Circadian Clock Regulation of Hepatic Lipid Metabolism by Modulation of m6A mRNA Methylation.

Transcriptional regulation of circadian rhythms is essential for lipid metabolic homeostasis, disruptions of which can lead to metabolic diseases. Whether N6-methyladenosine (m6A) mRNA methylation impacts circadian regulation of lipid metabolism is unclear. Here, we show m6A mRNA methylation oscillations in murine liver depend upon a functional circadian clock. Hepatic deletion of Bmal1 increases m6A mRNA methylation, particularly of PPaRα. Inhibition of m6A methylation via knockdown of m6A methyltransferase METTL3 decreases PPaRα m6A abundance and increases PPaRα mRNA lifetime and expression, reducing lipid accumulation in cells in vitro. Mechanistically, YTHDF2 binds to PPaRα to mediate its mRNA stability to regulate lipid metabolism. Induction of reactive oxygen species both in vitro and in vivo increases PPaRα transcript m6A levels, revealing a possible mechanism for circadian disruption on m6A mRNA methylation. These data show that m6A RNA methylation is important for circadian regulation of downstream genes and lipid metabolism, impacting metabolic outcomes.

Plasma microbiome-modulated indole- and phenyl-derived metabolites associate with advanced atherosclerosis and postoperative outcomes.

OBJECTIVE: Multiple studies have shown that gut microbes contribute to atherosclerosis, and there is mounting evidence that microbial metabolism of dietary nutrients influences pathophysiology. We hypothesized that indole- and phenyl-derived metabolites that originate solely or in part from bacterial sources would differ between patients with advanced atherosclerosis and age- and sex-matched controls without clinically apparent atherosclerosis. METHODS: Plasma from the advanced atherosclerosis cohort (n = 100) was from patients who underwent carotid endarterectomy, open infrainguinal leg revascularization, or major leg amputation for critical limb ischemia. The controls (n = 22) were age- and sex-matched participants who had no peripheral arterial disease or history of stroke or myocardial infarction. Patients with chronic kidney disease were excluded. Metabolites and internal standards were measured using high-performance liquid chromatography and tandem mass spectrometry. RESULTS: Plasma metabolite concentrations differed significantly between the advanced atherosclerosis and control cohorts. After adjustment for traditional atherosclerosis risk factors, indole (odds ratio [OR], 0.84; 95% confidence interval [CI], 0.75-0.95; P = .004), tryptophan (OR, <0.001; 95% CI, <0.001-0.003; P < .001), indole-3-propionic acid (OR, 0.27; 95% CI, 0.019-0.91; P = .02), and indole-3-aldehyde (OR, 0.12; 95% CI, 0.014-0.92; P = .04) concentrations negatively associated with advanced atherosclerosis, whereas the kynurenine/tryptophan ratio (OR, 61.7; 95% CI, 1.9->999; P = .02) was positively associated. Furthermore, tryptophan and indole-3-propionic acid concentrations (Spearman coefficients of 0.63 and 0.56, respectively; P < .001) correlated with the ankle-brachial index, a surrogate for overall atherosclerotic disease burden. Fourteen patients experienced a major postoperative cardiac complication within 30 days in the advanced atherosclerosis cohort, which was associated with baseline kynurenine/tryptophan ratio (P = .001) and hippuric acid (P = .03). In a multivariate analysis, only the kynurenine/tryptophan ratio remained significantly associated with a postoperative cardiac complication (OR, 44.1; 95% CI, 3.3-587.1; P = .004). Twenty patients in the advanced atherosclerosis cohort experienced a major adverse cardiac event during the follow-up period, which was associated with hippuric acid (P = .002) and the kynurenine/tryptophan ratio (P < .001) at baseline. Both hippuric acid and the kynurenine/tryptophan ratio were independently associated with a major adverse cardiac event in multivariate analyses that included diabetes mellitus. CONCLUSIONS: Specific microbe-derived metabolite signatures associate with advanced human atherosclerosis and postoperative cardiac complications. We suggest that these metabolites are potential novel biomarkers for atherosclerotic disease burden and that further investigation into mechanistic links between defined microbial metabolic pathways and cardiovascular disease is warranted.

Early Transcriptomic Changes in the Ileal Pouch Provide Insight into the Molecular Pathogenesis of Pouchitis and Ulcerative Colitis.

