No filters, no fridges: a method for preservation of water samples for eDNA analysis

Background: Advancements in the detection of environmental DNA (eDNA) for detecting species of interest will likely allow for expanded use of these techniques in the field. One obstacle that continues to hinder applications in the field is the requirement of a cold chain of storage for water samples containing eDNA. While eDNA has been successfully preserved using Longmire’s lysis buffer applied to filters, it has yet to be tried with freshwater samples collected for eDNA detection of an invasive species. We tested the utility of Longmire’s solution (100 mM Tris, 100 mM EDTA, 10 mM NaCl, 0.5 % SDS, 0.2 % sodium azide) as an additive to freshwater samples for preservation of eDNA. Results: Environmental DNA was effectively preserved in 15 mL water samples with Longmire’s solution added; eDNA positive detection was comparable to freezing the samples at −80 °C and occurred out to 56 days at the highest concentration (5 mL Longmire’s solution: 15 mL sample water). Medium and low concentrations of Longmire’s solution added to 15 mL of sample water generally preserved eDNA out to 56 days but not as well as did freezing or application of the highest concentration of Longmire’s lysis buffer. Treatment and degradation time had a significant effect on average DNA concentration of samples, although not the interaction of treatment and time. Perfect detection occurred out to 56 days with the high Longmire’s treatment group but DNA concentration was significantly lower at this time point compared to 28 days. Conclusion: We conclude that Longmire’s lysis buffer is a viable alternative to cold chain storage that can simplify the collection of eDNA by eliminating the need for filtering and allow more time for sample collection when added at our highest concentration (1 part Longmire’s:3 parts water sample), which could translate to an increase in the chances of detecting a rare or elusive species

Pupillometry via smartphone for low-resource settings

The photopupillary reflex regulates the pupil reaction to changing light conditions. Being controlled by the autonomic nervous system, it is a proxy for brain trauma and for the conditions of patients in critical care. A prompt evaluation of brain traumas can save lives. With a simple penlight, skilled clinicians can do that, whereas less specialized ones have to resort to a digital pupilometer. However, many low-income countries lack both specialized clinicians and digital pupilometers. This paper presents the early results of our study aiming at designing, prototyping and validating an app for testing the photopupillary reflex via Android, following the European Medical Device Regulation and relevant standards. After a manual validation, the prototype underwent a technical validation against a commercial Infrared pupilometer. As a result, the proposed app performed as well as the manual measurements and better than the commercial solution, with lower errors, higher and significant correlations, and significantly better Bland-Altman plots for all the pupillometry-related measures. The design of this medical device was performed based on our expertise in low-resource settings. This kind of environments imposes more stringent design criteria due to contextual challenges, including the lack of specialized clinicians, funds, spare parts and consumables, poor maintenance, and harsh environmental conditions, which may hinder the safe operationalization of medical devices. This paper provides an overview of how these unique contextual characteristics are cascaded into the design of an app in order to contribute to the Sustainable Development Goal 3 of the World Health Organization: Good health and well-being

Picky eaters are rare: DNA-based blood meal analysis of \u3ci\u3eCulicoides\u3c/i\u3e (Diptera: Ceratopogonidae) species from the United States

Background: Biting midges in the genus Culicoides (Diptera; Ceratopogonidae) have been implicated in the transmission of a number of parasites and highly pathogenic viruses. In North America, the complete transmission cycles of many of these pathogens need further elucidation. One way to increase our knowledge about the evolution and ecology of Culicoides species and the pathogens they transmit is to document the diversity of vertebrate hosts that Culicoides feed upon. Our objective was to identify the diversity of Culicoides hosts in the United States. Results: We sequenced two vertebrate mitochondrial genes (cytochrome c oxidase subunit 1 and cytochrome b) from blood-engorged Culicoides to identify Culicoides species and their blood meals. We detected the mitochondrial DNA of 12 host species from seven different Culicoides species from three states. The majority of the identified blood meals were from the C. variipennis species complex in California. The hosts included both mammals and birds. We documented new host records for some of the Culicoides species collected. The majority of the mammalian hosts were large ungulate species but we also detected a lagomorph and a carnivore. The bird species that were detected included house finch and emu; the latter is evidence that the species in the C. variipennis species complex are not strictly mammalophilic. Conclusions: These results demonstrate that Culicoides will feed on multiple classes of vertebrates and may be more opportunistic in regards to host choice than previously thought. This knowledge can help with identification of susceptible host species, pathogen reservoirs, and new vector species which, in turn, will improve disease outbreak risk assessments

