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Objective: Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze-thaw cycles on qPCR reagents for eDNA analysis across six assays., Results: Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of - 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations., (© 2024. The Author(s).)

Objective: Silica gel beads have promise as a non-toxic, cost-effective, portable method for storing environmental DNA (eDNA) immobilized on filter membranes. Consequently, many ecological surveys are turning to silica bead filter desiccation rather than ethanol preservation. However, no systematic evaluation of silica bead storage conditions or duration past 1 week has been published. The present study evaluates the quality of filter-immobilized eDNA desiccated with silica gel under different storage conditions for over a year using targeted quantitative real-time polymerase chain reaction (qPCR)-based assays., Results: While the detection of relatively abundant eDNA target was stable over 15 months from either ethanol- or silica gel-preserved filters at - 20 and 4 °C, silica gel out-performed ethanol preservation at 23 °C by preventing a progressive decrease in eDNA sample quality. Silica gel filter desiccation preserved low abundance eDNA equally well up to 1 month regardless of storage temperature (18, 4, or - 20 °C). However only storage at - 20 °C prevented a noticeable decrease in detectability at 5 and 12 months. The results indicate that brief storage of eDNA filters with silica gel beads up to 1 month can be successfully accomplished at a range of temperatures. However, longer-term storage should be at - 20 °C to maximize sample integrity.

The coastal tailed frog (Ascaphus truei) is endemic to the Pacific Northwest of North America and is listed as a species of Special Concern under the Canadian Species at Risk Act. Its range is limited to British Columbia where it occurs widely west of the Coast Mountain Ranges extending north almost to the Alaskan Panhandle. The present study focused on surveying within the Cayoosh, Bridge (Shulaps), Seton, Anderson, Carpenter, and Downton Lake drainages. Four years of previous inventory efforts using conventional time-constrained search (TCS) methods detected tailed frog at 23/292 discrete sites (7.9% detection rate) in seven watersheds. Non-invasive environmental DNA (eDNA) methods hold promise for cryptic and low-abundance species detection. We rigorously validated a quantitative real-time polymerase chain reaction (qPCR)-based tool for detecting coastal tailed frog eDNA in water samples. This eASTR4 test is highly specific and sensitive. We applied a two-step targeted eDNA analysis approach on duplicate filtered water samples from a total of 72 sites collected over five days. The first IntegritE-DNA step mitigates false negative results and tests all DNA samples for the ability to support amplification from endogenous plant chloroplast DNA as a measure of sample viability. Three DNA samples failed this step even after inhibitor clean up suggesting that these samples were poor quality and not reliable for targeted species' DNA analyses. All other DNA samples were deemed viable and were then tested for species-specific DNA. Coastal tailed frog eDNA was detected in 55/72 (76%) discrete stream reaches; nine sites with historical known occurrence were all eDNA positive. The false negative rate for TCS compared to eDNA methods was 58%. The results expand known coastal tailed frog distribution to 24 watersheds effectively more than tripling extant occurrences and confirm a previously suspected, apparently isolated coastal tailed frog metapopulation in the Shulaps drainage., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: JH is an employee of Hemmera Envirochem Inc. JH was involved with all aspects of the study with the exception of qPCR assay design, validation, data collection, and analysis. A patent is pending on some of the concepts presented in the manuscript (US provisional application number 62/406,802). The patent is owned by the University of Victoria who will handle commercial and academic licensing agreements. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: To determine the possible association between insomnia and risk of type 2 diabetes mellitus (T2DM) in the naturalistic clinical setting., Research Design and Methods: We conducted a retrospective cohort study to examine the risk of developing T2DM among patients with pre-diabetes with and without insomnia. Participants with pre-diabetes (identified by a physician or via two laboratory tests) between January 1, 2007 and December 31, 2015 and without sleep apnea were followed until December 31, 2016. Patients were determined to have T2DM when two of the following occurred within a 2-year window: physician-entered outpatient T2DM diagnosis (International Classification of Diseases [ICD]-9 250.00; ICD-10 E11), dispensing of an antihyperglycemia agent, and hemoglobin A1c (A1c) >6.5% (48 mmol/mol) or fasting plasma glucose (FPG) >125 mg/dL. One hospital inpatient stay with an associated T2DM diagnosis was also sufficient for classification of T2DM., Results: Our cohort consisted of 81 233 persons with pre-diabetes, 24 146 (29.7%) of whom had insomnia at some point during the 4.3-year average observation period. After adjustment for traditional risk factors, those with insomnia were 28% more likely to develop T2DM than those without insomnia (HR 1.28; 95% CI 1.24 to 1.33). The estimate was essentially unchanged after adjusting for baseline A1c level (HR 1.32; 95% CI 1.25 to 1.40) or FPG (HR 1.28; 95% CI 1.23 to 1.33)., Conclusions: Insomnia imparts an increased risk of T2DM comparable with that conferred by traditional risk factors (eg, overweight, non-white race, cardiovascular risk factors). This association could have clinical importance because it suggests a new potentially modifiable risk factor that could be targeted to prevent diabetes., Competing Interests: Competing interests: ESL has had unrelated funding from Merck in the last years. GAN has unrelated grant funding from Boehringer-Ingelheim, Merck & Co, Sanofi, Janssen Pharmaceuticals and Amarin.

