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Abstract 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.

The recovery of DNA preserved in the sediment of aquatic systems (sedDNA) has provided short-term and long-term data on biological groups (e.g., bacteria, phytoplankton, zooplankton, and fishes) and has advanced our understanding of how environmental changes have affected aquatic communities. Here, we demonstrate the use of fish sedDNA from lake sediment cores in reconstructing the history of fish occurrences in four lakes within the Oil Sands region in Alberta, Canada. A general fish and eight targeted qPCR-based eDNA assays for freshwater fish species (cisco, lake whitefish, northern pike, chain pickerel, burbot, rainbow trout, lake trout, and walleye) were rigorously designed and validated. Utilizing these assays, we detected sedDNA from several fish species in sediment cores spanning over a century, which aligned with conventional fish surveys and historical records of human-mediated introductions. The use of fish sedDNA provided greater temporal resolution into the historical fish faunal records, bridging the knowledge gap that spanned from 100- to 150-year-old data. The present study also allowed documentation of human-mediated introductions of fish populations to the examined lakes. These findings can be used to refine native freshwater fish ranges and clarify the influence of human-mediated introductions on fish diversity in lakes, providing essential baseline data for conservation management and environmental impact assessments.

Background: This paper describes the development of low-cost, effective, non-invasive machine learning-based prediction models for Down Syndrome in the first two trimesters of pregnancy in Vietnam. These models are adaptable to different situations with limited screening capacities at community-based healthcare facilities. Method: Ultrasound and biochemical testing alone and in combination, from both trimesters were employed to build prediction models based on k-Nearest Neighbor, Support Vector Machine, Random Forest, and Extreme Gradient Boosting algorithms. Results: A total of 7,076 pregnant women from a single site in Northern Vietnam were included, and 1,035 had a fetus with Down Syndrome. Combined ultrasound and biochemical testing were required to achieve the highest accuracy in trimester 2, while models based only on biochemical testing performed as well as models based on combined testing during trimester 1. In trimester 1, Extreme Gradient Boosting produced the best model with 94% accuracy and 88% AUC, while Support Vector Machine produced the best model in trimester 2 with 89% accuracy and 84% AUC. Conclusions: This study explored a range of machine learning models under different testing scenarios. Findings point to the potential feasibility of national screening, especially in settings without enough equipment and specialists, after additional model validation and fine tuning is performed.

Summary: Background: Approximately 20% of adults in Vietnam have hypertension, and management of this chronic condition remains challenging. This study aimed to assess the effectiveness of a multicomponent intervention in reducing blood pressure (BP) in adults with uncontrolled hypertension. Methods: This cluster randomised controlled trial was conducted in sixteen communities (8 intervention and 8 comparison) in a rural setting in Vietnam (2017–2022). Consenting adults (aged ≥ 18 years) with uncontrolled hypertension were enrolled. Sixteen communities were equally randomised to an intervention or comparison group stratified by district. Consenting adults were assigned to study groups by community. The comparison arm received training sessions about hypertension prevention and management for health workers, and patient education materials. The intervention arm received training sessions about hypertension prevention and management for health workers, and patient education materials and three enhancement components including a storytelling intervention, home BP self-monitoring, and expanded community health worker services. The primary outcome was the difference in changes in patient's levels of systolic BP between the study groups over a 12-month follow-up period. Patients and outcome assessors were masked. Findings: A total of 671 patients (340: intervention, 331: comparison) were enrolled in the trial. The mean age was 66 years and 45% were men. At the 12-month follow-up, the mean systolic BP declined by 18.4 mmHg in the intervention group and 3.7 mmHg in the comparison group (differential decline of 14.7 mmHg [95% CI: 11.8–17.6]). The intervention group also achieved better BP control and medication adherence than the comparison group. There were no serious adverse events related to study participation. Interpretation: The results of this trial demonstrate that a multicomponent intervention can effectively reduce elevated BP in individuals with uncontrolled hypertension in Vietnam. Trial registration: This trial was registered at ClinicalTrials.gov, NCT03590691. Funding: National Heart, Lung, and Blood Institute.