Detection and persistence of environmental DNA from an invasive, terrestrial mammal

Invasive Sus scrofa, a species commonly referred to as wild pig or feral swine, is a destructive invasive species with a rapidly expanding distribution across the United States. We used artificial wallows and small waterers to determine the minimum amount of time needed for pig eDNA to accumulate in the water source to a detectable level. We removed water from the artificial wallows and tested eDNA detection over the course of 2 weeks to understand eDNA persistence. We show that our method is sensitive enough to detect very low quantities of eDNA shed by a terrestrial mammal that has limited interaction with water. Our experiments suggest that the number of individuals shedding into a water system can affect persistence of eDNA. Use of an eDNA detection technique can benefit management efforts by providing a sensitive method for finding even small numbers of individuals that may be elusive using other methods

Accounting for observation processes across multiple levels of uncertainty improves inference of species distributions and guides adaptive sampling of environmental DNA

Understanding factors that influence observation processes is critical for accurate assessment of underlying ecological processes. When indirect methods of detection, such as environmental DNA, are used to determine species presence, additional levels of uncertainty from observation processes need to be accounted for. We conducted a field trial to evaluate observation processes of a terrestrial invasive species (wild pigs‐ Sus scrofa) from DNA in water bodies. We used a multi‐scale occupancy analysis to estimate different levels of observation processes (detection, p): the probability DNA is available per sample (θ), the probability of capturing DNA per extraction (γ), and the probability of amplification per qPCR run (δ). We selected four sites for each of three water body types and collected 10 samples per water body during two months (September and October 2016) in central Texas. Our methodology can be used to guide sampling adaptively to minimize costs while improving inference of species distributions. Using a removal sampling approach was more efficient than pooling samples and was unbiased. Availability of DNA varied by month, was considerably higher when water pH was near neutral, and was higher in ephemeral streams relative to wildlife guzzlers and ponds. To achieve a cumulative detection probability \u3e90% (including availability, capture, and amplification), future studies should collect 20 water samples per site, conduct at least two extractions per sample, and conduct five qPCR replicates per extraction. Accounting for multiple levels of uncertainty of observation processes improved estimation of the ecological processes and provided guidance for future sampling designs

Vigilancia epidemiológica en mujeres embarazadas para control de riesgos en el consumo de tabaco en la ciudad de Gualeguaychú

Objetivo: Determinar el nivel de cotinina urinaria en embarazadas fumadoras activas y pasivas en centros de salud públicos (cpub) y privados (cpri) de Gualeguaychú para conocer su riesgo de exposición y contribuir a mejorar el diseño de las intervenciones en la prevención del hábito tabáquico durante el embarazo. Materiales y métodos: Se trabajó con 443 embarazadas que concurrieron a cpub y cpri de Gualeguaychú para su control prenatal, solicitándoles a las que manifestaron ser fumadoras activas o estar expuestas al hat una muestra de orina para el dosaje de cotinina. Se aplicó un diseño de tipo no experimental, retrospectivo y de corte transversal. El dosaje de cotinina se realizó en orina, empleando una metodología quimioluminiscente. Previamente se obtuvo un valor referencial de cotinina urinaria inferior a los 15,2 ng/ml para el 98 % de sujetos no fumadores no expuestos al hat. Resultados: El 97,3 % de las embarazadas que declararon ser fumadoras activas presentaron valores de cotinina superiores a los 100 ng/ml y el 66,2 % de las que expresaron ser fumadoras pasivas presentaron un nivel superior a 15,2 ng/ml. Discusión y conclusiones: Los resultados obtenidos demuestran la utilidad de la cotinina como indicador para obtener datos fidedignos frente a la exposición al tabaco

Diabetes promotes invasive pancreatic cancer by increasing systemic and tumour carbonyl stress in Kras G12D/+mice

