Combined analysis of microbial metagenomic and metatranscriptomic sequencing data to assess in situ physiological conditions in the premature infant gut.

Microbes alter their transcriptomic profiles in response to the environment. The physiological conditions experienced by a microbial community can thus be inferred using meta-transcriptomic sequencing by comparing transcription levels of specifically chosen genes. However, this analysis requires accurate reference genomes to identify the specific genes from which RNA reads originate. In addition, such an analysis should avoid biases in transcript counts related to differences in organism abundance. In this study we describe an approach to address these difficulties. Sample-specific meta-genomic assembled genomes (MAGs) were used as reference genomes to accurately identify the origin of RNA reads, and transcript ratios of genes with opposite transcription responses were compared to eliminate biases related to differences in organismal abundance, an approach hereafter named the "diametric ratio" method. We used this approach to probe the environmental conditions experienced by Escherichia spp. in the gut of 4 premature infants, 2 of whom developed necrotizing enterocolitis (NEC), a severe inflammatory intestinal disease. We analyzed twenty fecal samples taken from four premature infants (4-6 time points from each infant), and found significantly higher diametric ratios of genes associated with low oxygen levels in samples of infants later diagnosed with NEC than in samples without NEC. We also show this method can be used for examining other physiological conditions, such as exposure to nitric oxide and osmotic pressure. These study results should be treated with caution, due to the presence of confounding factors that might also distinguish between NEC and control infants. Nevertheless, together with benchmarking analyses, we show here that the diametric ratio approach can be applied for evaluating the physiological conditions experienced by microbes in situ. Results from similar studies can be further applied for designing diagnostic methods to detect NEC in its early developmental stages

Epithelial NAD+ depletion drives mitochondrial dysfunction and contributes to intestinal inflammation

IntroductionWe have previously demonstrated that a pathologic downregulation of peroxisome proliferator-activated receptor–gamma coactivator 1-alpha (PGC1α) within the intestinal epithelium contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanism underlying downregulation of PGC1α expression and activity during IBD is not yet clear.MethodsMice (male; C57Bl/6, Villincre/+;Pgc1afl/fl mice, and Pgc1afl/fl) were subjected to experimental colitis and treated with nicotinamide riboside. Western blot, high-resolution respirometry, nicotinamide adenine dinucleotide (NAD+) quantification, and immunoprecipitation were used to in this study.ResultsWe demonstrate a significant depletion in the NAD+ levels within the intestinal epithelium of mice undergoing experimental colitis, as well as humans with ulcerative colitis. While we found no decrease in the levels of NAD+-synthesizing enzymes within the intestinal epithelium of mice undergoing experimental colitis, we did find an increase in the mRNA level, as well as the enzymatic activity, of the NAD+-consuming enzyme poly(ADP-ribose) polymerase-1 (PARP1). Treatment of mice undergoing experimental colitis with an NAD+ precursor reduced the severity of colitis, restored mitochondrial function, and increased active PGC1α levels; however, NAD+ repletion did not benefit transgenic mice that lack PGC1α within the intestinal epithelium, suggesting that the therapeutic effects require an intact PGC1α axis.DiscussionOur results emphasize the importance of PGC1α expression to both mitochondrial health and homeostasis within the intestinal epithelium and suggest a novel therapeutic approach for disease management. These findings also provide a mechanistic basis for clinical trials of nicotinamide riboside in IBD patients

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Evidence that RASSF1C stimulation of lung cancer cell proliferation depends on IGFBP-5 and PIWIL1 expression levels.

RASSF1C is a major isoform of the RASSF1 gene, and is emerging as an oncogene. This is in contradistinction to the RASSF1A isoform, which is an established tumor suppressor. We have previously shown that RASSF1C promotes lung cancer cell proliferation and have identified RASSF1C target genes with growth promoting functions. Here, we further report that RASSF1C promotes lung cancer cell migration and enhances lung cancer cell tumor sphere formation. We also show that RASSF1C over-expression reduces the inhibitory effects of the anti-cancer agent, betulinic acid (BA), on lung cancer cell proliferation. In previous work, we demonstrated that RASSF1C up-regulates piwil1 gene expression, which is a stem cell self-renewal gene that is over-expressed in several human cancers, including lung cancer. Here, we report on the effects of BA on piwil1 gene expression. Cells treated with BA show decreased piwil1 expression. Also, interaction of IGFBP-5 with RASSF1C appears to prevent RASSF1C from up-regulating PIWIL1 protein levels. These findings suggest that IGFBP-5 may be a negative modulator of RASSF1C/ PIWIL1 growth-promoting activities. In addition, we found that inhibition of the ATM-AMPK pathway up-regulates RASSF1C gene expression

PCR array screen.

