Protocol for Metatranscriptomic analysis of Intestinal Microbiota

The objective of this publication is to provide the detailed protocol for metartancriptomisc studies of animal intestinal microbiota. The protocol describes isolation of high quality microbial community RNA from the mammalian intestinal content, subsequent mRNA enrichment, cDNA synthesis and sequencing. Twelve libraries were prepared, pooled in equimolar concentrations into a single library and sequenced on one GS Titanium 70×75 picotiter plate, following this protocol. The total number of reads obtained for 12 libraries was 1,155,062 (average 96,000 per library) and the combined size of 12 libraries was 521 million bases (average 43 million bases per library). The reported size of non-ribosomal RNA library fraction is ~15%, the fraction of non-ribosomal reads is ~17%. Hence we described a robust technique for metranscriptomic studies of animal intestinal microbiota. The double stranded cDNAs, prepared following this protocol, are suitable for pyrosequencing (454, Illumina), clone library construction or could be used to archive and store metaranscriptomic samples

ARHGEF2 (rho/rac guanine nucleotide exchange factor (GEF) 2)

Review on ARHGEF2 (rho/rac guanine nucleotide exchange factor (GEF) 2), with data on DNA, on the protein encoded, and where the gene is implicated

Media from macrophages co-incubated with <i>Enterococcus faecalis</i> induces epithelial cell monolayer reassembly and altered cell morphology

Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis

Exosomes and metabolic functionin mice exposed to alternating dark-light cycles mimicking night shift work schedules

Sleep is an important modulator of metabolic function. Disruptions of sleep in circadian rhythm are common in modern societies and are associated with increased risk of developing cardiometabolic disorders. Exosomes are ubiquitous extracellular vesicles that may play a mechanistic role in metabolic derangements. We hypothesized that alternating dark-light cycles mimicking shift work in mice would alter fecal microbiota and colonic epithelium permeability and alter plasma exosome cargo and metabolic function. C57BL/6 mice were randomly assigned to (i) control day light (CL), or (ii) inverted dark-light every 2 weeks for 8 weeks (IN). Body weight, fat mass and HOMA-IR were measured, along with Tregs, metabolic, and resident macrophages in visceral white adipose tissue (vWAT). Fecal water samples were incubated with confluent colonic epithelium cell cultures in electric cell-substrate impedance sensing (ECIS) arrays, and plasma exosomes were added to differentiated adipocytes and insulin-induced pAKT/AKT expression changes were assessed by western blots. Mice exposed to IN showed elevated HOMA-IR, and their fecal samples showed altered microbiota which promote increased permeability of the colonic epithelial cell barrier. Plasma exosomes decreased pAKT/AKT responses to exogenous insulin compared to CL, and altered expression of circadian clock genes. Inflammatory macrophages (Ly-6chigh) were increased in IN-exposed vWAT, while Tregs were decreased. Thus, gut microbiota and the cargo of plasma exosomes are altered by periodic shifts in environmental lighting, and effectively alter metabolic function, possibly via induction of systemic inflammation and altered clock expression in target tissues. Further exploration of exosomal miRNA signatures in shift workers and their putative metabolic organ cell targets appears warranted

Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosa can be achieved without iron provision by maintaining local phosphate abundance: role of pH

<p>Abstract</p> <p>Background</p> <p>During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal <it>P. aeruginosa </it>to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation.</p> <p>Results</p> <p>In this study we examined the role of pH in the protective effect of [Pi] supplementation as it has been shown to be increased in the distal gut following surgical injury. Surgically injured mice drinking 25 mM [Pi] at pH 7.5 and intestinally inoculated with <it>P. aeruginosa </it>had increased mortality compared to mice drinking 25 mM [Pi] at pH 6.0 (p < 0.05). This finding was confirmed in <it>C. elegans</it>. Transcriptional analysis of <it>P. aeruginosa </it>demonstrated enhanced expression of various genes involved in media alkalization at pH 6.0 and a global increase in the expression of all iron-related genes at pH 7.5. Maintaining the pH at 6.0 via phosphate supplementation led to significant attenuation of iron-related genes as demonstrated by microarray and confirmed by QRT-PCR analyses.</p> <p>Conclusion</p> <p>Taken together, these data demonstrate that increase in pH in distal intestine of physiologically stressed host colonized by <it>P. aeruginosa </it>can lead to the expression of siderophore-related virulence in bacteria that can be prevented without providing iron by maintaining local phosphate abundance at pH 6.0. This finding is particularly important as provision of exogenous iron has been shown to have untoward effects when administered to critically ill and septic patients. Given that phosphate, pH, and iron are near universal cues that dictate the virulence status of a broad range of microorganisms relevant to serious gut origin infection and sepsis in critically ill patients, the maintenance of phosphate and pH at appropriate physiologic levels to prevent virulence activation in a site specific manner can be considered as a novel anti-infective therapy in at risk patients.</p

Chronic sleep disruption alters gut microbiota, induces systemic and adipose tissue inflammation and insulin resistance in mice.

Chronic sleep fragmentation (SF) commonly occurs in human populations, and although it does not involve circadian shifts or sleep deprivation, it markedly alters feeding behaviors ultimately promoting obesity and insulin resistance. These symptoms are known to be related to the host gut microbiota. Mice were exposed to SF for 4 weeks and then allowed to recover for 2 weeks. Taxonomic profiles of fecal microbiota were obtained prospectively, and conventionalization experiments were performed in germ-free mice. Adipose tissue insulin sensitivity and inflammation, as well as circulating measures of inflammation, were assayed. Effect of fecal water on colonic epithelial permeability was also examined. Chronic SF-induced increased food intake and reversible gut microbiota changes characterized by the preferential growth of highly fermentative members of Lachnospiraceae and Ruminococcaceae and a decrease of Lactobacillaceae families. These lead to systemic and visceral white adipose tissue inflammation in addition to altered insulin sensitivity in mice, most likely via enhanced colonic epithelium barrier disruption. Conventionalization of germ-free mice with SF-derived microbiota confirmed these findings. Thus, SF-induced metabolic alterations may be mediated, in part, by concurrent changes in gut microbiota, thereby opening the way for gut microbiome-targeted therapeutics aimed at reducing the major end-organ morbidities of chronic SF

Analysis of Muscle and Ovary Transcriptome of Sus scrofa: Assembly, Annotation and Marker Discovery

Pig (Sus scrofa) is an important organism for both agricultural and medical purpose. This study aims to investigate the S. scrofa transcriptome by the use of Roche 454 pyrosequencing. We obtained a total of 558 743 and 528 260 reads for the back-leg muscle and ovary tissue each. The overall 1 087 003 reads give rise to 421 767 341 bp total residues averaging 388 bp per read. The de novo assemblies yielded 11 057 contigs and 60 270 singletons for the back-leg muscle, 12 204 contigs and 70 192 singletons for the ovary and 18 938 contigs and 102 361 singletons for combined tissues. The overall GC content of S. scrofa transcriptome is 42.3% for assembled contigs. Alternative splicing was found within 4394 contigs, giving rise to 1267 isogroups or genes. A total of 56 589 transcripts are involved in molecular function (40 916), biological process (38 563), cellular component (35 787) by further gene ontology analyses. Comparison analyses showed that 336 and 553 genes had significant higher expression in the back-leg muscle and ovary each. In addition, we obtained a total of 24 214 single-nucleotide polymorphisms and 11 928 simple sequence repeats. These results contribute to the understanding of the genetic makeup of S. scrofa transcriptome and provide useful information for functional genomic research in future

Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity

Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6–24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity