Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Analysis of Human Accelerated DNA Regions Using Archaic Hominin Genomes

Several previous comparisons of the human genome with other primate and vertebrate genomes identified genomic regions that are highly conserved in vertebrate evolution but fast-evolving on the human lineage. These human accelerated regions (HARs) may be regions of past adaptive evolution in humans. Alternatively, they may be the result of non-adaptive processes, such as biased gene conversion. We captured and sequenced DNA from a collection of previously published HARs using DNA from an Iberian Neandertal. Combining these new data with shotgun sequence from the Neandertal and Denisova draft genomes, we determine at least one archaic hominin allele for 84% of all positions within HARs. We find that 8% of HAR substitutions are not observed in the archaic hominins and are thus recent in the sense that the derived allele had not come to fixation in the common ancestor of modern humans and archaic hominins. Further, we find that recent substitutions in HARs tend to have come to fixation faster than substitutions elsewhere in the genome and that substitutions in HARs tend to cluster in time, consistent with an episodic rather than a clock-like process underlying HAR evolution. Our catalog of sequence changes in HARs will help prioritize them for functional studies of genomic elements potentially responsible for modern human adaptations

Ecoregional Analysis of Nearshore Sea-Surface Temperature in the North Pacific

The quantification and description of sea surface temperature (SST) is critically important because it can influence the distribution, migration, and invasion of marine species; furthermore, SSTs are expected to be affected by climate change. To better understand present temperature regimes, we assembled a 29-year nearshore time series of mean monthly SSTs along the North Pacific coastline using remotely-sensed satellite data collected with the Advanced Very High Resolution Radiometer (AVHRR) instrument. We then used the dataset to describe nearshore (<20 km offshore) SST patterns of 16 North Pacific ecoregions delineated by the Marine Ecoregions of the World (MEOW) hierarchical schema. Annual mean temperature varied from 3.8°C along the Kamchatka ecoregion to 24.8°C in the Cortezian ecoregion. There are smaller annual ranges and less variability in SST in the Northeast Pacific relative to the Northwest Pacific. Within the 16 ecoregions, 31–94% of the variance in SST is explained by the annual cycle, with the annual cycle explaining the least variation in the Northern California ecoregion and the most variation in the Yellow Sea ecoregion. Clustering on mean monthly SSTs of each ecoregion showed a clear break between the ecoregions within the Warm and Cold Temperate provinces of the MEOW schema, though several of the ecoregions contained within the provinces did not show a significant difference in mean seasonal temperature patterns. Comparison of these temperature patterns shared some similarities and differences with previous biogeographic classifications and the Large Marine Ecosystems (LMEs). Finally, we provide a web link to the processed data for use by other researchers

Barriers and facilitators to the implementation of osteoarthritis management programmes in primary or community care settings: a systematic review and qualitative framework synthesis protocol.

Despite consistent international guidelines for osteoarthritis (OA) management, evidence-based treatments are underutilised. OA management programmes (OAMPs) are being implemented internationally to address this evidence-practice gap. An OAMP is defined as a 'model of evidence-based, non-surgical OA care that has been implemented in a real-world setting'. Our objective is to identify, synthesise and appraise qualitative research identifying anticipated or experienced micro (individual/behavioural), meso (organisational) or macro (context/system) level barriers or facilitators to the implementation of primary or community care-based OAMPs. Five electronic databases will be searched for papers published between 2010 and 2021. Qualitative or mixed-methods studies that include qualitative data on the anticipated or experienced barriers or facilitators to the implementation of primary or community care-based OAMPs, from the perspective of service users or service providers, will be included. The review will be reported using the PRISMA and ENTREQ guidelines. A data extraction form will be used to provide details of the included studies. Data will be analysed and identified barriers and facilitators will be mapped onto an appropriate implementation framework, such as the Theoretical Domains Framework. The appropriate JBI critical appraisal tools will be used to assess methodological quality, while the GRADE-CERQual approach will be used to assess confidence in the findings. Translation of evidence-based guidelines into practice is challenging and reliant on the quality of implementation. By comparing and contrasting anticipated and experienced barriers, this review will determine the extent of congruence between the two, and provide valuable insights into the views and experiences of key stakeholders involved in the implementation of OAMPs. The mapping of identified barriers and facilitators to behaviour change theory will enhance the applicability and construct validity of our findings and will offer significant utility for future development and implementation of OAMPs. Registration: This protocol was registered with PROSPERO (CRD42021255698) on 15/07/21