<p>MCF7 cells were treated with 90 chemical inhibitors and screened with RASSF1C gene specific primers. The PCR array screen identified several chemical inhibitors that up- and down-regulated RASSF1C gene expression. The two top modulators of RASSF1C gene expression are listed. Dorsomorphin (an inhibitor of AMPK) and KU60019 (an inhibitor of ATM kinase) up-regulate and Trichostatin A (an inhibitor of HDAC) down-regulates RASSF1C gene expression. The PCR array data analysis was performed using the Qiagen software program as directed.</p

Betulinic acid effects on Piwil1 gene expression.

<p><i>Piwil1</i> mRNA expression was assessed in the presence and absence of serum and BA in the lung cancer cell lines A549 (A) and NCI-H1299 (B). RT-PCR data show that <i>piwil1</i> expression was down-regulated by BA in the absence and presence of serum. PCR reactions were set in triplicates and Cyclophillin was used as an internal loading control and used to normalize the relative expression levels using the 2^−ΔΔ method (26). RT-PCR reaction were run at three independent times.</p

Evidence that RASSF1C Stimulation of Lung Cancer Cell Proliferation Depends on IGFBP-5 and PIWIL1 Expression Levels - Figure 2

<p>. <b>RASSF1C promotes lung cancer cell migration.</b> (A) The BD BioCoat Matrigel Invasion Chamber was used to assess cell invasion /migration of NCI-H1299 cells stably transduced with empty MLV-backbone (BB) or MLV-HA-RASSF1C (1C). Cells treated with doxycycline at 1ug/ml were co-incubated with serum-containing media. After 24 h, the lower sides of the filters were fixed and stained, and cells in four microscopic fields were counted. The average cell number count was plotted. (B) NCI-H1299 cells over-expressing RASSF1C showed a higher number of cells invading the Matrigel chamber and migrating to the other side of the filter compared to control NCI-H1299-BB cells. Data is representative of at least 3 independent experiments, and the values represent the average cell colony count. * P<0.05 as determined by t-test.</p

RASSF1C stimulates lung cancer cell proliferation.

<p>NCI-H1299 cells stably transduced with MLV-backbone (BB) or MLV-HA-RASSF1C (1C) were treated with 1 µg/ml doxycycline for 72 h, and then cells were counted. RASSF1C over-expression (1C) increased cell proliferation by 2.5 fold compared to control (BB). Data is representative of at least 3 independent experiments, and the values represent the mean±SEM. * P<0.05.</p

Dorsomorphin and Trichostatin A have opposite effects on RASSF1C and PIWIL1 gene expression.

<p>H1299 lung cancer cells were treated with Dorsomorphin and Trichostatin A at a concentration of 10 µM to validate the PCR array data. RT-PCR analysis shows that Dorsomorphin significantly up-regulated RASSF1C gene expression while slightly up-regulating PIWIL1 gene expression. Trichostatin A treatment significantly down-regulated RASSF1C and PIWIL1 mRNA levels. The data presented is an average of three independent RT-PCR experiments done in triplicate, *  =  P<0.05.</p

Proposed RASSF1C mechanistic pathway for lung cancer growth and progression.

<p>This model is based on our (12) and others previous published work and current data presented in this article. RASSF1C activates the MEK-ERK1/2 pathway (12), which controls a wide variety of genes that promote cell division and proliferation. Over-expression of RASSF1C up-regulates Piwil1 gene expression (12). Treatment of H1299 lung cancer cells with the MEK-ERK1/2 pathway inhibitor CI1040 resulted in down-regulation of Piwil1 mRNA levels (12). PIWIL1 is widely over-expressed in tumors compared to normal tissue, and it may have important functions in cancer initiation, maintenance, or progression (increased epigenetic gene modification, proliferation and cell migration, and reduced apoptosis). PIWI-like proteins have been shown to interact with PIWI-interacting RNAs (piRNAs) to form complexes that regulate transcriptional and translational repression leading to inhibition of apoptosis, stimulation of cell division and proliferation, and down-regulation of cyclin inhibitors and tumor suppressors (18). Over-expression of PIWIL1 down-regulates IGFBP-5 (18), which is an interacting partner and inhibitor of RASSF1C (19). Thus, co-expression of RASSF1C and IGFBP-5 abrogates the up-regulation of PIWIL1 expression in response to increased RASSF1C, suggesting that IGFBP-5 could block RASSF1C actions through the ERK1/2 pathway resulting in reduced PIWIL1 expression and function. Also, RASSF1C mRNA expression is regulated by AMP and ATM kinases. Various key components of the proposed RASSF1C pathway will be tested to validate this model.</p