Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, ...Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l
d
.
ObjectiveOmega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter ...the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22).DesignA randomised, open-label, cross-over trial of 8 weeks’ treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week ‘washout’ period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry.ResultsBoth omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or β diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects.ConclusionOmega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure.Trial registration numberISRCTN18662143.
Imine reductases (IREDs) Mangas-Sanchez, Juan; France, Scott P; Montgomery, Sarah L ...
Current opinion in chemical biology,
April 2017, 2017-Apr, 2017-04-00, 20170401, Letnik:
37
Journal Article
Recenzirano
Odprti dostop
•Imine reductases (IREDs) enable the asymmetric synthesis of amines from imines.•The development of annotated sequence databases has led to the discovery of new IREDs.•IREDs have been implemented in ...multi-enzyme cascades.•Structural studies have provided insights into their mechanism of action.•Reductive amination represents an important area of investigation for the future.
Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field.
Rupture of the fetal membranes is a common harbinger of imminent labor and delivery. Telomere shortening is a surrogate for oxidative stress (OS) and senescence. Fetal leukocyte and placental ...membrane DNA telomere lengths were evaluated to determine their association with preterm prelabor rupture of the membranes (pPROM) or spontaneous preterm births with intact membranes (PTB), compared to term birth.
Telomere lengths were quantified in cord blood leukocytes (n = 133) from three major groups: 1) pPROM (n = 28), 2) PTB (n = 69) and 3) uncomplicated full term births (controls, n = 35), using real-time quantitative PCR. Placental membrane specimens (n = 18) were used to correlate fetal leukocyte and placental telomere lengths. Telomere length differences among the groups were analyzed by ANOVA. Pearson correlation coefficients determined relationships between leukocyte and placental membrane telomere lengths.
In pregnancies with intact membranes, fetal leukocyte telomere length was inversely proportional to gestational age. The mean telomere length decreased as gestation progressed, with the shortest at term. pPROM had telomere lengths (9962 ± 3124 bp) that were significantly shorter than gestational age-matched PTB (11546 ± 4348 bp, p = 0.04), but comparable to term births (9011 ± 2497 bp, p = 0.31). Secondary analyses revealed no effects of race (African American vs. Caucasian) or intraamniotic infection on telomere length. A strong Pearson's correlation was noted between fetal leukocyte and placental membrane telomere lengths (ρ = 0.77; p<0.01).
Fetal leukocyte telomere length is reduced in pPROM compared to PTB but is similar to term births. pPROM represents a placental membrane disease likely mediated by OS-induced senescence.
Background
Expansion of access to surgical care can improve health outcomes, although the impact that scale-up of the surgical workforce will have on child mortality is poorly defined. In this study, ...we estimate the number of child deaths potentially avertable by increasing the surgical workforce globally to meet targets proposed by the Lancet Commission on Global Surgery.
Methods
To estimate the number of deaths potentially avertable through increases in the surgical workforce, we used log-linear regression to model the association between surgeon, anesthetist and obstetrician workforce (SAO) density and surgically amenable under-5 mortality rate (U5MR), infant mortality rate (IMR), and neonatal mortality rate (NMR) for 192 countries adjusting for potential confounders of childhood mortality, including the non-surgical workforce (physicians, nurses/midwives, community health workers), gross national income per capita, poverty rate, female literacy rate, health expenditure per capita, percentage of urban population, number of surgical operations, and hospital bed density. Surgically amenable mortality was determined using mortality estimates from the UN Inter-agency Group for Child Mortality Estimation adjusted by the proportion of deaths in each country due to communicable causes unlikely to be amenable to surgical care. Estimates of mortality reduction due to upscaling surgical care to support the Lancet Commission on Global Surgery (LCoGS) minimum target of 20–40 SAO/100,000 were calculated accounting for potential increases in surgical volume associated with surgical workforce expansion.
Results
Increasing SAO workforce density was independently associated with lower surgically amenable U5MR as well as NMR (
p
< 0.01 for each model). When accounting for concomitant increases in surgical volume, scale-up of the surgical workforce to 20–40 SAO/100,000 could potentially prevent between 262,709 (95% CI 229,643–295,434) and 519,629 (465,046–573,919) under 5 deaths annually. The majority (61%) of deaths averted would be neonatal deaths.
Conclusion
Scale up of surgical workforce may substantially decrease childhood mortality rates around the world. Our analysis suggests that scale-up of surgical delivery through increase in the SAO workforce could prevent over 500,000 children from dying before the age of 5 annually. This would represent significant progress toward meeting global child mortality reduction targets.
αKlotho is the prototypic member of the Klotho family and is most highly expressed in the kidney. αKlotho has pleiotropic biologic effects, and in the kidney, its actions include regulation of ion ...transport, cytoprotection, anti-oxidation and anti-fibrosis. In rodent models of chronic kidney disease (CKD), αKlotho deficiency has been shown to be an early biomarker as well as a pathogenic factor. The database for αKlotho in human CKD remains controversial even after years of study.