BackgroundIntrauterine inflammation and the requirement for mechanical ventilation independently increase the risk of perinatal brain injury and adverse neurodevelopmental outcomes. We aimed to investigate the effects of mechanical ventilation for 24 h, with and without prior exposure to intrauterine inflammation, on markers of brain inflammation and injury in the preterm sheep brain.MethodsChronically instrumented fetal sheep at ~115 days of gestation were randomly allocated to receive a single intratracheal dose of 1 mg lipopolysaccharide (LPS) or isovolumetric saline, then further randomly allocated 1 h after to receive mechanical ventilation with room air or no mechanical ventilation (unventilated control + saline [UVC, n = 7]; in utero mechanical ventilation + saline [VENT, n = 8], unventilated control + intratracheal LPS [UVC + LPS, n = 7]; in utero ventilation + intratracheal LPS [VENT + LPS, n = 7]). Serial fetal blood and plasma samples were collected throughout the experimental protocol for assessment of blood biochemistry and plasma interleukin (IL)-6 levels. After 24 h of mechanical ventilation, fetal brains were collected for RT-qPCR and immunohistochemical analyses.ResultsLPS exposure increased numbers of microglia and upregulated pro-inflammatory related genes within the cortical gray matter (GM) and subcortical white matter (SCWM) (pLPS

Background: Predictive biomarkers for immune checkpoint blockade in the second-line treatment of metastatic renal cell carcinoma (mRCC) are lacking. Materials and methods: Patients with histologically confirmed RCC who started nivolumab after at least 4 months of tyrosine kinase inhibitors (TKIs) were recruited for this study. Serial tissue and blood samples were collected for immune biomarker evaluation. The primary endpoint was to determine the association of specific T-cell subsets with clinical outcomes tested using Wilcoxon rank sum for clinical benefit rate (CBR) and log-rank test for progression-free survival (PFS). Results: Twenty patients were included in this trial with a median age of 64 years and followed-up for a median of 12 months. The median PFS for patients who received TKI was 13.8 months, while for those subsequently treated with nivolumab following TKI therapy, the median PFS was 2.6 months. CBR of nivolumab was 20% with two partial responses. Functionally active programmed cell death protein 1+ CD4+ T cells were enriched in non-responders (q = 0.003) and associated with worse PFS on nivolumab (P = 0.04). Responders showed a significant reduction in the effector CD4+T-cell (TEF) fraction compared to non-responders at 3 months on nivolumab (0.40 versus 0.80, P = 0.0005). CD127+CD4+ T cells were enriched in patients who developed immune-related adverse effects (q = 0.003). Using in-house validated multiplex immunohistochemistry for six markers, we measured tumour-associated immune cell densities in tissue samples. Responders to nivolumab showed a significantly higher mean of immune cell densities in tissue samples compared to non-responders (346 versus 87 cells/mm2, P = 0.04). Conclusions: In this small study, analysis of tissue-based and peripheral blood immune cell subsets predicted clinical outcomes of nivolumab. Further studies are warranted with larger populations to validate these observations.

Abstract Targeted environmental DNA (eDNA) studies mainly rely on quantitative real‐time polymerase chain reaction (qPCR) to amplify extremely low concentrations of DNA present in environmental samples. Understanding factors that influence targeted eDNA assay performance and detection in field samples, such as Taq DNA polymerase enzyme type and thermocycle protocol, is critical for the interpretation of qPCR results. We completed a systematic performance evaluation of five distinct targeted eDNA assays (eANFI6, eFISH1, eANBO5, eGLIN1, and eLICA3 targeting sablefish, general fish, Boreal toad, wood turtle, and the American bullfrog) by subjecting the same samples to analysis by five different Taq enzyme reagent mixes (Immolase, Environmental Master Mix (EMM), Amplitaq, QIAcuity, and QuantiNova) and the commonly used 2‐step (95°C, 60°C) and 3‐step (95°C, 64°C, 72°C) thermocycle protocols. We evaluated assay performance using a standardized dilution series of synthetic dsDNA target sequences and calculated limits of detection (LOD) and quantification (LOQ) and 95% confidence intervals for each combination of Taq enzyme and thermocycle protocol. All assays performed within acceptable performance criteria as defined by the Canadian national standard for targeted eDNA assays. Based on data generated by synthetic dsDNA fragments, the eDNA assays performed comparably regardless of the enzyme reagent mix and protocol used, except for eANFI6 and eGLIN1 using EMM and the 3‐step protocol, where no amplification was observed. On freshwater field samples, EMM and Immolase performed best. On marine field samples, Immolase, EMM, Qiacuity, and QuantiNova performed equally well, although EMM failed to amplify some samples. The work reveals that an enzyme reaction mix or thermocycle protocol can affect the result of an eDNA assay, but the appropriate choice also depends on the nature of the field sample. It is therefore imperative that these are considered when selecting appropriate reaction conditions and that they are clearly reported.

