Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types

Developing and validating subjective and objective risk-assessment measures for predicting mortality after major surgery: An international prospective cohort study

Background: Preoperative risk prediction is important for guiding clinical decision-making and resource allocation. Clinicians frequently rely solely on their own clinical judgement for risk prediction rather than objective measures. We aimed to compare the accuracy of freely available objective surgical risk tools with subjective clinical assessment in predicting 30-day mortality. Methods and findings: We conducted a prospective observational study in 274 hospitals in the United Kingdom (UK), Australia, and New Zealand. For 1 week in 2017, prospective risk, surgical, and outcome data were collected on all adults aged 18 years and over undergoing surgery requiring at least a 1-night stay in hospital. Recruitment bias was avoided through an ethical waiver to patient consent; a mixture of rural, urban, district, and university hospitals participated. We compared subjective assessment with 3 previously published, open-access objective risk tools for predicting 30-day mortality: the Portsmouth-Physiology and Operative Severity Score for the enUmeration of Mortality (P-POSSUM), Surgical Risk Scale (SRS), and Surgical Outcome Risk Tool (SORT). We then developed a logistic regression model combining subjective assessment and the best objective tool and compared its performance to each constituent method alone. We included 22,631 patients in the study: 52.8% were female, median age was 62 years (interquartile range [IQR] 46 to 73 years), median postoperative length of stay was 3 days (IQR 1 to 6), and inpatient 30-day mortality was 1.4%. Clinicians used subjective assessment alone in 88.7% of cases. All methods overpredicted risk, but visual inspection of plots showed the SORT to have the best calibration. The SORT demonstrated the best discrimination of the objective tools (SORT Area Under Receiver Operating Characteristic curve [AUROC] = 0.90, 95% confidence interval [CI]: 0.88–0.92; P-POSSUM = 0.89, 95% CI 0.88–0.91; SRS = 0.85, 95% CI 0.82–0.87). Subjective assessment demonstrated good discrimination (AUROC = 0.89, 95% CI: 0.86–0.91) that was not different from the SORT (p = 0.309). Combining subjective assessment and the SORT improved discrimination (bootstrap optimism-corrected AUROC = 0.92, 95% CI: 0.90–0.94) and demonstrated continuous Net Reclassification Improvement (NRI = 0.13, 95% CI: 0.06–0.20, p < 0.001) compared with subjective assessment alone. Decision-curve analysis (DCA) confirmed the superiority of the SORT over other previously published models, and the SORT–clinical judgement model again performed best overall. Our study is limited by the low mortality rate, by the lack of blinding in the ‘subjective’ risk assessments, and because we only compared the performance of clinical risk scores as opposed to other prediction tools such as exercise testing or frailty assessment. Conclusions: In this study, we observed that the combination of subjective assessment with a parsimonious risk model improved perioperative risk estimation. This may be of value in helping clinicians allocate finite resources such as critical care and to support patient involvement in clinical decision-making

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

Family physicians\u27 responses to personal protective equipment shortages in four regions in Canada: a qualitative study.

BACKGROUND: Despite well-documented increased demands and shortages of personal protective equipment (PPE) during previous disease outbreaks, health systems in Canada were poorly prepared to meet the need for PPE during the COVID-19 pandemic. In the primary care sector, PPE shortages impacted the delivery of health services and contributed to increased workload, fear, and anxiety among primary care providers. This study examines family physicians\u27 (FPs) response to PPE shortages during the first year of the COVID-19 pandemic to inform future pandemic planning. METHODS: As part of a multiple case study, we conducted semi-structured qualitative interviews with FPs across four regions in Canada. During the interviews, FPs were asked to describe the pandemic-related roles they performed over different stages of the pandemic, facilitators and barriers they experienced in performing these roles, and potential roles they could have filled. Interviews were transcribed and a thematic analysis approach was employed to identify recurring themes. For the current study, we examined themes related to PPE. RESULTS: A total of 68 FPs were interviewed across the four regions. Four overarching themes were identified: 1) factors associated with good PPE access, 2) managing PPE shortages, 3) impact of PPE shortages on practice and providers, and 4) symbolism of PPE in primary care. There was a wide discrepancy in access to PPE both within and across regions, and integration with hospital or regional health authorities often resulted in better access than community-based practices. When PPE was limited, FPs described rationing and reusing these resources in an effort to conserve, which often resulted in anxiety and personal safety concerns. Many FPs expressed that PPE shortages had come to symbolize neglect and a lack of concern for the primary care sector in the pandemic response. CONCLUSIONS: During the COVID-19 pandemic response, hospital-centric plans and a lack of prioritization for primary care led to shortages of PPE for family physicians. This study highlights the need to consider primary care in PPE conservation and allocation strategies and to examine the influence of the underlying organization of primary care on PPE distribution during the pandemic

Family physician leadership during the COVID-19 pandemic: roles, functions and key supports.

