Preparative Synthesis of dTDP-L-Rhamnose Through Combined Enzymatic Pathways

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy- D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4- keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6- dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1- phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identifiedbyHPLC, ESI-MS,andNMR,showingabout95%purity

Characterization of GDP-mannose Pyrophosphorylase from Escherichia Coli O157:H7 EDL933 and Its Broad Substrate Specificity

GDP-mannose pyrophosphorylase gene (ManC) of Escherichia coli (E. coli) O157 was cloned and expressed as a highly soluble protein in E. coli BL21 (DE3). The enzyme was subsequently purified using hydrophobic and ion exchange chromatographies. ManC showed very broad substrate specificities for four nucleotides and various hexose-1-phosphates, yielding ADP-mannose, CDP-mannose, UDP-mannose, GDP-mannose, GDP-glucose and GDP-2-deoxy-glucose

Long-term outcome of vertebral artery origin stenosis in patients with acute ischemic stroke

BACKGROUND: Vertebral artery origin (VAO) stenosis is occasionally observed in patients who have acute ischemic stroke. We investigated the long-term outcomes and clinical significance of VAO stenosis in patients with acute ischemic stroke. METHODS: We performed a prospective observational study using a single stroke center registry to investigate the risk of recurrent stroke and vascular outcomes in patients with acute ischemic stroke and VAO stenosis. To relate the clinical significance of VAO stenosis to the vascular territory of the index stroke, patients were classified into an asymptomatic VAO stenosis group and a symptomatic VAO stenosis group. RESULTS: Of the 774 patients who had acute ischemic stroke, 149 (19.3%) of them had more than 50% stenosis of the VAO. During 309 patient-years of follow-up (mean, 2.3 years), there were 7 ischemic strokes, 6 hemorrhagic strokes, and 2 unknown strokes. The annual event rates were 0.97% for posterior circulation ischemic stroke, 4.86% for all stroke, and 6.80% for the composite cardiovascular outcome. The annual event rate for ischemic stroke in the posterior circulation was significantly higher in patients who had symptomatic VAO stenosis than in patients who had asymptomatic stenosis (1.88% vs. 0%, p = 0.046). In a multivariate analysis, the hazard ratio, per one point increase of the Essen Stroke Risk Score (ESRS) for the composite cardiovascular outcome, was 1.46 (95% CI, 1.02-2.08, p = 0.036). CONCLUSIONS: Long-term outcomes of more than 50% stenosis of the VAO in patients with acute ischemic stroke were generally favorable. Additionally, ESRS was a predictor for the composite cardiovascular outcome. Asymptomatic VAO stenosis may not be a specific risk factor for recurrent ischemic stroke in the posterior circulation. However, VAO stenosis may require more clinical attention as a potential source of recurrent stroke when VAO stenosis is observed in patients who have concurrent ischemic stroke in the posterior circulation

Multidimensional Approaches to Examining Digital Literacies in the Contemporary Global Society

Literacy scholars have offered compelling theories about and methods for understanding the digital literacy practices of youth. However, little work has explored the possibility of an approach that would demonstrate how different perspectives on literacies might intersect and interconnect in order to better describe the multifaceted nature of youth digital literacies. In this conceptual article, we adopt the idea of theoretical triangulation in interpretive inquiry and explore how multiple perspectives can jointly contribute to constructing a nuanced description of young people’s literacies in today’s digitally mediated global world. For this purpose, we first suggest a triangulation framework that integrates sociocultural, affective, and cognitive perspectives on digital literacies, focusing on recent developments in these perspectives. We then use an example of discourse data from a globally connected online affinity space and demonstrate how our multidimensional framework can lead to a complex analysis and interpretation of the data. In particular, we describe the substance of one specific case of youth digital literacies from each of the three perspectives on literacy, which in turn converge to provide a complex account of such literacy practices. In conclusion, we discuss the promise and limitations of our integrative approach to studying the digital literacy practices of youth

One-pot Enzymatic Synthesis of Deoxy-thymidine-diphosphate (TDP)-2-deoxy-∝-d-glucose Using Phosphomannomutase

Production of deoxy-thymidine-diphosphate (TDP)-sugars as substrates of glycosyltransferases, has been one of main hurdles for combinatorial antibiotic biosynthesis, which combines sugar moiety with aglycon of various antibiotics. Here, we report the one-pot enzymatic synthesis of TDP-2-deoxy-glucose employing high efficient TMP kinase (TMK; E.C. 2.7.2.12), acetate kinase (ACK; E.C. 2.7.1.21), and TDP-glucose synthase (TGS; E.C. 2.7.7.24) with phosphomannomutase (PMM; E.C. 5.4.2.8). In this study, replacing phosphoglucomutase (PGM; E.C. 5.4.2) by PMM from Escherichia coli gave four times higher specific activity on 2-deoxy-6-phosphate glucose, suggesting that the activity on 2-deoxy-glucose-6-phosphate was mainly affected by PMM activity, not PGM activity. Using an in vitro system starting from TMP and 2-deoxy-glucose-6-phosphate glucose, TDP-2-deoxy-glucose (63% yield) was successfully synthesized. Considering low productivity of NDP-sugars from cheap starting materials, this paper showed how production of NDP-sugars could be enhanced by controlling mutase activity

Safety and Usability Guidelines of Clinical Information Systems Integrating Clinical Workflow: A Systematic Review.

OBJECTIVES: The usability of clinical information systems (CISs) is known to be an essential consideration in ensuring patient safety as well as integrating clinical flow. This study aimed to determine how usability and safety guidelines of CIS consider clinical workflow through a systematic review in terms of the target systems, methodology, and guideline components of relevant articles. METHODS: A literature search was conducted for articles published from 2000 to 2015 in PubMed, Cochrane, EMBASE, Web of Science, and CINAHL. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement method was employed. Articles containing recommendations, principles, and evaluation items for CIS usability and safety were included. The selected articles were classified according to article type, methodology, and target systems. Taking clinical workflow into consideration, the components of guidelines were extracted and classified. RESULTS: A total of 7,401 articles were identified by keyword search. From the 76 articles remaining after abstract screening, 15 were selected through full-text review. Literature review (n = 7) was the most common methodology, followed by expert opinions (n = 6). Computerized physician order entry (n = 6) was the most frequent system. Four articles considered the entire process of clinical tasks, and two articles considered the principles of the entire process of user interface affecting clinical workflow. Only two articles performed heuristic evaluations of CISs. CONCLUSIONS: The usability and safety guidelines of CISs need improvement in guideline development methodology and with consideration of clinical workflow

Design and Implementation of a Storage Management Method for Content Distribution

The SMART system is a special purpose server developed by ETRI and designed for efficient streaming services over high speed networks. The SMART server has one or more special purpose NS (Network-Storage) card. The NS card has several disks that store multimedia contents. However all of the multimedia contents to be serviced cannot be stored at the server. In this paper, we will describe the storage management mechanism in design and implementation aspects. With this storage management mechanism, the SMART server can provide effectiveness in managing storage and distributing some contents from a source station to streaming service servers

Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with Gαq

Background Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. Methods Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. Results Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 μM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. Conclusions These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03930458)