BACKGROUND: Ulcerative colitis (UC) only involves the colonic mucosa. Yet, nearly 50% of patients with UC who undergo total proctocolectomy with ileal pouch anal anastomosis develop UC-like inflammation of the ileal pouch (pouchitis). By contrast, patients with familial adenomatous polyposis (FAP) with ileal pouch anal anastomosis develop pouchitis far less frequently. We hypothesized that pathogenic events associated with the development of UC are recapitulated by colonic-metaplastic transcriptomic reprogramming of the UC pouch. METHODS: We prospectively sampled pouch and prepouch ileum mucosal biopsies in patients with UC with ileal pouch anal anastomosis 4, 8, and 12 months after their pouch was in continuity. Mucosal samples were also obtained from patients with FAP. Transcriptional profiles of the UC and FAP pouch and prepouch ileum were investigated via RNA sequencing and compared with data from a previously published microarray study. RESULTS: Unlike patients with FAP, subjects with UC exhibited a large set of differentially expressed genes between the pouch and prepouch ileum as early as 4 months after pouch functionalization. Functional pathway analysis of differentially expressed genes in the UC pouch revealed an enhanced state of immune/inflammatory response and extracellular matrix remodeling. Moreover, >70% of differentially expressed genes mapped to published inflammatory bowel diseases microarray data sets displayed directional changes consistent with active UC but not with Crohn's disease. CONCLUSIONS: The UC pouch, well before histologic inflammation, already displays a systems-level gain of colon-associated genes and loss of ileum-associated genes. Patients with UC exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease and may in part explain their susceptibility to the development of pouchitis.

Diet, gut microbes, and the pathogenesis of inflammatory bowel diseases.

The rising incidence of inflammatory bowel diseases in recent decades has notably paralleled changing lifestyle habits in Western nations, which are now making their way into more traditional societies. Diet plays a key role in IBD pathogenesis, and there is a growing appreciation that the interaction between diet and microbes in a susceptible person contributes significantly to the onset of disease. In this review, we examine what is known about dietary and microbial factors that promote IBD. We summarize recent findings regarding the effects of diet in IBD epidemiology from prospective population cohort studies, as well as new insights into IBD-associated dysbiosis. Microbial metabolism of dietary components can influence the epithelial barrier and the mucosal immune system, and understanding how these interactions generate or suppress inflammation will be a significant focus of IBD research. Our knowledge of dietary and microbial risk factors for IBD provides important considerations for developing therapeutic approaches through dietary modification or re-shaping the microbiota. We conclude by calling for increased sophistication in designing studies on the role of diet and microbes in IBD pathogenesis and disease resolution in order to accelerate progress in response to the growing challenge posed by these complex disorders.

Nutritional modulation of gut microbiota - the impact on metabolic disease pathophysiology.

The obesity epidemic afflicts over one third of the United States population. With few therapies available to combat obesity, a greater understanding of the systemic causes of this and other metabolic disorders is needed to develop new, effective treatments. The mammalian intestinal microbiota contributes to metabolic processes in the host. This review summarizes the research demonstrating the interplay of diet, intestinal microbiota and host metabolism. We detail the effects of diet-induced modifications in microbial activity and resultant impact on (1) sensory perception of macronutrients and total energy intake; (2) nutrient absorption, transport and storage; (3) liver and biliary function; (4) immune-mediated signaling related to adipose inflammation; and (5) circadian rhythm. We also discuss therapeutic strategies aimed to modify host-microbe interactions, including prebiotics, probiotics and postbiotics, as well as fecal microbiota transplantation. Elucidating the role of gut microbes in shaping metabolic homeostasis or dysregulation provides greater insight into disease development and a promising avenue for improved treatment of metabolic dysfunction.

The effect of laminar air flow and door openings on operating room contamination.

We evaluate the association of laminar airflow (LAF) and OR traffic with intraoperative contamination rates. Two sterile basins were placed in each room during 81 cases, one inside and one outside the LAF. One Replicate Organism Detection and Counting (RODAC) plate from each basin was sent for culture at successive 30-minute intervals from incision time until wound closure. At successive 30-minute intervals more plates were contaminated outside than inside the LAF. A negative binomial model showed that the bacteria colony forming units (CFU) depended on whether there were any door openings (P=0.02) and the presence of LAF (P=0.003). LAF decreases CFU by 36.6%. LAF independently reduces the risk of contamination and microbial counts for surgeries lasting 90 minutes or less.

