLP-tolerised cells. Overexpression of miR-146a induces a state analogous to tolerance in BLP-stimulated cells and therefore may represent a future target for exogenous modulation of tolerance during microbial infection and sepsis.