A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa

Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1–7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2–3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution

Use of mixed methods designs in substance research: a methodological necessity in Nigeria

The utility of mixed methods (qualitative and quantitative) is becoming increasingly accepted in health sciences, but substance studies are yet to substantially benefit from such utilities. While there is a growing number of mixed methods alcohol articles concerning developed countries, developing nations are yet to embrace this method. In the Nigerian context, the importance of mixed methods research is yet to be acknowledged. This article therefore, draws on alcohol studies to argue that mixed methods designs will better equip scholars to understand, explore, describe and explain why alcohol consumption and its related problems are increasing in Nigeria. It argues that as motives for consuming alcohol in contemporary Nigeria are multiple, complex and evolving, mixed method approaches that provide multiple pathways for proffering solutions to problems should be embraced

Genomic organisation analysis of novel immunoglobulin-like transcripts in Atlantic salmon (Salmo salar) reveals a tightly clustered and multigene family

Copyright 2011 Elsevier B.V., All rights reserved.Peer reviewedPublisher PD

Mortality after Hospitalization for Pneumonia among Individuals with HIV, 1995–2008: A Danish Cohort Study

BACKGROUND: HIV-infected persons are at increased risk of pneumonia, even with highly active antiretroviral treatment (HAART). We examined the impact of pneumonia on mortality and identified prognostic factors for death among HIV-infected. METHODOLOGY/PRINCIPAL FINDINGS: In a nationwide, population-based cohort of individuals with HIV, we included persons hospitalized with pneumonia from the Danish National Hospital Registry and obtained mortality data from the Danish Civil Registration System. Comparing individuals with and without pneumonia, we used Poisson regression to estimate relative mortality and logistic regression to examine prognostic factors for death following pneumonia. From January 1, 1995, to July 1, 2008, we observed 699 episodes of first hospitalization for pneumonia among 4,352 HIV patients. Ninety-day mortality after pneumonia decreased from 22.4% (95% confidence interval [CI]: 16.5%-28.9%) in 1995-1996 to 8.4% (95% CI: 6.1%-11.6%) in 2000-2008. Mortality remained elevated for more than a year after hospitalization for pneumonia: adjusted mortality rate ratio 5.38 (95% CI: 4.27-6.78), 1.80 (95% CI: 1.36-2.37), and 1.62 (95% CI: 1.32-2.00) for days 0-90, 91-365, and 366+, respectively. The following variables predicted mortality within 90 days following hospitalization for pneumonia (adjusted Odds Ratios): male sex (3.77, 95% CI: 1.37-10.4), Charlson Comorbidity Index score > or = 2 (3.86, 95% CI: 2.19-6.78); no current HAART (3.58, 95% CI: 1.83-6.99); history of AIDS (2.46, 95% CI: 1.40-4.32); age per 10 year increase (1.43, 95% CI: 1.11-1.85); and CD4+ cell count < or = 200 (2.52, 95% CI: 1.37-4.65). CONCLUSIONS/SIGNIFICANCE: The first hospitalization for pneumonia among HIV-infected individuals was associated with elevated risk of death up to more than a year later. Use of HAART decreased the risk, independent of current CD4+ cell count. Prognosis following pneumonia improved over calendar time

BOLD Correlates of Trial-by-Trial Reaction Time Variability in Gray and White Matter: A Multi-Study fMRI Analysis

Reaction time (RT) is one of the most widely used measures of performance in experimental psychology, yet relatively few fMRI studies have included trial-by-trial differences in RT as a predictor variable in their analyses. Using a multi-study approach, we investigated whether there are brain regions that show a general relationship between trial-by-trial RT variability and activation across a range of cognitive tasks.The relation between trial-by-trial differences in RT and brain activation was modeled in five different fMRI datasets spanning a range of experimental tasks and stimulus modalities. Three main findings were identified. First, in a widely distributed set of gray and white matter regions, activation was delayed on trials with long RTs relative to short RTs, suggesting delayed initiation of underlying physiological processes. Second, in lateral and medial frontal regions, activation showed a "time-on-task" effect, increasing linearly as a function of RT. Finally, RT variability reliably modulated the BOLD signal not only in gray matter but also in diffuse regions of white matter.The results highlight the importance of modeling trial-by-trial RT in fMRI analyses and raise the possibility that RT variability may provide a powerful probe for investigating the previously elusive white matter BOLD signal

CD6 and Syntaxin Binding Protein 6 Variants and Response to Tumor Necrosis Factor Alpha Inhibitors in Danish Patients with Rheumatoid Arthritis

<div><h3>Background</h3><p>TNFα inhibitor therapy has greatly improved the treatment of patients with rheumatoid arthritis, however at least 30% do not respond. We aimed to investigate insertions and deletions (INDELS) associated with response to TNFα inhibitors in patients with rheumatoid arthritis (RA).</p> <h3>Methodology and Principal Findings</h3><p>In the DANBIO Registry we identified 237 TNFα inhibitor naïve patients with RA (81% women; median age 56 years; disease duration 6 years) who initiated treatment with infliximab (n = 160), adalimumab (n = 56) or etanercept (n = 21) between 1999 and 2008 according to national treatment guidelines. Clinical response was assessed at week 26 using EULAR response criteria. Based on literature, we selected 213 INDELS potentially related to RA and treatment response using the GeneVa® (Compugen) <em>in silico</em> database of 350,000 genetic variations in the human genome. Genomic segments were amplified by polymerase chain reaction (PCR), and genotyped by Sanger sequencing or fragment analysis. We tested the association between genotypes and EULAR good response versus no response, and EULAR good response versus moderate/no response using Fisher’s exact test. At baseline the median DAS28 was 5.1. At week 26, 68 (29%) patients were EULAR good responders, while 81 (34%) and 88 (37%) patients were moderate and non-responders, respectively. A 19 base pair insertion within the CD6 gene was associated with EULAR good response vs. no response (OR = 4.43, 95% CI: 1.99–10.09, p = 7.211×10⁻⁵) and with EULAR good response vs. moderate/no response (OR = 4.54, 95% CI: 2.29–8.99, p = 3.336×10⁻⁶). A microsatellite within the syntaxin binding protein 6 (STXBP6) was associated with EULAR good response vs. no response (OR = 4.01, 95% CI: 1.92–8.49, p = 5.067×10⁻⁵).</p> <h3>Conclusion</h3><p>Genetic variations within CD6 and STXBP6 may influence response to TNFα inhibitors in patients with RA.</p> </div

Biologics registers in RA: methodological aspects, current role and future applications

The beginning of the 21st century saw a biopharmaceutical revolution in the treatment of inflammatory rheumatic diseases, particularly rheumatoid arthritis. The fast-evolving use of biologic therapies highlighted the need to develop registers at national and international levels with the aim of collecting long-term data on patient outcomes. Over the past 15 years, many biologics registers have contributed a wealth of data and provided robust and reliable evidence on the use, effectiveness and safety of these therapies. The unavoidable challenges posed by the continuous introduction of new therapies, particularly with regard to understanding their long-term safety, highlights the importance of learning from experience with established biologic therapies. In this Perspectives article, the role of biologics registers in bridging the evidence gap between efficacy in clinical trials and real-world effectiveness is discussed, with a focus on methodological aspects of registers, their unique features and challenges and their role going forward