Abstract We conducted a statistical analysis of local phase space density (PSD) minima across a wide energy range (∼20 keVs to ∼10 MeV), using observations from the Van Allen Probes and the Geostationary Operational Environmental Satellite. We identified deepening minima in PSD profiles of multi‐MeV (∼5% occurrence) and of “seed” electrons (up to 15% occurrence, corresponding to ∼70–100 s keV) and compared their distribution with a 3D diffusion model using the Versatile Electron Radiation Belts (VERB) code. The comparison of the observed and modeled distributions suggests that the PSD minima of seed electrons are likely associated with hiss waves and the corresponding L‐shell dependent electron lifetimes. However, the observed distribution was not fully reproduced by the model, potentially indicating other fast loss mechanisms of seed electrons.

Objectives: We tested an adapted version of an effective U.S.-based peer-texting intervention to promote Quitline use and smoking cessation among rural participants in Vietnam.Methods: We conducted a two-arm randomized trial with participants recruited at four rural community centers. The intervention included peer messages sent for six months that promoted Quitline use and smoking cessation. Additionally, biweekly two-way text messages assessed participants’ interest in Quitline referral and current smoking status. Comparison participants received only the bi-weekly text message assessment of their current smoking status. At six months, we assessed Quitline use and smoking cessation. Smoking cessation was assessed using the 7-day point prevalence question and verified with a carbon monoxide breath monitor (

IntroductionFetal growth restriction (FGR) is a common pregnancy complication, caused by placental insufficiency, with serious adverse consequences for development in utero and postnatal wellbeing. There are no antenatal treatments to improve growth or organ development in FGR, and animal models are essential to mimic the physiological adaptations in FGR and to assess potential interventions. This study aimed to identify the temporal nature of reduced developmental trajectory in fetuses with FGR, and to examine the effects of common factors that may mediate differential growth such as glucocorticoid treatment. We hypothesised that the trajectory of growth would be adversely impacted by FGR.MethodsFGR was induced via surgical placental insufficiency in fetal sheep (89 days gestation/0.6 gestation; n=135) and compared to age-matched controls over the last third of gestation and into neonatal life (n=153).ResultsBody weight of FGR fetuses/lambs was significantly reduced compared to controls (p

Abstract Environmental DNA (eDNA) is revolutionizing species monitoring in nature. At the heart of any eDNA approach is the reliance upon sufficient DNA sequence information to satisfy the demands of eDNA assay specificity and sensitivity. The most common source of this information has been restricted to short barcoding regions of the mitochondrial genome (mitogenome) and marker genes. The use of these limited regions for assay design has often resulted in substantial trade‐offs in assay performance. With increased accessibility of full mitogenome assemblies, the potential for designing more robust eDNA assays is considerably enhanced. However, this also poses a new challenge to effectively identify suitable regions for assay design using considerably larger sequences. We present unikseq, a utility that uses words of length k (k‐mers) to identify unique regions in a reference sequence relative to tolerated (ingroup) and not‐tolerated (outgroup or non‐target) sequence sets, quickly and with low memory that can yield highly specific assays. We illustrate its application within an assay development workflow through use‐case examples for the design and validation of four quantitative real‐time polymerase chain reaction (qPCR)‐based assays selective for American bullfrog (Rana [Lithobates] catesbeiana), Burbot (Lota lota), Lake trout (Salvelinus namaycush), and Quillback rockfish (Sebastes maliger). The chosen target species vary in range, habitat, and degree of relatedness to their sympatric species that, consequently, impact eDNA assay design difficulty. We demonstrate the effectiveness of unikseq through assay validation and characterization using DNA from voucher specimens, synthetic DNA, and, where possible, field samples, to verify the specificity and sensitivity of the newly designed assays. By facilitating whole mitogenome sequence comparison, the creation of high‐performing eDNA assays is substantially enhanced. Having several adjustable parameters for specifying user requirements within unikseq, this approach can facilitate the identification of suitable regions for a broad range of applications requiring nucleotide sequence comparisons.

