Partial fusion of a cytochrome P450 system by carboxy-terminal attachment of putidaredoxin reductase to P450cam (CYP101A1)

Cytochrome P450 (CYP) enzymes catalyze the insertion of oxygen into carbon–hydrogen bonds and have great potential for enzymatic synthesis. Application development of class I CYPs is hampered by their dependence on two redox partners (a ferredoxin and ferredoxin reductase), slowing catalysis compared to self-sufficient CYPs such as CYP102A1 (P450BM3). Previous attempts to address this have fused all three components in several permutations and geometries, with much reduced activity compared to the native system. We report here the new approach of fusing putidaredoxin reductase (PdR) to the carboxy-terminus of CYP101A1 (P450cam) via a linker peptide and reconstituting camphor hydroxylase activity with free putidaredoxin (Pdx). Initial purification of a P450cam–PdR fusion yielded 2.0% heme incorporation. Co-expression of E. coli ferrochelatase, lengthening the linker from 5 to 20 residues, and altering culture conditions for enzyme production furnished 85% heme content. Fusion co-expression with Pdx gave a functional system with comparable in vivo camphor oxidation activity as the native system. In vitro, the fused system's steady state NADH oxidation rate was two-fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme showed non-hyperbolic dependence on Pdx concentration, suggesting a role for the PdR domain; these data were consistent with a kinetic model based on two-site binding of Pdx by P450cam–PdR and inactive dimer formation of the fusion. P450cam–PdR is the first example of a class I P450 fusion that exhibits significantly more favorable behavior than that of the native system

Diameters of left gastric vein and its originating vein on magnetic resonance imaging in liver cirrhosis patients with hepatitis B : Association with endoscopic grades of esophageal varices

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108624/1/hepr12246.pd

Identifying critically important vascular access outcomes for trials in haemodialysis : an international survey with patients, caregivers and health professionals

BACKGROUND: Vascular access outcomes reported across haemodialysis (HD) trials are numerous, heterogeneous and not always relevant to patients and clinicians. This study aimed to identify critically important vascular access outcomes. METHOD: Outcomes derived from a systematic review, multi-disciplinary expert panel and patient input were included in a multilanguage online survey. Participants rated the absolute importance of outcomes using a 9-point Likert scale (7-9 being critically important). The relative importance was determined by a best-worst scale using multinomial logistic regression. Open text responses were analysed thematically. RESULTS: The survey was completed by 873 participants [224 (26%) patients/caregivers and 649 (74%) health professionals] from 58 countries. Vascular access function was considered the most important outcome (mean score 7.8 for patients and caregivers/8.5 for health professionals, with 85%/95% rating it critically important, and top ranked on best-worst scale), followed by infection (mean 7.4/8.2, 79%/92% rating it critically important, second rank on best-worst scale). Health professionals rated all outcomes of equal or higher importance than patients/caregivers, except for aneurysms. We identified six themes: necessity for HD, applicability across vascular access types, frequency and severity of debilitation, minimizing the risk of hospitalization and death, optimizing technical competence and adherence to best practice and direct impact on appearance and lifestyle. CONCLUSIONS: Vascular access function was the most critically important outcome among patients/caregivers and health professionals. Consistent reporting of this outcome across trials in HD will strengthen their value in supporting vascular access practice and shared decision making in patients requiring HD

Positive Effects of Exercise Intervention without Weight Loss and Dietary Changes in NAFLD-Related Clinical Parameters: A Systematic Review and Meta-Analysis

One of the focuses of non-alcoholic fatty liver disease (NAFLD) treatment is exercise. Randomized controlled trials investigating the effects of exercise without dietary changes on NAFLD-related clinical parameters (liver parameters, lipid metabolism, glucose metabolism, gut microbiota, and metabolites) were screened using the PubMed, Scopus, Web of Science, and Cochrane databases on 13 February 2020. Meta-analyses were performed on 10 studies with 316 individuals who had NAFLD across three exercise regimens: aerobic exercise, resistance training, and a combination of both. No studies investigating the role of gut microbiota and exercise in NAFLD were found. A quality assessment via the (RoB)2 tool was conducted and potential publication bias, statistical outliers, and influential cases were identified. Overall, exercise without significant weight loss significantly reduced the intrahepatic lipid (IHL) content (SMD: -0.76, 95% CI: -1.04, -0.48) and concentrations of alanine aminotransaminase (ALT) (SMD: -0.52, 95% CI: -0.90, -0.14), aspartate aminotransaminase (AST) (SMD: -0.68, 95% CI: -1.21, -0.15), low-density lipoprotein cholesterol (SMD: -0.34, 95% CI: -0.66, -0.02), and triglycerides (TG) (SMD: -0.59, 95% CI: -1.16, -0.02). The concentrations of high-density lipoprotein cholesterol, total cholesterol (TC), fasting glucose, fasting insulin, and glycated hemoglobin were non-significantly altered. Aerobic exercise alone significantly reduced IHL, ALT, and AST; resistance training alone significantly reduced TC and TG; a combination of both exercise types significantly reduced IHL. To conclude, exercise overall likely had a beneficial effect on alleviating NAFLD without significant weight loss. The study was registered at PROSPERO: CRD42020221168 and funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 813781

Comparison of Remote Sensing and Fixed-Site Monitoring Approaches for Examining Air Pollution and Health in a National Study Population

