Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue *

Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate

Vortex Plastic Motion in Twinned Superconductors

We present simulations, without electrodynamical assumptions, of $B(x,y,H(t)), M(H(t))$, and $J_c(H(t))$, in hard superconductors, for a variety of twin-boundary pinning potential parameters, and for a range of values of the density and strength of the pinning sites. We numerically solve the overdamped equations of motion of up to 10^4 flux-gradient-driven vortices which can be temporarily trapped at $\sim 10^6$ pinning centers. These simulations relate macroscopic measurements (e.g., M(H), ``flame'' shaped $B(x,y)$ profiles) with the underlying microscopic pinning landscape and the plastic dynamics of individual vortices

Critical currents, flux-creep activation energy and potential barriers for the vortex motion from the flux creep experiments

We present an experimental study of thermally activated flux creep in a superconducting ring-shaped epitaxial YBCO film as well as a new way of analyzing the experimental data. The measurements were made in a wide range of temperatures between 10 and 83 K. The upper temperature limit was dictated by our experimental technique and at low temperatures we were limited by a crossover to quantum tunneling of vortices. It is shown that the experimental data can very well be described by assuming a simple thermally activated hopping of vortices or vortex bundles over potential barriers, whereby the hopping flux objects remain the same for all currents and temperatures. The new procedure of data analysis also allows to establish the current and temperature dependencies of the flux-creep activation energy U, as well as the temperature dependence of the critical current Ic, from the flux-creep rates measured at different temperatures. The variation of the activation energy with current, U(I/Ic), is then used to reconstruct the profile of the potential barriers in real space.Comment: 12 pages, 13 Postscript figures, Submitted to Physical Review

Equity in health care financing: The case of Malaysia

Background: Equitable financing is a key objective of health care systems. Its importance is evidenced in policy documents, policy statements, the work of health economists and policy analysts. The conventional categorisations of finance sources for health care are taxation, social health insurance, private health insurance and out-of-pocket payments. There are nonetheless increasing variations in the finance sources used to fund health care. An understanding of the equity implications would help policy makers in achieving equitable financing. Objective: The primary purpose of this paper was to comprehensively assess the equity of health care financing in Malaysia, which represents a new country context for the quantitative techniques used. The paper evaluated each of the five financing sources (direct taxes, indirect taxes, contributions to Employee Provident Fund and Social Security Organization, private insurance and out-of-pocket payments) independently, and subsequently by combined the financing sources to evaluate the whole financing system. Methods: Cross-sectional analyses were performed on the Household Expenditure Survey Malaysia 1998/99, using Stata statistical software package. In order to assess inequality, progressivity of each finance sources and the whole financing system was measured by Kakwani's progressivity index. Results: Results showed that Malaysia's predominantly tax-financed system was slightly progressive with a Kakwani's progressivity index of 0.186. The net progressive effect was produced by four progressive finance sources (in the decreasing order of direct taxes, private insurance premiums, out-of-pocket payments, contributions to EPF and SOCSO) and a regressive finance source (indirect taxes). Conclusion: Malaysia's two tier health system, of a heavily subsidised public sector and a user charged private sector, has produced a progressive health financing system. The case of Malaysia exemplifies that policy makers can gain an in depth understanding of the equity impact, in order to help shape health financing strategies for the nation

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

A Randomized Trial to Compare a Tailored Web-Based Intervention and Tailored Phone Counseling to Usual Care for Increasing Colorectal Cancer Screening

Background: Colorectal cancer mortality could be decreased with risk-appropriate cancer screening. We examined the efficacy of three tailored interventions compared with usual care for increasing screening adherence. Methods: Women (n = 1,196) ages 51 to 74, from primary care networks and nonadherent to colorectal cancer guidelines, were randomized to (1) usual care, (2) tailored Web intervention, (3) tailored phone intervention, or (4) tailored Web + phone intervention. Average-risk women could select either stool test or colonoscopy, whereas women considered at higher than average risk received an intervention that supported colonoscopy. Outcome data were collected at 6 months by self-report, followed by medical record confirmation (attrition of 23%). Stage of change for colorectal cancer screening (precontemplation or contemplation) was assessed at baseline and 6 months. Results: The phone (41.7%, P < 0.0001) and combined Web + phone (35.8%, P < 0.001) interventions significantly increased colorectal cancer screening by stool test compared with usual care (11.1%), with ORs ranging from 5.4 to 6.8 in models adjusted for covariates. Colonoscopy completion did not differ between groups except that phone significantly increased colonoscopy completion compared with usual care for participants in the highest tertile of self-reported fear of cancer. Conclusions: A tailored phone with or without a Web component significantly increased colorectal cancer screening compared with usual care, primarily through stool testing, and phone significantly increased colonoscopy compared with usual care but only among those with the highest levels of baseline fear. Impact: This study supports tailored phone counseling with or without a Web program for increasing colorectal cancer screening in average-risk women

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue

Blood pressure and cholesterol level checks as dynamic interrelated screening examinations

This study analysed the determinants of screening uptake for blood pressure and cholesterol level checks. Furthermore, it investigated the presence of possible spillover effects from one type of cardiovascular screening to another type of cardiovascular screening. A dynamic random effects bivariate panel probit model with initial conditions (Wooldridge-type estimator) was adopted for the estimation. The outcome variables were the participation in blood pressure and cholesterol level checks by individuals in a given year. The balanced panel sample of 21,138 observations was constructed from 1,626 individuals from the British Household Panel Survey (BHPS) between 1996 and 2008. The analysis showed the significance of past screening behaviour for both cardiovascular screening examinations. For both cardiovascular screening examinations state dependence exist. The study also shows a significant spillover effect of the cholesterol level check on the blood pressure check and vice versa. Also a poorer health status led to a higher uptake for both types of screening examinations. Changes in recommendations have to consider the fact that taking part in one type of cardiovascular screening examination can influence the decision to take part in the other type of cardiovascular screening examination

An RCT to Increase Breast and Colorectal Cancer Screening

Introduction Adherence to breast and colorectal cancer screenings reduce mortality from these cancers, yet screening rates remain suboptimal. This 2 × 2 RCT compared 3 theory-based interventions to usual care to simultaneously increase breast and colon cancer screening in women who were nonadherent to both screenings at study entry. Design RCT. Setting/participants Women (n=692) who were nonadherent to both breast and colon cancer screenings and aged 51–75 years were recruited. Enrollment, intervention delivery, and data collection were completed between 2013 and 2017, and data analyzed in 2018. Intervention The randomized intervention included the following 4 groups: 3 intervention arms (personally tailored messages using a web-based intervention, phone delivery by a trained navigator, or both) compared with usual care. Women at an average risk for colon cancer were allowed to select either colonoscopy or stool test as their preferred colon cancer screening. Mammography was promoted for breast cancer screening. Main outcome measures Outcome data at 6 months included self-report and medical records for screening activity. Results All intervention arms significantly increased receipt of either a mammogram or stool test compared with control (web: p<0.0249, phone: p<0.0001, web + phone: p<0.0001). When considering receipt of both mammogram and stool test, all intervention arms were significantly different from usual care (web: p<0.0249, phone: p<0.0003, web + phone: p<0.0001). In addition, women who were adherent to mammography had a 4.5 times greater odds of becoming adherent to colonoscopy. Conclusions The tailored intervention simultaneously supporting both breast and colon cancer screenings significantly improved rates of obtaining one of the screenings and increased receipt of both tests