Properties and stabilization of an extracellular α-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN 1: enzyme activity at 130°C

An extracellular α-glucosidase from the thermophilic archaebacterium Thermococcus strain AN1 was purified 875-fold in five steps (Hiload Q-Sepharose, phenyl Sepharose, HPHT-hydroxyapatite, gel filtration and Mono Q chromatography) with a yield of 4%. It is a monomer with a molecular mass of about 60 kDa and a pI around 5. At 98°C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of l% (w/v) dithiothreitol and 1% (w/v) BSA. Dithiothreitol (1%, w/v) and BSA (0.4%, w/v) also substantially increase the enzyme activity. The Km at 75°C is 0.41 mM with pNP-α- -glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-α-D-glucoside > nigerose > panose > palatinose > isomaltose > maltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and β-gentiobiose. A variety of techniques including immobilization (e.g., on epoxy and glass beads), chemical modification (cross- and cocross-linking) and the use of additives (including polyhydroxylic molecules, BSA, salts, etc.) were applied to enhance stability at temperatures above 100°C. The half-life could be increased from about 4 min at 110°C to 30–60 min at 130°C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and l% (w/v) BSA, and by cocross-linking with BSA in the presence of 90% (w/v) sorbitol. The stabilized enzyme showed good activity at 130°C

Spatial and temporal aspects of visual backward masking in children and young adolescents

We thank Marc Repnow for his help setting up the experiments. In addition, we thank two anonymous reviewers for their very thoughtful and helpful comments. This work was supported by the Volkswagen Foundation project “Between Europe and the Orient—A Focus on Research and Higher Education in/on Central Asia and the Caucasus” and by the VELUX Foundation project “Perception, Cognition and Healthy Brain Aging.”Peer reviewedPublisher PD

Characterization of the murine cytomegalovirus genes encoding the major DNA binding protein and the ICP18.5 homolog

In several herpesviruses the genes for the major DNA binding protein (MDBP), a putative assembly protein, the glycoprotein B (gB), and the viral DNA polymerase (pol) coliocate. In murine cytomegalovirus (MCMV), two members of this gene block, pol (Elliott, Clark, Jaquish, and Spector, 1991, Virology 185, 169-186) and gB (Rapp, Messerle, BOhler, Tannheimer, Keil, and Koszinowski, 1992, J. Virol., 66,4399-4406) have been characterized. Here the two other MCMV genes are characterized, the gene encoding the MDBP and the ICP18.5 homolog encoding a putative assembly protein. Like in human cytomegalovirus (HCMV) the genes order is pol, gB, ICP18.5, and MDBP. The 4.2-kb MDBP mRNA is expressed first in the early phase, whereas the 3.0-kb ICP18.5 mRNA is a late transcript. The open reading frame of the MDBP gene has the capacity of encoding a protein of 1191 amino acids with a predicted molecular mass of 131.7 kDa. The MCMV ICP18.5 ORF is translated into a polypeptide of 798 amino acids with a calculated molecular mass of 89.1 kDa. Comparison of the amino acid sequences of the predicted proteins of MCMV with the respective proteins of HCMV, Epstein-Barr virus (EBV), and herpes simplex virus type-1 (HSV-1) reveals a striking homology ranging from 72% (HCMV), 50% (EBV), to 45% (HSV-1) for the MDBP sequence and from 74% (HCMV), 51 % (EBV), to 49% (HSV-1) for the ICP18.5 sequence. These results establish the elose relationship of the two cytomegaloviruses, and underline the usefulness of the murine model for studies on the biology of the CMV infection

Retention of Low Income Children in Three Dental Studies Investigating Early Childhood Caries

Background: To our knowledge no dental studies have looked closely at subject retention, which is crucial to better understand oral health disparities. In this paper, we report retention rates and review and attempt to assess which retention strategies utilized in 3 dental research studies investigating ECC were effective for retaining WIC-enrolled children. The purpose of this paper is to discuss challenges that were encountered when working with these populations, describe characteristics of those not retained, and summarize some recommendations for future dental studies working at WIC sites. Methods: Three dental studies were conducted at WIC clinics in Iowa. Retention strategies focused on maintenance of contact over time, persistence in rescheduling appointments, utilization of incentives, high recruitment, and frequent communication with parents and program staff. Results: Retention rates in the studies ranged from 60 to 75 percent at the final research interventions. Studies were challenged by frequent moves of subjects, missed appointments, disconnected phones, busy schedules of parents, transportation problems, loss of child custody, family illness, and lack of interest. Those not retained in the studies were more likely to be younger, single, and less educated, with a lower household income and a non-Caucasian child. Lower retention was also associated with the presence of carious lesions. Conclusions: Despite many challenges, studies had good retention rates and benefited from the retention strategies. Future dental studies at WIC clinics may also benefit from arranging transportation, obtaining a free, 800 callback number, and offering after-hours appointments for working parents

Evidence, process or context? Examining the factors that drive coverage decisions of pharmaceuticals by health technology assessment bodies in Europe

In Europe, Health Technology Assessment (HTA) bodies produce coverage decisions that guide public funding of pharmaceuticals. This thesis examines and weights those factors that drive HTA coverage decisions, focusing on the National Institute for Health and Clinical Excellence (NICE) in England and Wales, the Scottish Medicines Consortium (SMC), the Dutch College voor Zorgverzekeringen (CVZ), and the French Haute Autorité de Sante (HAS). To address the research question, a dataset of approximately 1000 HTA coverage decisions by NICE, SMC, CVZ and HAS from the period 2004-2009 was created, containing more than 30 clinical, economic, process and socio-economic factors extracted from published HTA reports. A three-category outcome variable was used, defined as the decision to ‘recommend’, ‘restrict’ or ‘not recommend’ a technology. Multivariate analyses were conducted to assess the relative contribution of the explanatory variables on coverage decisions both within and between HTA bodies. Results demonstrate that different combinations of clinical/economic evidence, process and socio-economic factors drive HTA coverage decisions by NICE, SMC, CVZ and HAS. In addition, the same factor may behave differently according to the nature of the coverage decision. The analysis further suggests there is a significant difference between HTA bodies in the probability of reaching a ‘restrict’ or ‘not recommend’ decision outcome relative to a ‘recommend’ outcome, adjusted for evidence, process and context factors. This thesis contributes to the understanding of factors driving HTA coverage decisions by examining multiple European HTA bodies, enhancing the comprehensiveness of the factors examined through descriptive and multivariate analyses and by identifying and weighting the key drivers of the coverage decisions made by the four HTA bodies between 2004 and 2009. This research further provides relevant insights to variation among HTA bodies in the determination of patient access to pharmaceuticals, and implications for collaboration between European HTA bodies