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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cryptic, fossorial snake distribution is challenging to determine. • Environmental DNA (eDNA) may solve this but there are few terrestrial applications. • A new sharp-tailed snake eDNA assay was developed and validated. • eDNA was measured from swabs and soil under artificial cover objects. • Detection rate was an order of magnitude better than conventional surveys. Performing ecological surveys for secretive, fossorial snakes is challenging. Traditional survey methods involve visual observation under artificial cover objects (ACOs); this is labor-intensive and requires multiple consistent surveys of suitable habitats. Detection of snake DNA deposited under ACOs represents an innovative method for species detection. However, for terrestrial species, common issues with soil-based methods include the challenges of adequately removing enzyme inhibitors that reduce environmental DNA (eDNA) detection and potential photodegradation of DNA taken from surface samples. These issues may be circumvented by obtaining swabs and soil samples directly from the underside of ACOs for eDNA analysis. We demonstrate the application of this method in surveys of sharp-tailed snake (Contia tenuis), an endangered species under the Canadian Species at Risk Act. We describe the design and validation of a new quantitative real-time polymerase chain reaction (qPCR)-based eDNA eCOTE3 assay with high specificity and sensitivity for sharp-tailed snake. We developed a practical and robust protocol for obtaining eDNA samples by swabbing the underside of ACOs and collecting soil samples under ACOs. Traditional surveys were conducted over two successive years (2018–19) on 220 paired ACOs at 110 sites monitored between 12 and 30 times each. Of the 6,060 ACO visits, only 24 resulted in sharp-tailed snake observations (0.4% success rate) illustrating the considerable difficulty in detecting these snakes. During this same time, 109 swabs were taken directly from the undersides of ACOs and 78 soil samples were collected from a subset of these ACOs. Of the 24 occurrences where sharp-tailed snakes were visually observed, 13 of 23 ACO swabs (57%) and nine of 20 soil samples (45%) tested positive for DNA. eDNA deposition is likely low because of the small size and behavior of this cryptic species, yet DNA was detected from soil exposed to captured snakes for only 10 min. Nevertheless, sharp-tailed snake eDNA was detected at eight sites (9%) from ACO swabs (n = 86) and seven sites (13%) from soil samples (n = 56) where snakes were not observed. This is an overall detection rate of 25% (14/56) for swab and soil samples testing positive in sites where both were tested, representing a substantial reduction in the effort required for detection of this species. Given the time-consuming nature of traditional surveys, eDNA holds great promise as a complementary survey tool for this terrestrial species. While further work is needed to delineate DNA deposition rates, this work represents a significant advance in monitoring a challenging species. [ABSTRACT FROM AUTHOR]

A current challenge for environmental DNA (eDNA) applications is how to account for an environmental (or false-positive) background in surveys. We performed two controlled experiments in the Goldstream Hatchery in British Columbia using a validated coho salmon (Oncorhynchus kisutch) eDNA assay (eONKI4). In the density experiment at high copy number, eDNA in 2 L water samples was measured from four 10 kL tanks containing 1 to 65 juvenile coho salmon. At these densities, we obtained a strong positive 1:1 relationship between predicted copy number/L and coho salmon biomass (g/L). The dilution experiment simulated a situation where fish leave a pool environment, and water from upstream continues to flow through at rates of 141–159 L/min. Here, three coho salmon were placed in four 10 kL tanks, removed after nine days, and the amount of remaining eDNA was measured at times coinciding with dilutions of 20, 40, 80, 160, and 1000 kL. The dilution experiment demonstrates a novel method using Binomial–Poisson distributions to detect target species eDNA at low copy number in the presence of an environmental background. This includes determination of the limit of blank with background (LOB-B) with a controlled false positive rate, and limit of detection with background (LOD-B) with a controlled false negative rate, which provides a statistically robust "Detect" or "No Detect" assessment for eDNA surveys. [ABSTRACT FROM AUTHOR]

