Efficient CRISPR-Cas9 mediated gene disruption in primary erythroid progenitor cells

The study of isolated primary progenitor cells offers great insight into developmental biology and human disease. In particular, ex vivo culture of isolated primary erythroid progenitor cells replicates the differentiation events that occur during in vivo erythropoiesis. Herein we report a high-efficiency method for CRISPR-Cas9 mediated gene disruption in isolated primary erythroid progenitor cells. We use this method to generate the novel result that Lmna is required in terminal erythroid differentiation.Frederick Lovejoy (Research Grant)National Institutes of Health (U.S.) (grant NIH/NHLBI 2 P01 HL032262-25

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5′ and 3′ of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants

Exploring the work–life challenges and dilemmas faced by managers and professionals who live alone

This article aims to question the dominant understanding of work–life balance or conflict as primarily a ‘work–family’ issue. It does this by exploring the experiences of managers and professionals who live alone and do not have children – a group of employees traditionally overlooked in work–life policy and research but, significantly, a group on the rise within the working age population. Semi-structured interviews with 36 solo-living managers and professionals were carried out in the UK, spanning a range of occupations. In addition to previously identified work–life issues, four themes emerged that were pressing for and specific to solo-living managers and professionals. These are articulated here as challenges and dilemmas relating to: assumptions about work and non-work time; the legitimacy of their work–life balance; lack of support connected to financial and emotional well-being; and work-based vulnerabilities

Altered translation of GATA1 in Diamond-Blackfan anemia

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA),1,2 congenital asplenia,3 and T-cell leukemia.4 Yet how mutations in such ubiquitously expressed proteins result in cell-type and tissue specific defects remains a mystery.5 Here, we show that GATA1 mutations that reduce full-length protein levels of this critical hematopoietic transcription factor can cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can similarly reduce translation of GATA1 mRNA - a phenomenon that appears to result from this mRNA having a higher threshold for initiation of translation. In primary hematopoietic cells from patients with RPS19 mutations, a transcriptional signature of GATA1 target genes is globally and specifically reduced, confirming that the activity, but not the mRNA level, of GATA1 is reduced in DBA patients with ribosomal protein mutations. The defective hematopoiesis observed in DBA patients with ribosomal protein haploinsufficiency can be at least partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations in ubiquitous ribosomal proteins can result in human disease

Community-Guided Focus Group Analysis to Examine Cancer Disparities

Accountability for Cancer Care through Undoing Racism and Equity (ACCURE) is a systems-change intervention addressing disparities in treatment initiation and completion and outcomes for early stage Black and White breast and lung cancer patients. Using a community-based participatory research approach, ACCURE is guided by a diverse partnership involving academic researchers, a non-profit community-based organization, its affiliated broader-based community coalition, and providers and staff from two cancer centers

Phase Ia/b Study of Giredestrant ± Palbociclib and ± Luteinizing Hormone-Releasing Hormone Agonists in Estrogen Receptor–Positive, HER2-Negative, Locally Advanced/Metastatic Breast Cancer

Purpose: Giredestrant is an investigational next-generation, oral, selective estrogen receptor antagonist and degrader for the treatment of estrogen receptor-positive (ER+) breast cancer. We present the primary analysis results of the phase Ia/b GO39932 study (NCT03332797).Patients and Methods: Patients with ER+, HER2-negative locally advanced/metastatic breast cancer previously treated with endocrine therapy received single-agent giredestrant (10, 30, 90, or 250 mg), or giredestrant (100 mg) +/- palbociclib 125 mg +/- luteinizing hormone-releasing hormone (LHRH) agonist. Detailed cardiovascular assessment was conducted with giredestrant 100 mg. Endpoints included safety (primary), pharmacokinetics, pharmacodynamics, and efficacy.Results: As of January 28, 2021, with 175 patients enrolled, no dose-limiting toxicity was observed, and the MTD was not reached. Adverse events (AE) related to giredestrant occurred in 64.9% and 59.4% of patients in the single-agent +/- LHRH agonist and giredestrant + palbociclib +/- LHRH agonist cohorts, respectively (giredestrant-only-related grade 3/4 AEs were reported in 4.5% of patients across the single-agent cohorts and 3.1% of those with giredestrant + palbociclib). Dose-dependent asymptomatic bradycardia was observed, but no clinically significant changes in cardiac-related outcomes: heart rate, blood pressure, or exercise duration. Clinical benefit was observed in all cohorts (48.6% of patients in the single-agent cohort and 81.3% in the giredestrant + palbociclib +/- LHRH agonist cohort), with no clear dose relationship, including in patients with ESR1-mutated tumors.Conclusions: Giredestrant was well tolerated and clinically active in patients who progressed on prior endocrine therapy. Results warrant further evaluation of giredestrant in randomized trials in early- and late-stage ER+ breast cancer

Beyond adoption: A new framework for theorising and evaluating Non-adoption, Abandonment and challenges to Scale-up, Spread and Sustainability (NASSS) of health and care technologies

