Sequence variation in the ß7-ß8 loop of bacterial class A sortase enzymes alters substrate selectivity.

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this ß7-ß8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by ß7-ß8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.

Tolerance of Novel Toxins through Generalized Mechanisms: Simulating Gradual Host Shifts of Butterflies.

Organisms encounter a wide range of toxic compounds in their environments, from chemicals that serve anticonsumption or anticompetition functions to pollutants and pesticides. Although we understand many detoxification mechanisms that allow organisms to consume toxins typical of their diet, we know little about why organisms vary in their ability to tolerate entirely novel toxins. We tested whether variation in generalized stress responses, such as antioxidant pathways, may underlie variation in reactions to novel toxins and, if so, their associated costs. We used an artificial diet to present cabbage white butterfly caterpillars (Pieris rapae) with plant material containing toxins not experienced in their evolutionary history. Families that maintained high performance (e.g., high survival, fast development time, large body size) on diets containing one novel toxic plant also performed well when exposed to two other novel toxic plants, consistent with a generalized response. Variation in constitutive (but not induced) expression of genes involved in oxidative stress responses was positively related to performance on the novel diets. While we did not detect reproductive trade-offs of this generalized response, there was a tendency to have less melanin investment in the wings, consistent with the role of melanin in oxidative stress responses. Taken together, our results support the hypothesis that variation in generalized stress responses, such as genes involved in oxidative stress responses, may explain the variation in tolerance to entirely novel toxins and may facilitate colonization of novel hosts and environments.

Covalent Label Transfer between Peroxisomal Importomer Components Reveals Export-driven Import Interactions.

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.

Knowledge, Attitudes, and Risk for Sudden Unexpected Infant Death in Children of Adolescent Mothers: A Qualitative Study.

OBJECTIVE: To investigate practices, knowledge, attitudes, and beliefs regarding infant sleep among adolescent mothers, a demographic at high risk for sudden unexpected infant death, and to identify novel public health interventions targeting the particular reasons of this population. STUDY DESIGN: Seven targeted focus groups including 43 adolescent mothers were conducted at high school daycare centers throughout Colorado. Focus groups were recorded, transcribed, validated, and then analyzed in NVivo 10. Validation included coding consistency statistics and expert review. RESULTS: Most mothers knew many of the American Academy of Pediatrics recommendations for infant sleep. However, almost all teens reported bedsharing regularly and used loose blankets or soft bedding despite being informed of risks. Reasons for nonadherence to recommendations included beliefs that babies are safest and sleep more/better in bed with them, that bedsharing is a bonding opportunity, and that bedsharing is easier than using a separate sleep space. The most common justifications for blankets were infant comfort and concern that babies were cold. Participants' decision making was often influenced by their own mothers, with whom they often resided. Participants felt that their instincts trumped professional advice, even when in direct contradiction to safe sleep recommendations. CONCLUSIONS: Among focus group participants, adherence with safe sleep practices was poor despite awareness of the American Academy of Pediatrics recommendations. Many mothers expressed beliefs and instincts that infants were safe in various unsafe sleep environments. Future study should investigate the efficacy of alternative educational strategies, including education of grandmothers, who have significant influence over adolescent mothers.

Arginine:glycine amidinotransferase (AGAT) deficiency: Clinical features and long term outcomes in 16 patients diagnosed worldwide.

BACKGROUND: Arginine:glycine aminotransferase (AGAT) (GATM) deficiency is an autosomal recessive inborn error of creative synthesis. OBJECTIVE: We performed an international survey among physicians known to treat patients with AGAT deficiency, to assess clinical characteristics and long-term outcomes of this ultra-rare condition. RESULTS: 16 patients from 8 families of 8 different ethnic backgrounds were included. 1 patient was asymptomatic when diagnosed at age 3 weeks. 15 patients diagnosed between 16 months and 25 years of life had intellectual disability/developmental delay (IDD). 8 patients also had myopathy/proximal muscle weakness. Common biochemical denominators were low/undetectable guanidinoacetate (GAA) concentrations in urine and plasma, and low/undetectable cerebral creatine levels. 3 families had protein truncation/null mutations. The rest had missense and splice mutations. Treatment with creatine monohydrate (100-800 mg/kg/day) resulted in almost complete restoration of brain creatine levels and significant improvement of myopathy. The 2 patients treated since age 4 and 16 months had normal cognitive and behavioral development at age 10 and 11 years. Late treated patients had limited improvement of cognitive functions. CONCLUSION: AGAT deficiency is a treatable intellectual disability. Early diagnosis may prevent IDD and myopathy. Patients with unexplained IDD with and without myopathy should be assessed for AGAT deficiency by determination of urine/plasma GAA and cerebral creatine levels (via brain MRS), and by GATM gene sequencing.