We used a synthetic antibody library to identify a high-affinity human antigen-binding fragment that recognizes human, rat and mouse αKlotho primarily in its native, rather than denatured, form.
Using an immunoprecipitation-immunoblot (IP-IB) assay, we measured both serum and urinary levels of full-length soluble αKlotho in humans and established that human CKD is associated with αKlotho deficiency in serum and urine. αKlotho levels were detectably lower in early CKD preceding disturbances in other parameters of mineral metabolism and progressively declined with CKD stages. We also found that exogenously added αKlotho is inherently unstable in the CKD milieu suggesting that decreased production may not be the sole reason for αKlotho deficiency.
Synthetic antibody libraries harbor tremendous potential for a variety of biomedical and clinical applications. Using such a reagent, we furnish data in support of αKlotho deficiency in human CKD, and we set the foundation for the development of diagnostic and therapeutic applications of anti-αKlotho antibodies.
Children of obese mothers have increased risk of metabolic syndrome as adults. Here we report the effects of a high-fat diet in the absence of maternal obesity at conception on skeletal muscle ...metabolic and transcriptional profiles of adult male offspring. Female Sprague Dawley rats were fed a diet rich in saturated fat and sucrose high-fat diet (HFD): 23.5% total fat, 9.83% saturated fat, 20% sucrose wt:wt or a normal control diet (CD) 7% total fat, 0.5% saturated fat, 10% sucrose wt:wt for the 3 wk prior to mating and throughout pregnancy and lactation. Maternal weights were not different at conception; however, HFD-fed dams were 22% heavier than controls during pregnancy. On a normal diet, the male offspring of HFD-fed dams were not heavier than controls but demonstrated features of insulin resistance, including elevated plasma insulin concentration 40.1 ± 2.5 (CD) vs 56.2 ± 6.1 (HFD) mU/L; P = 0.023. Next-generation mRNA sequencing was used to identify differentially expressed genes in the offspring soleus muscle, and gene set enrichment analysis (GSEA) was used to detect coordinated changes that are characteristic of a biological function. GSEA identified 15 upregulated pathways, including cytokine signaling (P < 0.005), starch and sucrose metabolism (P < 0.017), inflammatory response (P < 0.024), and cytokine-cytokine receptor interaction (P < 0.037). A further 8 pathways were downregulated, including oxidative phosphorylation (P < 0.004), mitochondrial matrix (P < 0.006), and electron transport/uncoupling (P < 0.022). Phosphorylation of the insulin signaling protein kinase B was reduced 2.86 ± 0.63 (CD) vs 1.02 ± 0.27 (HFD); P = 0.027 and mitochondrial complexes I, II, and V protein were downregulated by 50-68% (P < 0.005). On a normal diet, the male offspring of HFD-fed dams did not become obese adults but developed insulin resistance, with transcriptional evidence of muscle cytokine activation, inflammation, and mitochondrial dysfunction. These data indicate that maternal overnutrition, even in the absence of prepregnancy obesity, can promote metabolic dysregulation and predispose offspring to type 2 diabetes.
IMPORTANCE: Patients with stable coronary artery disease (CAD) attribute greater benefit to percutaneous coronary interventions (PCI) than indicated in clinical trials. Little is known about how ...cardiologists’ presentation of the benefits and risks may influence patients’ perceptions. OBJECTIVES: To broadly describe the content of discussions between patients and cardiologists regarding angiogram and PCI for stable CAD, and to describe elements that may affect patients’ understanding. DESIGN, SETTING, AND PARTICIPANTS: Qualitative content analysis of encounters between cardiologists and patients with stable CAD who participated in the Verilogue Point-of-Practice Database between March 1, 2008, and August 31, 2012. Transcripts in which angiogram and PCI were discussed were retrieved from the database. Patients were aged 44 to 88 years (median, 64 years); 25% were women; 50% reported symptoms of angina; and 6% were taking more than 1 medication to treat angina. MAIN OUTCOMES AND MEASURES: Results of conventional and directed qualitative content analysis. RESULTS: Forty encounters were analyzed. Five major categories and subcategories of factors that may affect patients’ understanding of benefit were identified: (1) rationale for recommending angiogram and PCI (eg, stress test results, symptoms, and cardiologist’s preferences); (2) discussion of benefits (eg, accurate discussion of benefit 5%, explicitly overstated benefit 13%, and implicitly overstated benefit 35%); (3) discussion of risks (eg, minimization of risk); (4) cardiologist’s communication style (eg, humor, teach-back, message framing, and failure to respond to patient questions); and (5) patient and family member contributions to the discussion. CONCLUSIONS AND RELEVANCE: Few cardiologists discussed the evidence-based benefits of angiogram and PCI for stable CAD, and some implicitly or explicitly overstated the benefits. The etiology of patient misunderstanding is likely multifactorial, but if future quantitative studies support the findings of this hypothesis-generating analysis, modifications to cardiologists’ approach to describing the risks and benefits of the procedure may improve patient understanding.
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