PURPOSE: Strong leadership in primary care is necessary to coordinate an effective pandemic response; however, descriptions of leadership roles for family physicians are absent from previous pandemic plans. This study aims to describe the leadership roles and functions family physicians played during the COVID-19 pandemic in Canada and identify supports and barriers to formalizing these roles in future pandemic plans. DESIGN/METHODOLOGY/APPROACH: This study conducted semi-structured qualitative interviews with family physicians across four regions in Canada as part of a multiple case study. During the interviews, participants were asked about their roles during each pandemic stage and the facilitators and barriers they experienced. Interviews were transcribed and a thematic analysis approach was used to identify recurring themes. FINDINGS: Sixty-eight family physicians completed interviews. Three key functions of family physician leadership during the pandemic were identified: conveying knowledge, developing and adapting protocols for primary care practices and advocacy. Each function involved curating and synthesizing information, tailoring communications based on individual needs and building upon established relationships. PRACTICAL IMPLICATIONS: Findings demonstrate the need for future pandemic plans to incorporate formal family physician leadership appointments, as well as supports such as training, communication aides and compensation to allow family physicians to enact these key roles. ORIGINALITY/VALUE: The COVID-19 pandemic presents a unique opportunity to examine the leadership roles of family physicians, which have been largely overlooked in past pandemic plans. This study\u27s findings highlight the importance of these roles toward delivering an effective and coordinated pandemic response with uninterrupted and safe access to primary care

Development of a primary care pandemic plan informed by in-depth policy analysis and interviews with family physicians across Canada during COVID-19: A qualitative case study protocol

Introduction Given the recurrent risk of respiratory illness-based pandemics, and the important roles family physicians play during public health emergencies, the development of pandemic plans for primary care is imperative. Existing pandemic plans in Canada, however, do not adequately incorporate family physicians\u27 roles and perspectives. This policy and planning oversight has become increasingly evident with the emergence of the novel coronavirus disease, COVID-19, pandemic. This study is designed to inform the development of pandemic plans for primary care through evidence from four provinces in Canada: British Columbia, Newfoundland and Labrador, Nova Scotia, and Ontario. Methods and analysis We will employ a multiple-case study of regions in four provinces. Each case consists of a mixed methods design which comprises: (1) a chronology of family physician roles in the COVID-19 pandemic response; (2) a provincial policy analysis; and (3) qualitative interviews with family physicians. Relevant policy and guidance documents will be identified through targeted, snowball and general search strategies. Additionally, these policy documents will be analysed to identify gaps and/or emphases in existing policies and policy responses. Interviews will explore family physicians\u27 proposed, actual and potential roles during the pandemic, the facilitators and barriers they have encountered throughout and the influence of gender on their professional roles. Data will be thematically analysed using a content analysis framework, first at the regional level and then through cross-case analyses. Ethics and dissemination Approval for this study has been granted by the Research Ethics of British Columbia, the Health Research Ethics Board of Newfoundland and Labrador, the Nova Scotia Health Authority Research Ethics Board and the Western University Research Ethics Board. Findings will be disseminated via conferences and peer-reviewed publications. Evidence and lessons learnt will be used to develop tools for government ministries, public health units and family physicians for improved pandemic response plans for primary care

HDAC9 is implicated in atherosclerotic aortic calcification and affects vascular smooth muscle cell phenotype.

Aortic calcification is an important independent predictor of future cardiovascular events. We performed a genome-wide association meta-analysis to determine SNPs associated with the extent of abdominal aortic calcification (n = 9,417) or descending thoracic aortic calcification (n = 8,422). Two genetic loci, HDAC9 and RAP1GAP, were associated with abdominal aortic calcification at a genome-wide level (P &lt; 5.0 × 10-8). No SNPs were associated with thoracic aortic calcification at the genome-wide threshold. Increased expression of HDAC9 in human aortic smooth muscle cells promoted calcification and reduced contractility, while inhibition of HDAC9 in human aortic smooth muscle cells inhibited calcification and enhanced cell contractility. In matrix Gla protein-deficient mice, a model of human vascular calcification, mice lacking HDAC9 had a 40% reduction in aortic calcification and improved survival. This translational genomic study identifies the first genetic risk locus associated with calcification of the abdominal aorta and describes a previously unknown role for HDAC9 in the development of vascular calcification

Nutrition aspects in children receiving maintenance hemodialysis: impact on outcome

Children with end-stage renal disease (ESRD) have rates of mortality estimated to be 30-times higher than expected for age compared with those of healthy children. Physical manifestations of under-nutrition, such as body mass index (BMI) and low height standard deviation score (SDS), have been associated with increased risk of mortality. Traditional measures, such as height, weight and serum albumin concentration, may not be accurate indicators to assess the nutritional status of children receiving maintenance hemodialysis. Normalized protein catabolic rate (nPCR) has emerged as a better marker of nutritional status of such children. Meeting the special nutritional needs of these children often requires nutritional supplementation, by either the enteral or the parenteral route. Recently, in children receiving maintenance hemodialysis who are malnourished, intradialytic parenteral nutrition (IDPN) has been utilized as a means to provide additional protein and calories. This article is a state-of-the-art review of malnutrition in children receiving maintenance hemodialysis, with special focus on outcome, nPCR and IDPN