Abstract Background In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. Results Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. Conclusion Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Exercise enhances aspects of human cognition, but its intensity may matter. Recent animal research suggests that vigorous exercise, which releases greater amounts of lactate, activates more brain-derived neurotrophic factor (BDNF) in the hippocampus and, thus, may be optimal for supporting cognitive function. The cognitive benefits of exercise may be further augmented when combined with cognitive training. The sport of orienteering simultaneously combines exercise with spatial navigation and, therefore, may result in greater cognitive benefits than exercising only, especially at vigorous intensities. The present study aimed to examine the effects of an acute bout of orienteering at different intensities on cognition and BDNF compared to exercising only. We hypothesized that vigorous-intensity orienteering would increase lactate and BDNF and improve cognition more than moderate-intensity orienteering or vigorous exercise alone. Sixty-three recreationally active, healthy young adults (Mage = 21.10±2.75 years) with no orienteering experience completed a 1.3 km intervention course by navigating and exercising at a vigorous (80-85% of heart rate reserve) or moderate (40-50% of heart rate reserve) intensity or exercising vigorously without navigation. Exercise intensity was monitored using peak lactate, heart rate and rating of perceived exertion. Serum BDNF was extracted immediately before and after the intervention. Memory was assessed using the Mnemonic Similarity Task (high-interference memory) and the Groton Maze Learning Test (spatial memory). Both exercising and orienteering at a vigorous intensity elicited greater peak lactate and increases in BDNF than moderate-intensity orienteering, and individuals with higher peak lactate also had greater increases in BDNF. High-interference memory improved after both vigorous-intensity interventions but did not improve after the moderate-intensity intervention. Spatial memory only increased after vigorous-intensity orienteering, suggesting that orienteering at a vigorous intensity may particularly benefit spatial cognition. Overall, the results demonstrate the benefits of vigorous exercise on human cognition and BDNF.

Abstract Cannabinoids have been shown to induce anti‐tumor activity in a variety of carcinoma cells such as breast, prostate, and brain. The aim of the present study is to investigate the anti‐tumor activity of cannabinoids, CBD (cannbidiol), and CBG (cannabigerol) in ovarian carcinoma cells sensitive and resistant to chemotherapeutic drugs. Sensitive A2780 cells and resistant A2780/CP70 carcinoma cells and non‐carcinoma cells were exposed to varying concentrations of CBD, CBG, carboplatin or CB1 and CB2 receptor antagonists, AM251 and AM630, respectively, alone or in combination, at different exposure times and cytotoxicity was measured by MTT assay. The mechanism of action of CBD and CB in inducing cytotoxicity was investigated involving a variety of apoptotic and cell cycle assays. Treatment with CBD and CBG selectively, dose and time dependently reduced cell viability and induced apoptosis. The effect of CBD was stronger than CBG in all cell lines tested. Both CBD and CBG induced stronger cytotoxicity than afforded by carboplatin in resistant cells. The cytotoxicity induced by CBD was not CB1 or CB2 receptor dependent in both carcinoma cells, however, CBG‐induced cytotoxicity may involve CB1 receptor activity in cisplatin‐resistant carcinoma cells. A synergistic effect was observed when cannabinoids at sublethal doses were combined with carboplatin in both carcinoma cells. The apoptotic event may involve loss of mitochondrial membrane potential, Annexin V, caspase 3/7, ROS activities, and cell cycle arrest. Further studies are required to investigate whether these results are translatable in the clinic. Combination therapies with conventional cancer treatments using cannabinoids are suggested.

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.

BackgroundPreterm infants have immature respiratory drive and often require prolonged periods of mechanical ventilation. Prolonged mechanical ventilation induces systemic inflammation resulting in ventilation-induced brain injury, however its effect on brainstem respiratory centers is unknown. We aimed to determine the effects of 24 h of mechanical ventilation on inflammation and injury in brainstem respiratory centres of preterm fetal sheep.MethodsPreterm fetal sheep at 110 ± 1 days (d) gestation were instrumented to provide mechanical ventilation in utero. At 112 ± 1 d gestation, fetuses received either mechanical ventilation (VENT; n = 7; 3 ml/kg) for 24 h, or no ventilation (CONT; n = 6). At post-mortem, fetal brainstems were collected for assessment of mRNA and histological markers of inflammation and injury.ResultsIn utero ventilation (IUV) did not alter any blood-gas parameters. IUV significantly increased systemic IL-6 and IL-8 concentrations over the 24 h period compared to CONT. The number of ameboid microglia within the nucleus tractus solitarius and the raphe nucleus increased in VENT fetuses (p

