A simple DNA stretching method for fluorescence imaging of single DNA molecules

Stretching or aligning DNA molecules onto a surface by means of molecular combing techniques is one of the critical steps in single DNA molecule analysis. However, many of the current studies have focused on λ-DNA, or other large DNA molecules. There are very few studies on stretching methodologies for DNA molecules generated via PCR (typically smaller than 20 kb). Here we describe a simple method of stretching DNA molecules up to 18 kb in size on a modified glass surface. The very low background fluorescence allows efficient detection of single fluorescent dye labels incorporated into the stretched DNA molecules

Rapid DNA mapping by fluorescent single molecule detection

DNA mapping is an important analytical tool in genomic sequencing, medical diagnostics and pathogen identification. Here we report an optical DNA mapping strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs on the molecules. Individual genomic DNA molecules were labeled with fluorescent dyes at specific sequence motifs by the action of nicking endonuclease followed by the incorporation of dye terminators with DNA polymerase. The labeled DNA molecules were then stretched into linear form on a modified glass surface and imaged using total internal reflection fluorescence (TIRF) microscopy. By determining the positions of the fluorescent labels with respect to the DNA backbone, the distribution of the sequence motif recognized by the nicking endonuclease can be established with good accuracy, in a manner similar to reading a barcode. With this approach, we constructed a specific sequence motif map of lambda-DNA. We further demonstrated the capability of this approach to rapidly type a human adenovirus and several strains of human rhinovirus

The impact of classroom design on pupils' learning: Final results of a holistic, multi-level analysis

Assessments have been made of 153 classrooms in 27 schools in order to identify the impact of the physical classroom features on the academic progress of the 3766 pupils who occupied each of those specific spaces. This study confirms the utility of the naturalness, individuality and stimulation (or more memorably, SIN) conceptual model as a vehicle to organise and study the full range of sensory impacts experienced by an individual occupying a given space. In this particular case the naturalness design principle accounts for around 50% of the impact on learning, with the other two accounting for roughly a quarter each. Within this structure, seven key design parameters have been identified that together explain 16% of the variation in pupils' academic progress achieved. These are Light, Temperature, Air Quality, Ownership, Flexibility, Complexity and Colour. The muted impact of the whole-building level of analysis provides some support for the importance of “inside-out design”. The identification of the impact of the built environment factors on learning progress is a major new finding for schools' research, but also suggests that the scale of the impact of building design on human performance and wellbeing in general, can be isolated and that it is non-trivial. It is argued that it makes sense to capitalise on this promising progress and to further develop these concepts and techniques

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Development and Validation of a Symptom-Based Activity Index for Adults With Eosinophilic Esophagitis

Standardized instruments are needed to assess the activity of eosinophilic esophagitis (EoE), to provide endpoints for clinical trials and observational studies. We aimed to develop and validate a patient-reported outcome (PRO) instrument and score, based on items that could account for variations in patients’ assessments of disease severity. We also evaluated relationships between patients’ assessment of disease severity and EoE-associated endoscopic, histologic, and laboratory findings

Toward Understanding the Healthcare Value of Veterans Affairs' Primary Care – Mental Health Integration

Access to high-quality mental health specialty care for primary care patients has historically been problematic. Behavioral health care integration into the patient-centered medical homes has been shown to be effective, but dissemination and implementation of these team-based models of care remain challenging to healthcare systems. As such, lessons can be learned from VA's national implementation of Primary Care–Mental Health Integration (PC-MHI). Here we devise and validate a new metric for VA clinic engagement in PC-MHI (number of PC-MHI service users/number of primary care patients) during each year.This is a retrospective longitudinal cohort study of 112,737 primary care patients in 29 Southern California VA clinics from October 1, 2008 to September 30, 2013. First, we examined a subset of 66,638 patients with mental health diagnoses to understand access to mental health care and other healthcare services. Our multilevel regression models used clinic PC-MHI engagement to predict relative rates for the full-range of patient healthcare utilization and total VA costs, adjusting for year and clinic fixed effects, other clinic interventions, and patient characteristics. Then, we constructed quality metrics for 12,663 patients who were newly diagnosed to have depression. Our fully-adjusted regression models used clinic PC-MHI engagement to predict probabilities of follow-up within 84 and 180 days and receipt of minimally appropriate treatment for these patients. Greater clinic PC-MHI engagement was associated with significantly more mental health visits and less non-primary care based mental health specialty (MHS) visits, consistent with a substitution of PC-MHI visits for MHS visits. It was associated with less general MHS visits, rather than more specialized MHS visits. This reduction appeared targeted at patients with mild-to-moderate mental illnesses (i.e., depression), rather than with serious mental illness (i.e., schizophrenia, bipolar disorder). Despite shifting mental health care for Veterans with less complicated mental illnesses from specialty to primary care, our findings demonstrated no difference in depression care quality. We did not find adverse impacts on ED visits, hospitalizations, total patient costs, or mortality. Therefore, PC-MHI may improve mental healthcare value for primary care patients, as it may have improved realized accessibility to mental health care without necessarily increasing costs

Toward Understanding the Healthcare Value of Veterans Affairs' Primary Care – Mental Health Integration

Access to high-quality mental health specialty care for primary care patients has historically been problematic. Behavioral health care integration into the patient-centered medical homes has been shown to be effective, but dissemination and implementation of these team-based models of care remain challenging to healthcare systems. As such, lessons can be learned from VA's national implementation of Primary Care–Mental Health Integration (PC-MHI). Here we devise and validate a new metric for VA clinic engagement in PC-MHI (number of PC-MHI service users/number of primary care patients) during each year.This is a retrospective longitudinal cohort study of 112,737 primary care patients in 29 Southern California VA clinics from October 1, 2008 to September 30, 2013. First, we examined a subset of 66,638 patients with mental health diagnoses to understand access to mental health care and other healthcare services. Our multilevel regression models used clinic PC-MHI engagement to predict relative rates for the full-range of patient healthcare utilization and total VA costs, adjusting for year and clinic fixed effects, other clinic interventions, and patient characteristics. Then, we constructed quality metrics for 12,663 patients who were newly diagnosed to have depression. Our fully-adjusted regression models used clinic PC-MHI engagement to predict probabilities of follow-up within 84 and 180 days and receipt of minimally appropriate treatment for these patients. Greater clinic PC-MHI engagement was associated with significantly more mental health visits and less non-primary care based mental health specialty (MHS) visits, consistent with a substitution of PC-MHI visits for MHS visits. It was associated with less general MHS visits, rather than more specialized MHS visits. This reduction appeared targeted at patients with mild-to-moderate mental illnesses (i.e., depression), rather than with serious mental illness (i.e., schizophrenia, bipolar disorder). Despite shifting mental health care for Veterans with less complicated mental illnesses from specialty to primary care, our findings demonstrated no difference in depression care quality. We did not find adverse impacts on ED visits, hospitalizations, total patient costs, or mortality. Therefore, PC-MHI may improve mental healthcare value for primary care patients, as it may have improved realized accessibility to mental health care without necessarily increasing costs