The 16S rRNA gene has been a mainstay of sequence-based bacterial analysis for decades. However, high-throughput sequencing of the full gene has only recently become a realistic prospect. Here, we use in silico and sequence-based experiments to critically re-evaluate the potential of the 16S gene to provide taxonomic resolution at species and strain level. We demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene. We further demonstrate that full-length sequencing platforms are sufficiently accurate to resolve subtle nucleotide substitutions (but not insertions/deletions) that exist between intragenomic copies of the 16S gene. In consequence, we argue that modern analysis approaches must necessarily account for intragenomic variation between 16S gene copies. In particular, we demonstrate that appropriate treatment of full-length 16S intragenomic copy variants has the potential to provide taxonomic resolution of bacterial communities at species and strain level.

Long noncoding RNAs (lncRNAs) are potentially important regulators of cell differentiation and development, but little is known about their roles in B lymphocytes. Using RNA-seq and de novo transcript assembly, we identified 4516 lncRNAs expressed in 11 stages of B-cell development and activation. Most of these lncRNAs have not been previously detected, even in the closely related T-cell lineage. Comparison with lncRNAs previously described in human B cells identified 185 mouse lncRNAs that have human orthologs. Using chromatin immunoprecipitation-seq, we classified 20% of the lncRNAs as either enhancer-associated (eRNA) or promoter-associated RNAs. We identified 126 eRNAs whose expression closely correlated with the nearest coding gene, thereby indicating the likely location of numerous enhancers active in the B-cell lineage. Furthermore, using this catalog of newly discovered lncRNAs, we show that PAX5, a transcription factor required to specify the B-cell lineage, bound to and regulated the expression of 109 lncRNAs in pro-B and mature B cells and 184 lncRNAs in acute lymphoblastic leukemia.

The gut microbiome is a major determinant of host health, yet it is only in the last 2 decades that the advent of next-generation sequencing has enabled it to be studied at a genomic level. Shotgun sequencing is beginning to provide insight into the prokaryotic as well as eukaryotic and viral components of the gut community, revealing not just their taxonomy, but also the functions encoded by their collective metagenome. This revolution in understanding is being driven by continued development of sequencing technologies and in consequence necessitates reciprocal development of computational approaches that can adapt to the evolving nature of sequence datasets. In this review, we provide an overview of current bioinformatic strategies for handling metagenomic sequence data and discuss their strengths and limitations. We then go on to discuss key technological developments that have the potential to once again revolutionise the way we are able to view and hence understand the microbiome.

Oxygenated polyunsaturated fatty acids (oxylipins) are bioactive molecules established as important mediators during inflammation. Different classes of oxylipins have been found to have opposite effects, e.g., pro-inflammatory prostaglandins and anti-inflammatory resolvins. Production of the different classes of oxylipins occurs during distinct stages of development and resolution of inflammation. Chronic inflammation is involved in the progression of many pathophysiological conditions and diseases such as non-alcoholic fatty liver disease, insulin resistance, diabetes, and obesity. Determining oxylipin profiles before, during, and after inflammatory-related diseases could provide clues to the onset, development, and prevention of detrimental conditions. This review focusses on recent developments in our understanding of the role of oxylipins in inflammatory disease, and outlines novel technological advancements and approaches to study their action.

Weight loss improves metabolic dysfunction-associated steatohepatitis (MASH). We investigated whether there were associated changes in intestinal permeability, short-chain fatty acids (SCFAs), and gut microbiota, which are implicated in the pathophysiology of MASH. Sixteen adults with MASH, moderate fibrosis, and obesity received a low-energy total diet replacement program for 12 weeks and stepped food re-introduction over the following 12 weeks (ISRCTN12900952). Intestinal permeability, fecal SCFAs, and fecal microbiota were assessed at 0, 12, and 24 weeks. Data were analyzed using mixed-effects linear regression and sparse partial least-squares regression. Fourteen participants completed the trial, lost 15% (95% CI: 11.2-18.6%) of their weight, and 93% had clinically relevant reductions in liver disease severity markers. Serum zonulin concentrations were reduced at both 12 and 24 weeks (152.0 ng/ml, 95% CI: 88.0-217.4, p < 0.001). Each percentage point of weight loss was associated with a 13.2 ng/mL (95% CI: 3.8-22.5, p < 0.001) reduction in zonulin. For every 10 ng/mL reduction in zonulin, there was a 6.8% (95% CI: 3.5%-10.2, p < 0.001) reduction in liver fat. There were reductions in SCFA and alpha diversity evenness as well as increases in beta diversity of the gut microbiota at 12 weeks, but the changes did not persist at 24 weeks. In conclusion, substantial dietary energy restriction is associated with significant improvement in MASH markers alongside reduction in intestinal permeability. Changes in gut microbiota and SCFA were not maintained with sustained reductions in weight and liver fat, suggesting that microbiome modulation may not explain the relationship between weight loss and improvements in MASH.