Abstract Background Fetal growth restriction (FGR) is associated with deficits in the developing brain, including neurovascular unit (NVU) dysfunction. Endothelial colony forming cells (ECFC) can mediate improved vascular stability, and have demonstrated potential to enhance vascular development and protection. This investigation examined whether ECFCs from human umbilical cord blood (UCB) enhanced NVU development in FGR and appropriate for gestational age (AGA) fetal sheep. Methods Twin-bearing ewes had surgery performed at 88–90 days’ gestation, inducing FGR in one fetus. At 113 days, ECFCs (1 × 107 cells) cultured from human UCB were administered intravenously to fetal sheep in utero. At 127 days, ewes and their fetuses were euthanised, fetal brains collected, and NVU components analysed by immunohistochemistry. Results Twenty-four fetal lambs, arranged in four groups: AGA (n = 7), FGR (n = 5), AGA + ECFC (n = 6), and FGR + ECFC (n = 6), were included in analyses. FGR resulted in lower body weight than AGA (P = 0.002) with higher brain/body weight ratio (P = 0.003). ECFC treatment was associated with increased vascular density throughout the brain in both AGA + ECFC and FGR + ECFC groups, as well as increased vascular–astrocyte coverage and VEGF expression in the cortex (P = 0.003, P = 0.0006, respectively) and in the subcortical white matter (P = 0.01, P = 0.0002, respectively) when compared with the untreated groups. Conclusions ECFC administration enhanced development of NVU components in both the AGA and FGR fetal brain. Further investigation is required to assess how to optimise the enhanced angiogenic capabilities of ECFCs to provide a therapeutic strategy to protect the developing NVU against vulnerabilities associated with FGR.

Abstract The Earth’s magnetic field traps charged particles which are transported longitudinally around Earth, generating a near-circular current, known as the ring current. While the ring current has been measured on the ground and space for many decades, the enhancement of the ring current during geomagnetic storms is still not well understood, due to many processes contributing to its dynamics on different time scales. Here, we show that existing ring current models systematically overestimate electron flux observations of 10–50 keV on the nightside during storm onset. By analyzing electron drift trajectories, we show that this systematic overestimation of flux can be explained through a missing loss process which operates in the pre-midnight sector. Quantifying this loss reveals that the theoretical upper limit of loss has to be reached over a broad region of space in order to reproduce the observations. This missing loss may be attributed to inaccuracies in the parameterization of the loss due to chorus wave interactions, combined with the scattering by electrostatic electron cyclotron harmonic waves which is currently not included in ring current models.

Background: “Storytelling” interventions influence knowledge, attitudes and behavior to promote chronic disease management. We aimed to describe the development of a video “storytelling” intervention to increase gout knowledge and promote adherence to medications and follow-up care after an acute gout flare visit in the emergency department. Methods: We developed a direct-to-patient storytelling intervention to mitigate modifiable barriers to gout care and promote outpatient follow-up and medication adherence. We invited adult patients with gout as storytellers. We utilized a modified Delphi process involving gout experts to identify key themes to guide development of an intervention. Using a conceptual model, we selected stories to ensure delivery of evidence-based concepts and to maintain authenticity. Results: Our video-based storytelling intervention consisted of segments addressing modifiable barriers to gout care. Four diverse gout patients were recruited as storytellers and interviewed with questions that covered gout diagnosis and care. Eleven international gout experts from diverse geographic locations generated and ranked items they considered important messages to promote outpatient gout care follow-up and treatment adherence. Filmed videos were truncated into segments and coded thematically. Distinct segments that captured desired messages were combined to form a cohesive narrative story based on gout patient experiences that conveyed evidence-based strategies to manage gout. Conclusions: Using the Health Belief Model, we developed a culturally appropriate narrative intervention containing “storytelling” that can be tested as an approach to improve gout outcomes. The methods we describe may be generalizable to other chronic conditions requiring outpatient follow-up and medication adherence to improve outcomes.