Several magnetic resonance parallel imaging techniques require explicit estimates of the receive coil sensitivity profiles. These estimates must be accurate over both the object and its surrounding regions to avoid generating artifacts in the reconstructed images. Regularized estimation methods that involve minimizing a cost function containing both a data-fit term and a regularization term provide robust sensitivity estimates. However, these methods can be computationally expensive when dealing with large problems. In this paper, we propose an iterative algorithm based on variable splitting and the augmented Lagrangian method that estimates the coil sensitivity profile by minimizing a quadratic cost function. Our method, ADMM-Circ, reformulates the finite differencing matrix in the regularization term to enable exact alternating minimization steps. We also present a faster variant of this algorithm using intermediate updating of the associated Lagrange multipliers. Numerical experiments with simulated and real data sets indicate that our proposed method converges approximately twice as fast as the preconditioned conjugate gradient method over the entire field-of-view. These concepts may accelerate other quadratic optimization problems.

Background: Chronic obstructive pulmonary disease (COPD) is increasing worldwide and thus its associated morbidity and mortality. However, COPD often goes undiagnosed., Objectives: We evaluated the rate of non-diagnosed irreversible airway obstruction (AO) and characterized this patient group. We further assessed the possible effects of conducting targeted spirometry in a population sample in Salzburg, Austria, as part of the Burden of Obstructive Lung Disease (BOLD) study., Methods: 1,258 adults ≥40 years of age completed a questionnaire and performed spirometry before and after bronchodilator therapy (post-BD). Irreversible AO was defined as post-BD FEV(1)/FVC below the lower limit of normal; we used the FEV(1)% predicted (pred.) to further grade the disease. Participants without a physician diagnosis of COPD who reported respiratory symptoms and a history of risk factors (ever smoking or occupational risk) were defined as eligible for targeted spirometry., Results: 85.9% (171/199) of the participants with irreversible AO did not report a prior diagnosis of COPD. Non-diagnosed AO was inversely related to severity, age, self-reported prior respiratory diseases and cough as a respiratory symptom. 343 participants were eligible for targeted spirometry and irreversible AO was present in 86 (25.1%) participants. Therefore, targeted spirometry could reduce the underdiagnosis of irreversible AO of any severity by 50.3% (86 of 171). The diagnosis of 1 person with FEV(1) <80% pred. would require spirometry in 8.4 subjects (95% confidence interval 6.2-11.1)., Conclusion: Although several factors are associated with non-diagnosed AO, spirometry in individuals with respiratory symptoms and exposure to risk factors could reduce undiagnosed irreversible AO by half., (Copyright © 2010 S. Karger AG, Basel.)

Background: COPD is projected to be the third leading cause of death worldwide by 2020. The Burden of Obstructive Lung Disease initiative was started to measure the prevalence of COPD in a standardized way and to provide estimates of the social and economic burden of disease., Methods: We surveyed a gender-stratified, population-based sample of 2,200 adults >or= 40 years of age. The findings of prebronchodilator and postbronchodilator spirometry, as well as information on smoking and reported respiratory disease was recorded. Irreversible airflow obstruction was defined as a postbronchodilator FEV(1)/FVC ratio of < 0.70., Results: For 1,258 participants with good-quality postbronchodilator spirometry findings, the overall prevalence of COPD at stage I or higher was 26.1%, and was equal in men and women. The prevalence of COPD stage II or higher (FEV(1)/FVC ratio, < 0.7; FEV(1), < 80% predicted) was 10.7%. The prevalence of COPD stage I+, and COPD stage II+, increased with age and cigarette smoking. A doctor diagnosis of COPD was reported by only 5.6% of participants., Conclusion: One quarter of residents of Salzburg County (Austria) who were >or= 40 years of age had at least mild irreversible airflow obstruction. The high prevalence of COPD highlights the impending health-care crisis that will affect many countries as a result of this greatly underappreciated condition.

Most information concerning possible cancer risks attributable to lifetime exposure to diagnostic x-rays comes from studies in which x-ray history was ascertained by interview or questionnaire, but little is known about the accuracy of such information. The authors assessed agreement between medical x-ray histories obtained through interview and by review of medical records from thyroid cancer case-control studies conducted in Sweden (1985-1992; 123 cases and 123 controls) and from members of a prepaid health plan in the United States (1986-1991; 50 cases and 50 controls). In both studies, substantial disagreement was found between the numbers of x-ray examinations reported in the interview and in the medical records. There was an indication of relatively poorer reporting among controls, particularly for certain types of x-ray examinations and for large numbers of such examinations. Estimates of the risk associated with exposure to diagnostic x-rays were similar, regardless of whether interview or medical record data were used, even though ordinal dose classifications based on the two sources differed considerably. In populations with a high frequency of exposure, spurious associations with numbers of x-ray examinations or estimated thyroid dose might arise because of differences in recall. However, in the present data, reporting errors by cases and controls seemed to be largely nondifferential.