К вопросу формирования маркетинговой компетентности студента вуза

Interview topic guide. This document is the topic guide used by the researcher to structure all participant interviews. (DOCX 15 kb

More frequent, more costly? Health economic modelling aspects of monitoring glaucoma patients in England

BACKGROUND: Chronic open angle glaucoma (COAG) is an age-related eye disease causing irreversible loss of visual field (VF). Health service delivery for COAG is challenging given the large number of diagnosed patients requiring lifelong periodic monitoring by hospital eye services. Yet frequent examination better determines disease worsening and speed of VF loss under treatment. We examine the cost-effectiveness of increasing frequency of VF examinations during follow-up using a health economic model. METHODS: Two different VF monitoring schemes defined as current practice (annual VF testing) and proposed practice (three VF tests per year in the first 2 years after diagnosis) were examined. A purpose written health economic Markov model is used to test the hypothesis that cost effectiveness improves by implementing proposed practice on groups of patients stratified by age and severity of COAG. Further, a new component of the model, estimating costs of visual impairment, was added. Results were derived from a simulated cohort of 10000 patients with quality-adjusted life years (QALYs) and incremental cost-effectiveness ratios (ICERs) used as main outcome measures. RESULTS: An ICER of £21,392 per QALY was derived for proposed practice improving to a value of £11,382 once savings for prevented visual impairment was added to the model. Proposed practice was more cost-effective in younger patients. Proposed practice for patients with advanced disease at diagnosis generated ICERs > £60,000 per QALY; these cases would likely be on the most intensive treatment pathway making clinical information on speed of VF loss redundant. Sensitivity analysis indicated results to be robust in relation to hypothetical willingness to pay threshold identified by national guidelines, although greatest uncertainty was allied to estimates of implementation and visual impairment costs. CONCLUSION: Increasing VF monitoring at the earliest stages of follow-up for COAG appears to be cost-effective depending on reasonable assumptions about implementation costs. Our health economic model highlights benefits of stratifying patients to more or less monitoring based on age and stage of disease at diagnosis; a prospective study is needed to prove these findings. Further, this works highlights gaps in knowledge about long term costs of visual impairment

Etiological spectrum and treatment outcome of Obstructive jaundice at a University teaching Hospital in northwestern Tanzania: A diagnostic and therapeutic challenges

Obstructive jaundice poses diagnostic and therapeutic challenges to general surgeons practicing in resource-limited countries. This study was undertaken to highlight the etiological spectrum, treatment outcome of obstructive jaundice in our setting and to identify prognostic factors for morbidity and mortality. This was a descriptive prospective study which was conducted at Bugando Medical Centre between July 2006 and June 2010. All patients with a clinical diagnosis of obstructive jaundice were, after informed consent for the study, consecutively enrolled into the study. Data were collected using a pre-tested structured questionnaire and analyzed using SPSS computer software version 11.5. A total of 116 patients were studied. Females outnumbered males by a ratio of 1.3:1. Patients with malignant obstructive jaundice were older than those of benign type. Ca head of pancreas was the commonest malignant cause of jaundice where as choledocholithiasis was the commonest benign cause. Abdominal ultrasound was the only diagnostic imaging done in all patients and revealed dilated intra and extra-hepatic ducts, common bile stones and abdominal masses in 56.2%, 78.9%, 58.1% and 72.4% of the cases respectively. A total of 110 (94.8%) patients underwent surgical treatment and the remaining 6 (5.2%) patients were unfit for surgery. The complication rate was 22.4% mainly surgical site infections. The mean hospital stay and mortality rate were 14.54 days and 15.5% respectively. A low haematocrit and presence of postoperative sepsis were the main predictors of the hospital stay (P < 0.001), whereas age > 60 years, prolonged duration of jaundice, malignant causes and presence of postoperative complications mainly sepsis significantly predicted mortality (P < 0.001). Obstructive jaundice in our setting is more prevalent in females and the cause is mostly malignant. The result of this study suggests that early diagnosis and treatment plays an important role in the prognosis of patients with obstructive jaundice