A Patient-Centered Documentation Skills Curriculum for Preclerkship Medical Students in an Open Notes Era.

Introduction: New legislation allows patients (with permitted exceptions) to read their clinical notes, leading to both benefits and ethical dilemmas. Medical students need a robust curriculum to learn documentation skills within this challenging context. We aimed to teach note-writing skills through a patient-centered lens with special consideration for the impact on patients and providers. We developed this session for first-year medical students within their foundational clinical skills course to place bias-free language at the forefront of how they learn to construct a medical note. Methods: One hundred seventy-three first-year medical and dental students participated in this curriculum. They completed an asynchronous presession module first, followed by a 2-hour synchronous workshop including a didactic, student-led discussion and sample patient note exercise. Students were subsequently responsible throughout the year for constructing patient-centered notes, graded by faculty with a newly developed rubric and checklist of best practices. Results: On postworkshop surveys, learners reported increased preparedness in their ability to document in a patient-centered manner (presession M = 2.2, midyear M = 3.9, p < .001), as rated on a 5-point Likert scale (1 = not prepared at all, 5 = very prepared), and also found this topic valuable to learn early in their training. Discussion: This curriculum utilizes a multipart approach to prepare learners to employ clinical notes to communicate with patients and providers, with special attention to how patients and their care partners receive a note. Future directions include expanding the curriculum to higher levels of learning and validating the developed materials.

Developing Clinical Phenotype Data Collection Standards for Research in Africa.

Modern biomedical research is characterised by its high-throughput and interdisciplinary nature. Multiproject and consortium-based collaborations requiring meaningful analysis of multiple heterogeneous phenotypic datasets have become the norm; however, such analysis remains a challenge in many regions across the world. An increasing number of data harmonisation efforts are being undertaken by multistudy collaborations through either prospective standardised phenotype data collection or retrospective phenotype harmonisation. In this regard, the Phenotype Harmonisation Working Group (PHWG) of the Human Heredity and Health in Africa (H3Africa) consortium aimed to facilitate phenotype standardisation by both promoting the use of existing data collection standards (hosted by PhenX), adapting existing data collection standards for appropriate use in low- and middle-income regions such as Africa, and developing novel data collection standards where relevant gaps were identified. Ultimately, the PHWG produced 11 data collection kits, consisting of 82 protocols, 38 of which were existing protocols, 17 were adapted, and 27 were novel protocols. The data collection kits will facilitate phenotype standardisation and harmonisation not only in Africa but also across the larger research community. In addition, the PHWG aims to feed back adapted and novel protocols to existing reference platforms such as PhenX.

TBDBT: A TB DataBase Template for collection of harmonized TB clinical research data in REDCap, facilitating data standardisation for inter-study comparison and meta-analyses.

Clinical tuberculosis research, both within research groups and across research ecosystems, is often undertaken in isolation using bespoke data collection platforms and applying differing data conventions. This failure to harmonise clinical phenotype data or apply standardised data collection and storage standards in turn limits the opportunity to undertake meta-analyses using data generated across multiple research projects for the same research domain. We have developed the Tuberculosis DataBase Template (TBDBT), a template for the well-supported, free and commonly deployed clinical databasing platform, REDCap. This template can be used to set up a new tuberculosis research database with a built-in set of standardised data conventions, to ensure standardised data capture across research projects and programs. A modular design enables researchers to implement only the modules of the database template that are appropriate for their particular study. The template includes core modules for informed consent data, participant demographics, clinical symptoms and presentation, diagnostic imaging and laboratory tests. Optional modules have been designed for visit scheduling and calendar functionality, clinical trial randomisation, study logistics and operations, and pharmacokinetic data. Additional fields can be added as needed. This REDCap template can facilitate collection of high-quality data for tuberculosis research, providing a tool to ensure better data harmonisation, analysis and meta-analysis.

Implementation of a Brief Screening Tool to Identify Needs of Breast Cancer Survivors.