Background: Type 1 and 2 diabetes confer an increased risk of pancreatic cancer (PaC) of similar magnitude, suggesting a common mechanism. The recent finding that PaC incidence increases linearly with increasing fasting glucose levels supports a central role for hyperglycaemia, which is known to cause carbonyl stress and advanced glycation end-product (AGE) accumulation through increased glycolytic activity and non-enzymatic reactions. This study investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanisms involved. Methods: Pdx1-Cre;LSL-Kras G12D/+ mice were interbred with mitosis luciferase reporter mice, rendered diabetic with streptozotocin and treated or not with carnosinol (FL-926-16), a selective scavenger of reactive carbonyl species (RCS) and, as such, an inhibitor of AGE formation. Mice were monitored for tumour development by in vivo bioluminescence imaging. At the end of the study, pancreatic tissue was collected for histology/immunohistochemistry and molecular analyses. Mechanistic studies were performed in pancreatic ductal adenocarcinoma cell lines challenged with high glucose, glycolysis- and glycoxidation-derived RCS, their protein adducts AGEs and sera from diabetic patients. Results: Cumulative incidence of invasive PaC at 22 weeks of age was 75% in untreated diabetic vs 25% in FL-926-16-gtreated diabetic and 8.3% in non-diabetic mice. FL-926-16 treatment suppressed systemic and pancreatic carbonyl stress, extracellular signal-regulated kinases (ERK) 1/2 activation, and nuclear translocation of Yes-associated protein (YAP) in pancreas. In vitro, RCS scavenging and AGE elimination completely inhibited cell proliferation stimulated by high glucose, and YAP proved essential in mediating the effects of both glucose-derived RCS and their protein adducts AGEs. However, RCS and AGEs induced YAP activity through distinct pathways, causing reduction of Large Tumour Suppressor Kinase 1 and activation of the Epidermal Growth Factor Receptor/ERK signalling pathway, respectively. Conclusions: An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINs progression to invasive PaC, showing that hyperglycaemia promotes PaC mainly through increased carbonyl stress. In vitro experiments demonstrated that both circulating RCS/AGEs and tumour cell-derived carbonyl stress generated by excess glucose metabolism induce proliferation by YAP activation, hence providing a molecular mechanism underlying the link between diabetes and PaC (and cancer in general)

Effect of dexamethasone on newborn survival at different administration-to-birth intervals: A secondary analysis of the who action (Antenatal corticosteroids for improving outcomes in preterm newborn)-I trial

Background: The WHO ACTION-I trial demonstrated that dexamethasone significantly reduced neonatal mortality when administered to women at risk of early preterm birth in low-resource countries. We conducted a secondary analysis to determine how these benefits can be optimised, by evaluating the effect of dexamethasone compared to placebo on newborn mortality and severe respiratory distress outcomes at different administration-to-birth intervals, and identifying the interval with the greatest benefits.Methods: The WHO ACTION-I trial was a multi-country, individually-randomised, parallel-group, double-blind, placebo-controlled trial. It was conducted in 29 hospitals across Bangladesh, India, Kenya, Nigeria, and Pakistan. Women with a viable singleton or multiple pregnancy who presented to participating hospitals at a gestational age of 26 weeks 0 days-33 weeks 6 days and who were at risk of imminent preterm birth were eligible. In this secondary analysis, 2638 women and their newborns treated with single course of dexamethasone or placebo were analysed. Multivariate logistic regression was used to assess the effect of dexamethasone versus placebo on neonatal death, stillbirth or neonatal death, and severe respiratory distress at 24 h and at 168 h, by administration-to-birth interval (from 0 through 28 days), adjusting for gestational age at first dose. We used relative risks to identify the administration-to-birth interval with the greatest benefits of dexamethasone compared to placebo on the newborn outcomes.Findings: Between 24 December 2017 and 21 November 2019, 2852 women and their 3070 babies were enrolled in the WHO ACTION-I trial; 1332 women (1464 babies) in the dexamethasone group and 1306 women (1440 babies) in the placebo group were included in this secondary analysis. Neonatal mortality risk was lower with increasing time between initiating dexamethasone and birth, achieving peak mortality reduction by days 13 and 14 and then diminishing as the interval approached 28 days, regardless of gestational age at administration. For other outcomes, the overall pattern of risk reduction extending into the second week was consistent with that of neonatal death.Interpretation: In women at risk of preterm birth prior to 34 weeks\u27 gestation, the neonatal benefits of antenatal dexamethasone appear to increase with longer administration-to-birth intervals than previously thought. This knowledge can support clinical assessment and estimation of the risks of adverse preterm newborn outcomes at the time of birth, and the potential benefits of antenatal dexamethasone treatment for a known administration-to-birth interval.Funding: Bill and Melinda Gates Foundation; World Health Organization