Differential sensitivity of Src-family kinases to activation by SH3 domain displacement

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. © 2014 Moroco et al

Development of a core capability framework for qualified health professionals to optimise care for people with osteoarthritis: an OARSI initiative

Summary Objective Develop a generic trans-disciplinary, skills-based capability framework for health professionals providing care for people with OA. Design e-Delphi survey. An international inter-professional Delphi Panel (researchers; clinicians; consumer representatives) considered a draft framework (adapted from elsewhere) of 131 specific capabilities mapped to 14 broader capability areas across four domains (A: person-centred approaches; B: assessment, investigation and diagnosis; C: management, interventions and prevention; D: service and professional development). Over three rounds, the Panel rated their agreement (Likert or numerical rating scales) on whether each specific capability in Domains B and C was essential (core) for all health professionals when providing care for all people with OA. Those achieving consensus (=80% of Panel) rating of = seven out of ten (Round 3) were retained. Generic domains (A and D) were included in the final framework and amended based on Panel comments. Results 173 people from 31 countries, spanning 18 disciplines and including 26 consumer representatives, participated. The final framework comprised 70 specific capabilities across 13 broad areas i) communication; ii) person-centred care; iii) history-taking; iv) physical assessment; v) investigations and diagnosis; vi) interventions and care planning; vii) prevention and lifestyle interventions; viii) self-management and behaviour change; ix) rehabilitative interventions; x) pharmacotherapy; xi) surgical interventions; xii) referrals and collaborative working; and xiii) evidence-based practice and service development). Conclusion Experts agree that health professionals require an array of skills in person-centred approaches; assessment, investigation and diagnosis; management, interventions and prevention; and service and professional development to provide optimal care for people with OA

Estimating the lifetime economic burden of stroke according to the age of onset in South Korea: a cost of illness study

<p>Abstract</p> <p>Background</p> <p>The recently-observed trend towards younger stroke patients in Korea raises economic concerns, including erosion of the workforce. We compared per-person lifetime costs of stroke according to the age of stroke onset from the Korean societal perspective.</p> <p>Methods</p> <p>A state-transition Markov model consisted of three health states ('post primary stroke event', 'alive post stroke', and 'dead') was developed to simulate the natural history of stroke. The transition probabilities for fatal and non-fatal recurrent stroke by age and gender and for non-stroke causes of death were derived from the national epidemiologic data of the Korean Health Insurance Review and Assessment Services and data from the Danish Monitoring Trends in Cardiovascular Disease study. We used an incidence-based approach to estimate the long-term costs of stroke. The model captured stroke-related costs including costs within the health sector, patients' out-of-pocket costs outside the health sector, and costs resulting from loss of productivity due to morbidity and premature death using a human capital approach. Average insurance-covered costs occurring within the health sector were estimated from the National Health Insurance claims database. Other costs were estimated based on the national epidemiologic data and literature. All costs are presented in 2008 Korean currency values (Korean won = KRW).</p> <p>Results</p> <p>The lifetime costs of stroke were estimated to be: 200.7, 81.9, and 16.4 million Korean won (1,200 KRW is approximately equal to one US dollar) for men who suffered a first stroke at age 45, 55 and 65 years, respectively, and 75.7, 39.2, and 19.3 million KRW for women at the same age. While stroke occurring among Koreans aged 45 to 64 years accounted for only 30% of the total disease incidence, this age group incurred 75% of the total national lifetime costs of stroke.</p> <p>Conclusions</p> <p>A higher lifetime burden and increasing incidence of stroke among younger Koreans highlight the need for more effective strategies for the prevention and management of stroke especially for people between 40 and 60 years of ages.</p