Satellite remote sensing (RS) has emerged as a cutting edge approach for estimating ground level ambient air pollution. Previous studies have reported a high correlation between ground level PM2.5 and NO2 estimated by RS and measurements collected at regulatory monitoring sites. The current study examined associations between air pollution and adverse respiratory and allergic health outcomes using multi-year averages of NO2 and PM2.5 from RS and from regulatory monitoring. RS estimates were derived using satellite measurements from OMI, MODIS, and MISR instruments. Regulatory monitoring data were obtained from Canada's National Air Pollution Surveillance Network. Self-reported prevalence of doctor-diagnosed asthma, current asthma, allergies, and chronic bronchitis were obtained from the Canadian Community Health Survey (a national sample of individuals 12 years of age and older). Multi-year ambient pollutant averages were assigned to each study participant based on their six digit postal code at the time of health survey, and were used as a marker for long-term exposure to air pollution. RS derived estimates of NO2 and PM2.5 were associated with 6e10% increases in respiratory and allergic health outcomes per interquartile range (3.97 mg m3 for PM2.5 and 1.03 ppb for NO2) among adults (aged 20e64) in the national study population. Risk estimates for air pollution and respiratory/ allergic health outcomes based on RS were similar to risk estimates based on regulatory monitoring for areas where regulatory monitoring data were available (within 40 km of a regulatory monitoring station). RS derived estimates of air pollution were also associated with adverse health outcomes among participants residing outside the catchment area of the regulatory monitoring network (p < 0.05)

Glycan composition of serum alpha-fetoprotein in patients with hepatocellular carcinoma and non-seminomatous germ cell tumour

Although estimation of serum alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular carcinoma (HCC) and non-seminomatous germ cell tumours (NSGCT), the clinical usefulness of this test is limited by a low specificity. However, there exist glycoforms of AFP which may be more specific for particular tumours. Previously, detailed analysis has been prevented by the low levels of AFP in human serum. We report here the application of fluorescence labelling, sequential exoglycosidase digestion, high-performance liquid chromatography and matrix-assisted laser desorption ionization in time-of-flight mass spectrometry, to determine the glycan structures of purified serum AFP from patients with HCC and NSGCT. Eleven major glycans were found, of which seven were N-linked, and four were O-linked, to the protein backbone. The structure of the N-linked glycans (all of bi-antennary complex-type with varying degrees of sialylation, fucosylation and galactosylation) were consistent with those previously reported. The O-linked glycans (three mucin O-GalNAc type glycans with variable degrees of sialylation, one O-HexNAc monosaccharide glycan) have not previously been reported. The finding of mucin O-GalNAc type glycans was supported by the prediction of potential O-GalNAc glycosylation sites on the protein backbone by analysis of the AFP structure by molecular modelling. With knowledge of these structures it may be possible to develop more specific assays for the detection of HCC and NSGCT. © 1999 Cancer Research Campaign © 1999 Cancer Research Campaig

Mortality in Aransas-Wood Buffalo Whooping Cranes: Timing, Location, and Causes

The Aransas-Wood Buffalo Population (AWBP) of Whooping Cranes (Grus americana) has experienced a population growth rate of approximately 4% for multiple decades (Butler et al., 2014a; Miller et al., 1974). Population growth for long-lived species of birds is generally highly sensitive to variation in adult mortality rates (Sæther and Bakke, 2000). A population model for endangered Red-crowned Cranes (Grus japonensis) in Japan conforms to this pattern, where growth rate is most sensitive to adult mortality (Masatomi et al., 2007). Earlier analyses observed that the AWBP growth rate increased in the mid-1950s and that this increase was likely caused by reduced annual mortality rates, even while the population experienced slightly decreasing natality (Binkley and Miller, 1988; Miller et al., 1974). A more contemporary analysis of the AWBP determined that approximately 50% of variation in annual population growth could be explained by variation in annual mortality (Butler et al., 2014a). Therefore, as a vital rate, mortality is critical to the maintained growth of the AWBP

Microbial inactivation properties of a new antimicrobial/antithrombotic catheter lock solution (citrate/methylene blue/parabens)

Background. Microbial infections are the most serious complications associated with indwelling central venous catheters. A catheter lock solution that is both antibacterial and antithrombotic is needed. The goal of this study was to determine whether a new catheter lock solution containing citrate, methylene blue and parabens has antimicrobial properties against planktonic bacteria and against sessile bacteria within a biofilm. These effects were compared to the antimicrobial properties of heparin at 2500 units/ml

Macrophage metabolic reprogramming presents a therapeutic target in lupus nephritis.

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1β, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1β and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis

A discourse perspective on creating organizational knowledge: the case of strategizing

This chapter outlines a discursive epistemology of knowledge production through an analysis of the role of time and context in the social construction of organizational insights, outcomes and theories. While the role of time and context has been widely acknowledged in organizational discourse analysis, it has remained unclear what is specific to knowledge generation. Drawing upon a case study of an attempted company acquisition, we illustrate how knowledge is discursively produced and consumed during a process of strategizing. Our analysis shows how knowledge producing processes (e.g. strategizing, theorizing, conceptualizing and hypothesizing) extend both the time horizon of discourses that relate to the future, and the context horizon for discourse(s) that relate to the broader context. This reconstructs the tapestry of interwoven discourses that make up a local discourse and enable new managerial knowledge to be produced