© 2017 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be included.Background: Many promising technological innovations in health and social care are characterized by nonadoption or abandonment by individuals or by failed attempts to scale up locally, spread distantly, or sustain the innovation long term at the organization or system level. Objective: Our objective was to produce an evidence-based, theory-informed, and pragmatic framework to help predict and evaluate the success of a technology-supported health or social care program. Methods: The study had 2 parallel components: (1) secondary research (hermeneutic systematic review) to identify key domains, and (2) empirical case studies of technology implementation to explore, test, and refine these domains. We studied 6 technology-supported programs—video outpatient consultations, global positioning system tracking for cognitive impairment, pendant alarm services, remote biomarker monitoring for heart failure, care organizing software, and integrated case management via data sharing—using longitudinal ethnography and action research for up to 3 years across more than 20 organizations. Data were collected at micro level (individual technology users), meso level (organizational processes and systems), and macro level (national policy and wider context). Analysis and synthesis was aided by sociotechnically informed theories of individual, organizational, and system change. The draft framework was shared with colleagues who were introducing or evaluating other technology-supported health or care programs and refined in response to feedback. Results: The literature review identified 28 previous technology implementation frameworks, of which 14 had taken a dynamic systems approach (including 2 integrative reviews of previous work). Our empirical dataset consisted of over 400 hours of ethnographic observation, 165 semistructured interviews, and 200 documents. The final nonadoption, abandonment, scale-up, spread, and sustainability (NASSS) framework included questions in 7 domains: the condition or illness, the technology, the value proposition, the adopter system (comprising professional staff, patient, and lay caregivers), the organization(s), the wider (institutional and societal) context, and the interaction and mutual adaptation between all these domains over time. Our empirical case studies raised a variety of challenges across all 7 domains, each classified as simple (straightforward, predictable, few components), complicated (multiple interacting components or issues), or complex (dynamic, unpredictable, not easily disaggregated into constituent components). Programs characterized by complicatedness proved difficult but not impossible to implement. Those characterized by complexity in multiple NASSS domains rarely, if ever, became mainstreamed. The framework showed promise when applied (both prospectively and retrospectively) to other programs.Peer reviewe

A genome-wide association study of severe asthma exacerbations in Latino children and adolescents

Severe asthma exacerbations are a major cause of school absences and healthcare costs in children, particularly those in high-risk racial/ethnic groups. To identify susceptibility genes for severe asthma exacerbations in Latino children and adolescents, we conducted a meta-analysis of genome-wide association studies (GWAS) in 4010 Latino youth with asthma in four independent cohorts, including 1693 Puerto Ricans, 1019 Costa Ricans, 640 Mexicans, 256 Brazilians, and 402 members of other Latino subgroups. We then conducted methylation quantitative trait locus (mQTL), expression quantitative trait locus (eQTL), and expression quantitative trait methylation (eQTM) analyses to assess whether the top SNP in the meta-analysis is linked to DNA methylation and gene expression in nasal (airway) epithelium in separate cohorts of Puerto Rican and Dutch children and adolescents. In the meta-analysis of GWAS, a SNP in FLJ22447 (rs2253681) was significantly associated with 1.55 increased odds of severe asthma exacerbations (95% confidence interval=1.34 to 1.79, p=6.3×10-9). This SNP was significantly associated with DNA methylation of a CpG site (cg25024579) at the FLJ22447 locus, which was in turn associated with increased expression of KCNJ2-AS1 in nasal airway epithelium from Puerto Rican children and adolescents (β=0.10, p=2.18×10-7). Thus, SNP rs2253681 was significantly associated with both DNA methylation of a cis-CpG in FLJ22447 and severe asthma exacerbations in Latino youth. This may be partly explained by changes in airway epithelial expression of a gene recently implicated in atopic asthma in Puerto Rican children and adolescents (KCNJ2-AS1)

Status and prospects for renewable energy using wood pellets from the southeastern United States

The ongoing debate about costs and benefits of wood-pellet based bioenergy production in the southeastern United States (SE USA) requires an understanding of the science and context influencing market decisions associated with its sustainability. Production of pellets has garnered much attention as US exports have grown from negligible amounts in the early 2000s to 4.6 million metric tonnes in 2015. Currently, 98% of these pellet exports are shipped to Europe to displace coal in power plants. We ask, ‘How is the production of wood pellets in the SE USA affecting forest systems and the ecosystem services they provide?’ To address this question, we review current forest conditions and the status of the wood products industry, how pellet production affects ecosystem services and biodiversity, and what methods are in place to monitor changes and protect vulnerable systems. Scientific studies provide evidence that wood pellets in the SE USA are a fraction of total forestry operations and can be produced while maintaining or improving forest ecosystem services. Ecosystem services are protected by the requirement to utilize loggers trained to apply scientifically based best management practices in planning and implementing harvest for the export market. Bioenergy markets supplement incomes to private rural landholders and provide an incentive for forest management practices that simultaneously benefit water quality and wildlife and reduce risk of fire and insect outbreaks. Bioenergy also increases the value of forest land to landowners, thereby decreasing likelihood of conversion to nonforest uses. Monitoring and evaluation are essential to verify that regulations and good practices are achieving goals and to enable timely responses if problems arise. Conducting rigorous research to understand how conditions change in response to management choices requires baseline data, monitoring, and appropriate reference scenarios. Long-term monitoring data on forest conditions should be publicly accessible and utilized to inform adaptive management

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London