BACKGROUND: Innovation in health care delivery is needed to improve care for cancer survivors. We report our experience with adapting screening questions from the National Comprehensive Cancer Network (NCCN) guideline to evaluate the needs of breast cancer survivors. MATERIALS AND METHODS: We adapted the NCCN-recommended screening questions into a plain language, self-administered 1-page intake questionnaire. The tool was administered to a convenience sample of female breast cancer survivors at follow-up oncology and primary care visits. Domains included symptoms, lifestyle concerns, and financial issues. Frequency of concerns was assessed as "never," "rarely," "sometimes," "very frequently," and "always." We evaluated feasibility and utility of administration and the prevalence and frequency of patient-reported concerns. RESULTS: The questionnaire was highly acceptable to patients and enhanced visits for clinicians. Clinicians reported that it led to discussion of issues that may not otherwise be addressed in the visit and did not find it burdensome. The most commonly endorsed patient concerns were desire to improve fitness or nutrition, worry about cancer recurrence, and insomnia. A majority also reported feeling anxiety and aches or pains in joints or extremities. Several issues known to be underreported in clinic visits were frequently endorsed, included sexual dissatisfaction and memory impairments. Clinicians suggested incorporating the tool into the electronic health record to increase utility and awareness. CONCLUSION: Screening for individual needs among breast cancer survivors is feasible, efficient, and may identify prevalent issues that otherwise can be missed in routine survivorship care.

The influence of black race on treatment and mortality for early-stage breast cancer.

BACKGROUND: Black Americans have higher mortality from breast cancer than white Americans. This study explores the influence of socioeconomic factors and black race on treatment and mortality for early-stage breast cancer. METHODS: A cohort of 21,848 female black and white, non-Hispanic subjects from the Massachusetts Cancer Registry diagnosed with stage I or II breast cancer between 1999-2004 was studied. Subjects with tumors larger than 5 cm were excluded. We used mixed modeling methods to assess the impact of race on guideline concordant care (GCC), defined as receipt of mastectomy or breast conserving surgery plus radiation. Cox proportional hazard regression was used to assess disease-specific mortality. RESULTS: Blacks were less likely to receive GCC after adjusting for age and clinical variables (OR: 0.75; 95% CI: 0.61, 0.92). Marital status and insurance were predictors of receipt of GCC. After adjustment for all covariates, there were no longer significant differences between black and white women regarding the receipt of GCC. Nevertheless, black women were more likely to die of early-stage breast cancer than white women after adjusting for clinical, treatment, socioeconomic variables, and reporting hospital (HR: 1.6; 95% CI: 1.1-2.1). CONCLUSIONS: Socioeconomic factors are mediators of racial differences in treatment outcomes. Significant racial differences exist in disease-specific mortality for women with early-stage breast cancer. Attention to reducing socioeconomic barriers to care may influence racial differences in breast cancer treatment and mortality.

Proposed minimum information guideline for kidney disease-research and clinical data reporting: a cross-sectional study.

OBJECTIVE: This project aimed to develop and propose a standardised reporting guideline for kidney disease research and clinical data reporting, in order to improve kidney disease data quality and integrity, and combat challenges associated with the management and challenges of 'Big Data'. METHODS: A list of recommendations was proposed for the reporting guideline based on the systematic review and consolidation of previously published data collection and reporting standards, including PhenX measures and Minimal Information about a Proteomics Experiment (MIAPE). Thereafter, these recommendations were reviewed by domain-specialists using an online survey, developed in Research Electronic Data Capture (REDCap). Following interpretation and consolidation of the survey results, the recommendations were mapped to existing ontologies using Zooma, Ontology Lookup Service and the Bioportal search engine. Additionally, an associated eXtensible Markup Language schema was created for the REDCap implementation to increase user friendliness and adoption. RESULTS: The online survey was completed by 53 respondents; the majority of respondents were dual clinician-researchers (57%), based in Australia (35%), Africa (33%) and North America (22%). Data elements within the reporting standard were identified as participant-level, study-level and experiment-level information, further subdivided into essential or optional information. CONCLUSION: The reporting guideline is readily employable for kidney disease research projects, and also adaptable for clinical utility. The adoption of the reporting guideline in kidney disease research can increase data quality and the value for long-term preservation, ensuring researchers gain the maximum benefit from their collected and generated data.

The Arabidopsis ALDP protein homologue COMATOSE is instrumental in peroxisomal acetate metabolism.

The Arabidopsis acn (acetate non-utilizing) mutants were isolated by fluoroacetate-resistant germination and seedling establishment. We report the characterization of the acn2 mutant. Physiological analyses of acn2 showed that it possessed characteristics similar to those of the mutants cts (COMATOSE)-1 and pxa [peroxisomal ABC (ATP-binding-cassette) transporter]1. The acn2 locus was mapped to within 3 cM of the CTS gene on the bottom arm of chromosome IV using CAPS (cleavage amplification polymorphism) and SSLP (simple sequence-length polymorphism) markers. Crossing acn2 and cts-1 failed to restore the fluoroacetate-sensitive phenotype, suggesting that these mutations were allelic. Sequencing of the ACN2 locus revealed a C-->T nonsense mutation in exon 13, which would have resulted in the elimination of the C-terminal hemitransporter domain of the encoded protein. Neither the full-length CTS protein nor the truncated protein was detected on immunoblots using either C-terminal- or N-terminal-specific anti-CTS antibodies respectively, demonstrating the absence of the entire CTS protein in acn2 mutants. Emerged seedlings of both cts-1 and pxa1 alleles displayed increased resistance to FAc (monofluoroacetic acid) compared with the corresponding wild-type seedlings. Complementation studies showed that mutation of the CTS gene was responsible for the FAc-resistant phenotype, as when the wild-type protein was expressed in both the cts-1 and pxa1 mutant lines, the strains became FAc-sensitive. Feeding studies confirmed that both acn2 and cts-1 mutants were compromised in their ability to convert radiolabelled acetate into soluble carbohydrate. These results demonstrate a role for the ABC protein CTS in providing acetate to the glyoxylate cycle in developing seedlings.