Mortality in centenarians with hip fractures.

The proximal femur is one of the primary areas of weakness in the skeleton due to osteoporosis. With more than 50,000 Americans aged at least 100 years, an increasing number of these extremely elderly patients will present with hip fractures. A paucity of literature exists on functional outcomes and mortality rates in the centenarian population who sustain hip fractures. This study evaluated the mortality rate in centenarians who sustained hip fractures to determine whether operative intervention is safe and appropriate.The authors retrospectively reviewed 23 patients (22 women and 1 man) aged at least 100 years with hip fractures treated at their institution between 2003 and 2010. Twenty-one patients were treated operatively and 2 were treated nonoperatively. Mean Charlson comorbidity index was 2 (range, 0 to 5). The patients' medical charts or the Social Security Death Index was used to determine their dates of death. Average patient age was 101.9 years at injury and 102.8 years at death. Cumulative in-hospital, 30- and 90-day, 6- and 12-month, and 2-, 3-, and 6-year mortality rates for operatively treated patients were 15%, 20%, 30%, 45%, 60%, 70%, 90%, and 95%, respectively. Both patients treated nonoperatively died within 90 days. One patient is still alive 6 years postoperatively. Postoperative complications occurred in 9 (43%) patients.Although this patient population is a relatively small subset of the elderly population, the number of these patients is rapidly increasing. Operating on patients older than 100 years carries an acceptable mortality rate. Age alone should not preclude centenarians from undergoing operative treatment for hip fractures.

Crystal structures of SlyA protein, a master virulence regulator of Salmonella, in free and DNA-bound states.

SlyA is a master virulence regulator that controls the transcription of numerous genes in Salmonella enterica. We present here crystal structures of SlyA by itself and bound to a high-affinity DNA operator sequence in the slyA gene. SlyA interacts with DNA through direct recognition of a guanine base by Arg-65, as well as interactions between conserved Arg-86 and the minor groove and a large network of non-base-specific contacts with the sugar phosphate backbone. Our structures, together with an unpublished structure of SlyA bound to the small molecule effector salicylate (Protein Data Bank code 3DEU), reveal that, unlike many other MarR family proteins, SlyA dissociates from DNA without large conformational changes when bound to this effector. We propose that SlyA and other MarR global regulators rely more on indirect readout of DNA sequence to exert control over many genes, in contrast to proteins (such as OhrR) that recognize a single operator.

Genome wide screens in yeast to identify potential binding sites and target genes of DNA-binding proteins.

Knowledge of all binding sites for transcriptional activators and repressors is essential for computationally aided identification of transcriptional networks. The techniques developed for defining the binding sites of transcription factors tend to be cumbersome and not adaptable to high throughput. We refined a versatile yeast strategy to rapidly and efficiently identify genomic targets of DNA-binding proteins. Yeast expressing a transcription factor is mated to yeast containing a library of genomic fragments cloned upstream of the reporter gene URA3. DNA fragments with target-binding sites are identified by growth of yeast clones in media lacking uracil. The experimental approach was validated with the tumor suppressor protein p53 and the forkhead protein FoxI1 using genomic libraries for zebrafish and mouse generated by shotgun cloning of short genomic fragments. Computational analysis of the genomic fragments recapitulated the published consensus-binding site for each protein. Identified fragments were mapped to identify the genomic context of each binding site. Our yeast screening strategy, combined with bioinformatics approaches, will allow both detailed and high-throughput characterization of transcription factors, scalable to the analysis of all putative DNA-binding proteins.

Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions.

Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F(1) offspring of the infected fish. From F(1) fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes. By inbreeding fish carrying proviral integrations in 25 different genes, we were able to demonstrate that in approximately 50% of the gene "hits," the mRNA transcript levels were reduced by >/=70%, with the highest probability for mutation occurring if the integration was in an exon or first intron. Based on these data, the mutagenic frequency for the retrovirus is nearly one in five integrations. In addition, a strong mutagenic effect is seen when murine leukemia virus integrates specifically in the first intron of genes but not in other introns. Three of 19 gene inactivation events had embryonic defects. Using the strategy we outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F(1) fish. This is a 20- to 30-fold increase in efficiency when compared with the current resequencing approach [targeting induced local lesions in genomes (TILLING)] used in zebrafish for identifying mutations in genes. Combining this increase in efficiency with cryopreservation of sperm samples from the F(1) fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene.