Misfolding of vWF to Pathologically Disordered Conformations Impacts the Severity of von Willebrand Disease

The primary hemostatic von Willebrand factor (vWF) functions to sequester platelets from rheological blood flow and mediates their adhesion to damaged subendothelium at sites of vascular injury. We have surveyed the effect of 16 disease-causing mutations identified in patients diagnosed with the bleeding diathesis disorder, von Willebrand disease (vWD), on the structure and rheology of vWF A1 domain adhesiveness to the platelet GPIbα receptor. These mutations have a dynamic phenotypical range of bleeding from lack of platelet adhesion to severe thrombocytopenia. Using new rheological tools in combination with classical thermodynamic, biophysical, and spectroscopic metrics, we establish a high propensity of the A1 domain to misfold to pathological molten globule conformations that differentially alter the strength of platelet adhesion under shear flow. Rheodynamic analysis establishes a quantitative rank order between shear-rate-dependent platelet-translocation pause times that linearly correlate with clinically reported measures of patient platelet counts and the severity of thrombocytopenia. These results suggest that specific secondary structure elements remaining in these pathological conformations of the A1 domain regulate GPIbα binding and the strength of vWF-platelet interactions, which affects the vWD functional phenotype and the severity of thrombocytopenia.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Occupancy in dynamic systems: accounting for multiple scales and false positives using environmental DNA to inform monitoring

Occupancy is an important metric to understand current and future trends in populations that have declined globally. In addition, occupancy can be an efficient tool for conducting landscape-scale and long-term monitoring. A challenge for occupancy monitoring programs is to determine the appropriate spatial scale of analysis and to obtain precise occupancy estimates for elusive species. We used a multi-scale occupancy model to assess occupancy of Columbia spotted frogs in the Great Basin, USA, based on environmental DNA (eDNA) detections. We collected three replicate eDNA samples at 220 sites across the Great Basin. We estimated and modeled ecological factors that described watershed and site occupancy at multiple spatial scales simultaneously while accounting for imperfect detection. Additionally, we conducted visual and dipnet surveys at all sites and used our paired detections to estimate the probability of a false positive detection for our eDNA sampling. We applied the estimated false positive rate to our multi-scale occupancy dataset and assessed changes in model selection. We had higher naïve occupancy estimates for eDNA (0.37) than for traditional survey methods (0.20). We estimated our false positive detection rate per qPCR replicate at 0.023 (95% CI: 0.016–0.033). When the false positive rate was applied to the multi-scale dataset, we did not observe substantial changes in model selection or parameter estimates. Conservation and resource managers have an increasing need to understand species occupancy in highly variable landscapes where the spatial distribution of habitat changes significantly over time due to climate change and human impact. A multi-scale occupancy approach can be used to obtain regional occupancy estimates that can account for spatially dynamic differences in availability over time, especially when assessing potential declines. Additionally, this study demonstrates how eDNA can be used as an effective tool for improved occupancy estimates across broad geographic scales for long-term monitoring.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Construction of Aspergillus nidulans strains producing enzymes of potential use in enology

Two different Aspergillus nidulans recombinant strains producing either the Aspergillus nidulans a-L-arabinofuranosidase A or a Candida molischiana b-glucosidase have been constructed. Depending on the growing conditions, the modified strains produce up to 4 or 18 times more b-glucosidase or a-L-arabinofuranosidase activity levels, respectively, than the wild type strain.     

A Survey Investigating the Infection of Fusarium langsethiae and Production of HT‐2 and T‐2 Mycotoxins in UK Oat Fields

Fusarium langsethiae is a toxigenic fungal species that has been reported in European small‐grain cereal crops such as oats, wheat and barley. Although its relative contribution to fusarium head blight (FHB) symptoms is not well understood, it is reported to contaminate these cereals with high levels of HT‐2 and T‐2 trichothecenes mycotoxins that are currently under consideration for legislation by the European Commission. Ten commercial oat fields in Shropshire and Staffordshire (two adjacent counties in the Midlands) in the UK were surveyed in the 2006/2007 growing season. Samples were taken from predetermined field locations at Zadoks growth stages 32/33, 69, 77‐85 and 90‐92 for F. langsethiae biomass and HT‐2 and T‐2 toxins quantification. The results from this study showed that oats can be heavily infected with F. langsethiae and have high concentrations of HT‐2 and T‐2 toxins with no apparent FHB symptoms. The regression of HT‐2 + T‐2 toxins on F. langsethiae DNA concentration was highly significant (P < 0.001, r² = 0.55). The results indicated that although F. langsethiae had no direct effect on crop yield, it may result in indirect economic losses where the grain can be rejected or downgraded as a result of intolerable levels of HT‐2 and T‐2 toxins, which are of human food and animal feed safety concern. The influence of cultural field practices on the infection and HT‐2 and T‐2 toxins accumulation in oats was not clear and warrants further studies to identify the sources of F. langsethiae inoculum and conditions favourable for infection and mycotoxin production.     

Measures of Quality of Care for People with HIV: A Scoping Review of Performance Indicators for Primary Care

The healthcare of people with HIV is transitioning from specialty care to the primary healthcare (PHC) system. However, many of the performance indicators used to measure the quality of HIV care pre-date this transition. The goal of this work was to examine how existing HIV care performance indicators measure the comprehensive and longitudinal care offered in a PHC setting. A scoping review consisting of peer-reviewed and grey literature searches was performed. Two reviewers evaluated study eligibility and indicators in documents meeting inclusion criteria were extracted into a database. Indicators were matched to a PHC performance measurement framework to determine their applicability for evaluating quality of care in the PHC setting. The literature search identified 221 publications, of which 47 met inclusion criteria. 1184 indicators were extracted and removal of duplicates left 558 unique indicators. A majority of the 558 indicators fell under the ‘secondary prevention’ (12%) and ‘care of chronic conditions’ (33%) domains when indicators were matched to the PHC performance framework. Despite the imbalance, nearly all performance domains in the PHC framework were populated by at least one indicator with significant concentrations in domains such as patient-provider relationship, patient satisfaction, population and community characteristics, and access to care. Existing performance frameworks for the care of people with HIV provide a comprehensive set of indicators that align well with a PHC performance framework. Nonetheless, some important elements of care, such as patient-reported outcomes, are poorly covered by existing indicators. Advancing our understanding of how the experience of care for people with HIV is impacted by changes in health services delivery, specifically more care within the PHC system, will require performance indicators to capture this aspect of HIV care.