Mucosa-associated bacterial diversity in necrotizing enterocolitis

Background: Previous studies of infant fecal samples have failed to clarify the role of gut bacteria in the pathogenesis of NEC. We sought to characterize bacterial communities within intestinal tissue resected from infants with and without NEC. Methods: 26 intestinal samples were resected from 19 infants, including 16 NEC samples and 10 non-NEC samples. Bacterial 16S rRNA gene sequences were amplified and sequenced. Analysis allowed for taxonomic identification, and quantitative PCR was used to quantify the bacterial load within samples. Results: NEC samples generally contained an increased total burden of bacteria. NEC and non-NEC sample sets were both marked by high inter-individual variability and an abundance of opportunistic pathogens. There was no statistically significant distinction between the composition of NEC and non-NEC microbial communities. K-means clustering enabled us to identify several stable clusters, including clusters of NEC and midgut volvulus samples enriched with Clostridium and Bacteroides. Another cluster containing both NEC and non-NEC samples was marked by an abundance of Enterobacteriaceae and decreased diversity among NEC samples. Conclusions: The results indicate that NEC is a disease without a uniform pattern of microbial colonization, but that NEC is associated with an abundance of strict anaerobes and a decrease in community diversity

Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants

Background: The source inoculum of gastrointestinal tract (GIT) microbes is largely influenced by delivery mode in full-term infants, but these influences may be decoupled in very low birth weight (VLBW, <1,500 g) neonates via conventional broad-spectrum antibiotic treatment. We hypothesize the built environment (BE), specifically room surfaces frequently touched by humans, is a predominant source of colonizing microbes in the gut of premature VLBW infants. Here, we present the first matched fecal-BE time series analysis of two preterm VLBW neonates housed in a neonatal intensive care unit (NICU) over the first month of life.Results: Fresh fecal samples were collected every 3 days and metagenomes sequenced on an Illumina HiSeq2000 device. For each fecal sample, approximately 33 swabs were collected from each NICU room from 6 specified areas: sink, feeding and intubation tubing, hands of healthcare providers and parents, general surfaces, and nurse station electronics (keyboard, mouse, and cell phone). Swabs were processed using a recently developed 'expectation maximization iterative reconstruction of genes from the environment' (EMIRGE) amplicon pipeline in which full-length 16S rRNA amplicons were sheared and sequenced using an Illumina platform, and short reads reassembled into full-length genes. Over 24,000 full-length 16S rRNA sequences were produced, generating an average of approximately 12,000 operational taxonomic units (OTUs) (clustered at 97% nucleotide identity) per room-infant pair. Dominant gut taxa, including Staphylococcus epidermidis, Klebsiella pneumoniae, Bacteroides fragilis, and Escherichia coli, were widely distributed throughout the room environment with many gut colonizers detected in more than half of samples. Reconstructed genomes from infant gut colonizers revealed a suite of genes that confer resistance to antibiotics (for example, tetracycline, fluoroquinolone, and aminoglycoside) and sterilizing agents, which likely offer a competitive advantage in the NICU environment.Conclusions: We have developed a high-throughput culture-independent approach that integrates room surveys based on full-length 16S rRNA gene sequences with metagenomic analysis of fecal samples collected from infants in the room. The approach enabled identification of discrete ICU reservoirs of microbes that also colonized the infant gut and provided evidence for the presence of certain organisms in the room prior to their detection in the gut

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

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

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&lt;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 (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;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

Effect of selected thermal processes on antioxidant properties of berry fruit homogenates

