The conserved C-terminus of the PcrA/UvrD helicase interacts directly with RNA polymerase

Copyright: © 2013 Gwynn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a Wellcome Trust project grant to MD (Reference: 077368), an ERC starting grant to MD (Acronym: SM-DNA-REPAIR) and a BBSRC project grant to PM, NS and MD (Reference: BB/I003142/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Position of the Third Na+ Site in the Aspartate Transporter GltPh and the Human Glutamate Transporter, EAAT1

Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na+ ions, one H+ and the counter-transport of one K+ ion. Transport by an archaeal homologue of the human glutamate transporters, GltPh, whose three dimensional structure is known is also coupled to three Na+ ions but only two Na+ ion binding sites have been observed in the crystal structure of GltPh. In order to fully utilize the GltPh structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of GltPh and accurately determine the number and location of Na+ ions coupled to transport. Several sites have been proposed for the binding of a third Na+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for GltPh and reveal a new site for the third Na+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in GltPh, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na+ ion in GltPh and EAAT1

Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design

Systematic review on quality control for drug management programs: Is quality reported in the literature?

<p>Abstract</p> <p>Background</p> <p>Maintaining quality of care while managing limited healthcare resources is an ongoing challenge in healthcare. The objective of this study was to evaluate how the impact of drug management programs is reported in the literature and to identify potentially existing quality standards.</p> <p>Methods</p> <p>This analysis relates to the published research on the impact of drug management on economic, clinical, or humanistic outcomes in managed care, indemnity insurance, VA, or Medicaid in the USA published between 1996 and 2007. Included articles were systematically analyzed for study objective, study endpoints, and drug management type. They were further categorized by drug management tool, primary objective, and study endpoints.</p> <p>Results</p> <p>None of the 76 included publications assessed the overall quality of drug management tools. The impact of 9 different drug management tools used alone or in combination was studied in pharmacy claims, medical claims, electronic medical records or survey data from either patient, plan or provider perspective using an average of 2.1 of 11 possible endpoints. A total of 68% of the studies reported the impact on plan focused endpoints, while the clinical, the patient or the provider perspective were studied to a much lower degree (45%, 42% and 12% of the studies). Health outcomes were only accounted for in 9.2% of the studies.</p> <p>Conclusion</p> <p>Comprehensive assessment of quality considering plan, patient and clinical outcomes is not yet applied. There is no defined quality standard. Benchmarks including health outcomes should be determined and used to improve the overall clinical and economic effectiveness of drug management programs.</p

Secondary Endoleak Management Following TEVAR and EVAR.

Endovascular abdominal and thoracic aortic aneurysm repair and are widely used to treat increasingly complex aneurysms. Secondary endoleaks, defined as those detected more than 30 days after the procedure and after previous negative imaging, remain a challenge for aortic specialists, conferring a need for long-term surveillance and reintervention. Endoleaks are classified on the basis of their anatomic site and aetiology. Type 1 and type 2 endoleaks (EL1 and EL2) are the most common endoleaks necessitating intervention. The management of these requires an understanding of their mechanics, and the risk of sac enlargement and rupture due to increased sac pressure. Endovascular techniques are the main treatment approach to manage secondary endoleaks. However, surgery should be considered where endovascular treatments fail to arrest aneurysm growth. This chapter reviews the aetiology, significance, management strategy and techniques for different endoleak types

Millisecond dynamics of an unlabeled amino acid transporter

Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution

Potassium and Sodium Transport in Yeast

[EN] As the proper maintenance of intracellular potassium and sodium concentrations is vital for cell growth, all living organisms have developed a cohort of strategies to maintain proper monovalent cation homeostasis. In the model yeast Saccharomyces cerevisiae, potassium is accumulated to relatively high concentrations and is required for many aspects of cellular function, whereas high intracellular sodium/potassium ratios are detrimental to cell growth and survival. The fact that S. cerevisiae cells can grow in the presence of a broad range of concentrations of external potassium (10 M–2.5 M) and sodium (up to 1.5 M) indicates the existence of robust mechanisms that have evolved to maintain intracellular concentrations of these cations within appropriate limits. In this review, current knowledge regarding potassium and sodium transporters and their regulation will be summarized. 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Inorganic Mass Spectrometry

To establish a method for sensitive, accurate, and precise determination of Se in real samples, isotope dilution analysis using high-power nitrogen microwave-induced plasma mass spectrometry (N 2 MIP-IDMS) was conducted. In this study, freeze-dried human blood serum (Standard Reference Material, NIES No. 4) provided by NIES (National Institute for Environmental Studies) was used as a real sample. The measured isotopes of Se were 78 Se and 80 Se which are the major isotopes of Se. The appropriate amount of a Se spike solution was theoretically calculated by using an error multiplication factor (F) and was confirmed experimentally for the isotope dilution analysis. The mass discrimination effect was corrected for by using a standard Se solution for the measurement of Se isotope ratios in the spiked sample. However, the sensitivity for the detection of Se was not so good and the precision of the determination was not improved (2-3%) by N 2 MIP-IDMS with use of the conventional nebulizer. Therefore, a hydride generation system was connected to N 2 MIP-IDMS as a sample introduction system (HG-N 2 MIP-IDMS) in order to establish a more sensitive detection and a more precise determination of Se. A detection limit (3σ) of 10 pg mL -1 could be achieved, and the RSD was less than 1% at the concentration level of 5.0-10.0 ng mL -1 by HG-N 2 MIP-IDMS. The analytical results were found to be in a good agreement with those obtained by the standard addition method using conventional Ar ICPMS. It is well-known that Se is an essential element for all mammals. Se deficiency leads to deficiency syndromes, for example, Keshan disease, which is known for cardiac insufficiency that occurred in children and pregnant women in China. Problems also occur if the concentration of Se is too high; for example, gastroenteric disorders, dermatitis, and neurotic disorders are caused by excessive intake of Se. Moreover, it is well-known that the range of permissive intake amounts of Se is very narrow for human beings. Therefore, it is restricted as a toxic element in environmental standards. There are several sources of environmental Se pollution: the processes of Se refinement and the production processes of Se-containing products. For these reasons, the accurate and precise determination of trace levels of Se in environmental and biological samples is required, and studies of Se determination have been reported by several groups. [1][2][3][4][5][6][7][8][9][10][11] Because Ar ICPMS can measure multiple elements at a concentration range from ng mL -1 to fg mL -1 , it has widespread use in the determination of trace elements in various samples. 12-25 However