Combined use of sedimentary DNA (sedDNA) and Indigenous Knowledge in reconstructing historical fish records. [Display omitted] • Indigenous peoples have a long-term link with Cowpar Lake in the Canadian Oil Sands. • Sedimentary DNA (sedDNA) from sediment core acts as an archive of lake history. • qPCR-based environmental DNA methods reconstructed historical fish occupation. • SedDNA results aligned with Indigenous Knowledge (IK) on fish communities. • Combined sedDNA and IK give powerful insight into natural and human-mediated impact. Lake sediment serves as a natural archive of historical biological information. The use of sedimentary DNA (sedDNA), a form of environmental DNA (eDNA) shed by aquatic organisms and preserved in sediment, has been instrumental in reconstructing past faunal composition in aquatic communities. However, the low abundance of fish sedDNA and the often humic-rich nature of lake sediments create methodological challenges for the accurate detection of target sedDNA using quantitative polymerase chain reaction (qPCR)-based approaches. Herein, we present a consolidated qPCR-based eDNA workflow to reconstruct past and current fish fauna in Cowpar Lake located in the Oil Sands region in Alberta (Canada), which were then validated using Indigenous Knowledge from Chipewyan Prairie First Nation community members. The present study highlights the importance of combining column- and precipitation-based PCR inhibitor clean-up, nucleic acid concentration, incorporating endogenous chloroplast DNA as a sample integrity control. Robust qPCR-based eDNA assays were also useful in preventing the false-negative detection of low copies of target fish DNA. The presence of Northern pike (1905 to 2019) and Cisco (1919 to 1942) in Cowpar Lake was confirmed based on detected sedDNA from sediment core. The reconstructed fish records from sedDNA-inferred data aligned with the Indigenous accounts of natural and human-mediated changes in land use around the lake. Overall, the present study addresses common methodological concerns in processing lake sediment samples for fish eDNA detection and demonstrates the great potential of combined eDNA-inferred data and Indigenous Knowledge in reconstructing historical fish records in aquatic communities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Forage fish are keystones of the coastal marine food web. • Pacific Sand Lance and Surf Smelt habitat use data are difficult to obtain. • Environmental DNA (eDNA) may improve data from visual egg surveys from sand samples. • Novel eDNA assays for both species were developed and validated. • Detection rate shows promise for broad application of eDNA for forage fish. Many coastal forage fish species spend most of their life history using the water column, but some species such as Pacific Sand Lance (Ammodytes personatus) and Surf Smelt (Hypomesus pretiosus) have a requirement for intertidal sediments for spawning and or burying. Detection of suitable but typically uncommon intertidal habitats for spawning has historically relied on extensive visual surveys for eggs from large sand samples. In the present study, we developed and validated two new non-destructive qPCR-based tools for detecting Sand Lance and Surf Smelt eDNA from small sand samples. A total of 279 composite transect sand samples were collected from 101 beaches and were tested for eDNA sample integrity using the IntegritE-DNATM test. Initial testing showed that all but 42 of 46 samples passed after an inhibitor clean-up step. Of the 101 beaches sampled 275 times, there were 111 detections of Sand Lance on 52 beaches, and there were 13 detections of Surf Smelt on 10 beaches. One hundred and eighty-one samples were paired with visual egg counts and 76 (42%) of these tested positive for Sand Lance while 9 of 132 (7%) samples tested positive for Surf Smelt. For the 27 paired samples with two or more Sand Lance eggs visually detected there was a significant positive correlation (r = 0.694, p = 0.0001) with eDNA copies per liter suggesting that a larger Sand Lance presence is easily and reliably detected using eDNA. The methods and approaches described herein will serve as a foundation for broad application of eDNA approaches for forage fish species that utilize intertidal benthic habitats. [ABSTRACT FROM AUTHOR]

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 FEV1/FVC below the lower limit of normal; we used the FEV1% 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 FEV1 <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 [ABSTRACT FROM AUTHOR]

The article discusses the results of the Burden of Obstructive Lung Disease (BOLD) study in Salzburg, Austria. The BOLD study was started to provide estimates of the social and economic burden of chronic obstructive pulmonary disease. The study found that one quarter of residents of Salzburg who are ≥ 40 years of age had at least mild irreversible airflow obstruction.

Introduction: The Kaiser Permanente ASCVD Risk Estimator (KPARE) was developed to address the systematic overestimation of actual ASCVD risk by the ACC/AHA ASCVD Pooled Cohort Equation (ACC/AHA PCE) in contemporary populations. We evaluated the accuracy and generalizability of KPARE for estimating 10-year ASCVD risk in eligible primary prevention, community-based populations in the U.S. and Canada, as compared with the ACC/AHA PCE. Methods: We identified adults 40-79 years old in 3 contemporary U.S. populations (Kaiser Permanente Northern California (KPNC), Southern California (KPSC) and Northwest (KPNW), as well as adults 66-79 years old in Ontario, Canada who had LDL-C 70-189 mg/dL, no known ASCVD, diabetes or lipid-lowering therapy and had complete follow-up. Non-fatal and fatal ASCVD events were ascertained from health system electronic health records and death certificates using validated algorithms. We examined observed ASCVD event rates within deciles of predicted 10-year risk using the KPARE vs. the ACC/AHA PCE predicted risk, and calculated metrics of discrimination and calibration. Results: In eligible primary prevention validation cohorts of KPNC (N=151,409), KPSC (N=246,815), KPNW (N=46,784), and Ontario, Canada (N=60,612 adults), the KPARE showed both better discrimination and calibration (Figure) compared to the ACC/AHA PCE to predict 10-year risk of ASCVD events, with c-statistics of 0.78, 0.78, 0.79, and 0.65, respectively. Categorical net reclassification index showed the KPARE improved ASCVD risk prediction over the ACC/AHA PCE by 23% in the KPNC validation cohort, 24% in KPSC, 33% in KPNW, and 25% in Ontario, Canada. Results were similar when stratified by race-gender subgroups. Conclusions: The new Kaiser Permanente ASCVD Risk Estimator provides more accurate ASCVD risk estimates in various community populations than the ACC/AHA PCE and may provide greater utility in shared decision-making for primary prevention strategies. [ABSTRACT FROM AUTHOR]