Celem pracy było określenie wpływu różnych technik ogrzewania oraz zamrażania, rozmrażania i zamrażalniczego przechowywania na właściwości przeciwutleniające homogenatów z owoców jagodowych. Materiał do badań stanowiły homogenaty owocowe: truskawkowy, z czarnej porzeczki, aroniowy i żurawinowy. Surowce poddano obróbce termicznej: ogrzewaniu (z zastosowaniem kuchenki gazowej, mikrofalowej i urządzenia wielofunkcyjnego Thermomix) i zamrażaniu. Zamrożone próbki przechowywano w temp. -24 ºC: 1) przez 3 dni – w celu określenia wpływu procesu zamrażania na właściwości przeciwutleniające homogenatów, 2) przez 90 dni – w celu określenia wpływu zamrażalniczego przechowywania na te właściwości. Przed badaniem próbki rozmrażano w powietrzu (temp. ok. 21 ºC) i w kuchence mikrofalowej. W surowcach oznaczono zawartość witaminy C, siłę redukującą i zdolność neutralizowania wolnych rodników. Najwięcej witaminy C zawierał homogenat z czarnej porzeczki (6,4 mg/g s. m.), a najwyższą zdolnością redukującą charakteryzował się homogenat truskawkowy (670,2 mg kwasu askorbinowego/g s. m.). Homogenaty aroniowe poddane obróbce termicznej były najefektywniejszym neutralizatorem wolnych rodników (EC50 = 1,52 g s. m./g DPPH•). Temperatura powyżej 95 ºC spowodowała największe straty witaminy C w homogenacie z czarnej porzeczki, przy czym były one mniejsze w wyniku ogrzewania mikrofalowego niż tradycyjnego. Ogrzewanie wpłynęło na wzrost właściwości redukujących i zdolności neutralizowania wolnych rodników, przy czym był on większy podczas podgrzewania metodą tradycyjną. W wyniku zamrażalniczego składowania nastąpiło zmniejszenie zawartości witaminy C i zdolności neutralizowania wolnych rodników (DPPH•). W przypadku siły redukującej wynik przechowywania zależał od surowca. W homogenatach z czarnej porzeczki i truskawki zaobserwowano zmniejszenie siły redukującej, a w żurawinowych i aroniowych - jej wzrost.The objective of the research study was to determine the effect of various heating and freezing techniques, as well as of the defrosting and frozen storage on the antioxidant properties of berry fruit homogenates. The experimental material consisted of strawberry, blackcurrant, cranberry, and chokeberry homogenates. The raw material was thermally treated, i.e. it was heated (using a gas stove, a microwave oven, and a Thermomix multifunction device) and frozen. The frozen samples were stored at a temperature of -24 ºC: 1) for 3 days, to determine the effect of freezing process on the antioxidant properties of homogenates; 2) for 90 days to determine the effect of frozen storage on those properties. Prior to the analysis, the samples were defrosted in air (at a temperature of approx. 21 ºC) and in a microwave oven. In the raw material, there were assayed: content of vitamin C, reducing power, and ability to scavenge free radicals. The blackcurrant homogenate contained the highest amount of vitamin C (6.4 mg/g of dry matter), whereas the strawberry homogenate was characterized by the highest reducing capability (670.2 mg of ascorbic acid/g of dry matter). The thermally treated chokeberry homogenates were the most effective scavengers of free radicals (EC50 = 1.52 g of dry matter/g of DPPH). A temperature above 950C caused the highest losses in vitamin C in the blackcurrant homogenates; those losses were lower in the microwave-heated homogenates than in the conventionally heated ones. The heating caused the reducing properties and free radical-scavenging activity to increase; the increase was higher when heating by a traditional method. The frozen storage caused the content of vitamin C and the scavenging capability of free radicals to decrease. As for the reducing power, the effect of storage depended on the raw material. A decrease was reported in the reducing power of the blackcurrant and strawberry homogenates, while the reducing power of cranberry and chokeberry homogenates increased

Gametophyte-specific transposition of the maize Ds element in transgenic tobacco

To assess the potential advantages of a transposon-tagging system based on gametophyte-specific transposition a fusion between the anther-specific Arabidopsis thaliana apg promoter and the maize Ac transposase gene was constructed and introduced into tobacco. The ability of this transposase source to activate Ds transposition in a developmentally controlled manner was monitored by crossing to plants harbouring the cell autonomous excision marker gene construct, Ds—SPT. A number of fully green, streptomycin-resistant seedlings resulting from germinal transposition events were observed in the progeny of apg-TPase x Ds—SPT F1 plants. Streptomycin-resistant sectors were not observed in either F1 seedlings or F2 progeny, indicating a complete lack of somatic excision. Further crosses of apg—TPase sources to plants containing Ds—bar herbicide selection excision marker constructs gave reproducible gametophytic excision frequencies of up to 0.3%. Sequencing of Ds excision sites from F2 seedlings derived from single F1 plants revealed various sequence alterations in the original Ds insertion ‘footprint’ indicative of independent Ds excision events. Independent re-insertion was confirmed by Southern analysis of F2 siblings. It is concluded that apg-controlled Ac transposase expression activates male gametophyte-specific Ds transposition

High resolution studies of the Xenopus laevis ribosomal gene promoter in vivo and in vitro

Journal of Biological Chemistry2671610961-10967JBCH

The Staphylococcus aureus cidAB Operon: Evaluation of Its Role in Regulation of Murein Hydrolase Activity and Penicillin Tolerance

Recent studies have shown that expression of the Staphylococcus aureus lrgAB operon inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing. It was proposed that the lrgAB gene products function in a manner analogous to an antiholin, inhibiting a putative holin from transporting murein hydrolases out of the cell. In the present study the cidAB operon was identified and characterized based on the similarity of the cidA and cidB gene products to the products of the lrgAB operon. Zymographic and quantitative analyses of murein hydrolase activity revealed that mutation of the cidA gene results in decreased extracellular murein hydrolase activity compared to that of S. aureus RN6390, the parental strain. Complementation of cidA expression restored the wild-type phenotype, indicating that expression of the cidAB operon has a positive influence on extracellular murein hydrolase activity. The cidA mutant also displayed a significant decrease in sensitivity to the killing effects of penicillin. However, complementation of the cidA defect did not restore penicillin sensitivity to wild-type levels. Reverse transcriptase PCR also revealed that cidAB is maximally expressed during early exponential growth, opposite of what was previously observed for lrgAB expression. Based on these results, we propose that the cidAB operon encodes the holin-like counterpart of the lrgAB operon and acts in a manner opposite from that of lrgAB by increasing extracellular murein hydrolase activity and increasing sensitivity to penicillin-induced killing