Implementation of diagnostic pauses in the ambulatory setting.

BACKGROUND: Diagnostic errors result in preventable morbidity and mortality. The outpatient setting may be at increased risk, where time constraints, the indolent nature of outpatient complaints and single decision-maker practice models predominate. METHODS: We developed a self-administered diagnostic pause to address diagnostic error. Clinicians (physicians and nurse practitioners) in an academic primary care setting received the tool if they were seeing urgent care patients who had previously been seen in the past two weeks in urgent care. We used pre-post-intervention surveys, focus groups and chart audits 6 months after the urgent care visit to assess the impact of the intervention on participant perceptions and actions. RESULTS: We piloted diagnostic pauses in two phases (3 months and 6 months, respectively); 9 physicians participated in the first phase, and 16 physicians and 2 nurse practitioners in the second phase. Subjects received 135 alerts for diagnostic pauses and responded to 82 (61% response). Thirteen per cent of alerts resulted in clinicians reporting new actions as a result of the diagnostic pauses. Thirteen per cent of cases at a 6-month chart audit resulted in diagnostic discrepancies, defined as differences in diagnosis from the initial working diagnosis. Focus groups reported that the diagnostic pauses were brief and fairly well integrated into the overall workflow for evaluation but would have benefited as a real-time application for patients at higher risk for diagnostic error. CONCLUSION: This pilot represents the first known examination of diagnostic pauses in the outpatient setting, and this work potentially paves the way for more broad-based systems and/or electronic interventions to address diagnostic error.

Driving Skills Training for Older Adults: An Assessment of DriveSharp.

RÉSUMÉ Les procédures de formation cognitive informatique visent à augmenter la sécurité en améliorant les compétences relatives à la conduite, comme la vitesse-de-traitement et le Useful Field of View. L'étude actuelle a évalué l'efficacité du DriveSharp dans la formation des conducteurs âgés dans un cadre de classe réaliste. Les participants (n = 24) ont assisté à 10 heures de cours de DriveSharp pendant 5 semaines. Les séances pré- et post-test ont evalués améliorations sur un essai dynamique de la perception du risque, Trails A et Trails B. Un groupe de contrôle (n = 18) a terminé seulement les séances pré- et post-test. En classe, les temps de formation étaient plus bas que prévus. L'amélioration des participants aux jeux ont stabilisée après la première évaluation, et le groupe de DriveSharp n'a pas démontré une amélioration significative des performances sur les tests, par rapport au groupe de contrôle. Parmi plusieurs questions relatives à la facilité d'utilisation, les plus problématiques étaient le malentendudes objectifs de la tâche et la différence entre la formation et l'évaluation. Il y a plusieurs implications pour ceux qui utilisent DriveSharp pour améliorer la sécurité des conducteurs âgés.

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida.

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.

The use of meta-analysis or research synthesis to combine driving simulation or naturalistic study results on driver distraction.

Three important and inter-related topics are addressed in this paper. First, the importance of meta-analysis and research synthesis methods to combine studies on traffic safety, in general, and on driver distraction, in particular, is briefly reviewed. Second, naturalistic, epidemiologic, and driving simulation studies on driver distraction are used to illustrate convergent and divergent results that have accumulated thus far in this domain of research. In particular, mobile phone conversation, passenger presence, and text messaging naturalistic studies use meta-analyses and research syntheses to illustrate important patterns of results that are in need of more in-depth study. Third, a number of driver distraction study limitations such as poorly defined dependent variables, lack of methodological detail, and omission of statistical information prevent the integration of many studies into meta-analyses. In addition, the overall quality of road safety studies suffers from these same limitations and suggestions for improvement are made to guide researchers and reviewers. Practical Applications. The use of research synthesis and meta-analysis provide comprehensive estimates of the impact of distractions on driving performance, which can be used to guide public policy and future research.