A genome sequencing system for universal newborn screening, diagnosis, and precision medicine for severe genetic diseases.

Newborn screening (NBS) dramatically improves outcomes in severe childhood disorders by treatment before symptom onset. In many genetic diseases, however, outcomes remain poor because NBS has lagged behind drug development. Rapid whole-genome sequencing (rWGS) is attractive for comprehensive NBS because it concomitantly examines almost all genetic diseases and is gaining acceptance for genetic disease diagnosis in ill newborns. We describe prototypic methods for scalable, parentally consented, feedback-informed NBS and diagnosis of genetic diseases by rWGS and virtual, acute management guidance (NBS-rWGS). Using established criteria and the Delphi method, we reviewed 457 genetic diseases for NBS-rWGS, retaining 388 (85%) with effective treatments. Simulated NBS-rWGS in 454,707 UK Biobank subjects with 29,865 pathogenic or likely pathogenic variants associated with 388 disorders had a true negative rate (specificity) of 99.7% following root cause analysis. In 2,208 critically ill children with suspected genetic disorders and 2,168 of their parents, simulated NBS-rWGS for 388 disorders identified 104 (87%) of 119 diagnoses previously made by rWGS and 15 findings not previously reported (NBS-rWGS negative predictive value 99.6%, true positive rate [sensitivity] 88.8%). Retrospective NBS-rWGS diagnosed 15 children with disorders that had been undetected by conventional NBS. In 43 of the 104 children, had NBS-rWGS-based interventions been started on day of life 5, the Delphi consensus was that symptoms could have been avoided completely in seven critically ill children, mostly in 21, and partially in 13. We invite groups worldwide to refine these NBS-rWGS conditions and join us to prospectively examine clinical utility and cost effectiveness.

Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra- and inter-species model to predict the suitable formulation mix.

The ecotoxicity of anticoagulants used for rodent pests' management is a major concern, particularly with second generation anticoagulants, which are more persistent in the body of rodents and therefore more likely to cause secondary exposure in their predators. One of the solutions envisaged to mitigate this risk is to use stereoisomers of these anticoagulants, each of which has particular pharmacokinetics. However, the few studies published to date have considered only one species and one sex. Here, we study the pharmacokinetics of the 4 stereoisomers of 3.4 mg/kg of difethialone in rats (Rattus norvegicus) and 3 mg/kg in mice (Mus musculus) in both sexes and propose a model to choose the optimal stereoisomer efficacy/ecotoxicity mixture for the management of all these animals. Our results show that while the most persistent stereoisomer (E3-cis) is common to both species and sexes, the pharmacokinetics of the other stereoisomers show marked differences between sexes and species. Thus, the area under curve (AUC) of E4-trans in male rats is four times lower than in females or mice, making it a priori unusable in male rats. Conversely, our modeling seems to show that the E1-trans stereoisomer seems to offer the best compromise AUC persistence. In conclusion, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy and with regard to the ecotoxicity of these molecules.

Seasonal diet-based resistance to anticoagulant rodenticides in the fossorial water vole (Arvicola amphibius).

Anticoagulant rodenticides (AR) resistance has been defined as "a major loss of efficacy due to the presence of a strain of rodent with a heritable and commensurately reduced sensitivity to the anticoagulant". The mechanism that supports this resistance has been identified as based on mutations in the Vkorc1 gene leading to severe resistance in rats and mice. This study evaluates the validity of this definition in the fossorial water vole and explores the possibility of a non-genetic diet-based resistance in a strict herbivorous rodent species. Genetic support was explored by sequencing the Vkorc1 gene and the diet-based resistance was explored by the dosing of vitamins K in liver of voles according to seasons. From a sample of 300 voles, only 2 coding mutations, G71R and S149I, were detected in the Vkorc1 gene in the heterozygous state with low allele frequencies (0.5-1%). These mutations did not modify the sensitivity to AR, suggesting an absence of genetic Vkorc1-based resistance in the water vole. On the contrary, vitamin K1 was shown to be 5 times more abundant in the liver of the water vole compared to rats. This liver concentration was shown to seasonally vary, with a trough in late winter and a peak in late spring/early summer related to the growth profile of grass. This increase in concentration might be responsible for the increased resistance of water voles to AR. This study highlights a non-genetic, diet-related resistance mechanism in rodents to AR. This diet-based resistance might explain the different evolution of the Vkorc1 gene in the fossorial water vole compared to rats and mice.

Comparative biological properties of the four stereoisomers of difethialone, a second-generation anticoagulant rodenticide, in rats: development of a model allowing to choose the appropriate stereoisomeric ratio.

The current management of rodent pest populations is based on second-generation anticoagulant rodenticides (SGAR). These molecules, of which difethialone is part, are much more efficient than the first generation. Nevertheless, this efficiency comes with a major drawback, SGARs are tissue persistent that increases the exposure of rodent predators to them. According to its chemical structure, difethialone has four stereoisomers, whose specific inhibition potency and pharmacokinetic have never been described and might be useful to design new eco-friendly rodenticides. The study aimed to investigate the ability to inhibit anticoagulant target enzyme (VKORC1) and the pharmacokinetics in rats of the four difethialone stereoisomers in rats. We show that stereoisomers are all highly efficient to inhibit VKORC1 activity, but they have distinct initial half-life with 6.0 h, 25.4 h, 69.3 h, and 82.3 h for, respectively, E4-trans, E2-cis, E1-trans, and E3-cis stereoisomer. These results open the way of the development of eco-friendly and efficient rodenticide by mixing some of these stereoisomers. Preferential incorporation of the E4-trans stereoisomer (high inhibitory VKORC1 potency, relatively shorter liver half-life) into difethialone rodenticides baits might result in a more eco-friendly product than current commercially available difethialone formulations. In addition, we put forward modelling to help design bait according to the circumstance of use (presence of non-target species, food competition, etc.) by modulating the theorical AUC and and the theorical concentration of the product at the death of the rodent pest. Thus, this modeling might allow to diminish the use of laboratory animal in assay.

Thrombin generation test: A reliable tool to evaluate the pharmacodynamics of vitamin K antagonist rodenticides in rats.

Vitamin K antagonist rodenticide pharmacodynamics (PD) is studied in rodents with traditional laboratory tests. We wondered if thrombin generation test (TGT) could add value. Difethialone (10 mg/kg) was administered per os to 97 OFA-Sprague Dawley rats. PD was studied over a 72 h-period using the Calibrated Automated Thrombogram on platelet poor plasma before and after intoxication (3 female and 3 male rats for each 13 time points) and TGT parameters were compared with the prothrombin time (PT) and vitamin K dependent factor activities previously reported. Following intoxication, preliminary tests evidenced rapid and full inhibition of thrombin generation triggered with 5 or 20 pM human recombinant tissue factor. To study the evolution of TGT parameters following difethialone intake, we adapted the test by complementing intoxicated rat samples with pooled normal rat plasma (3/1, v/v). Adapted TGT confirmed the known higher procoagulant basal level in females compared to males through higher endogenous thrombin potential (ETP) and peak height (PH) (p < 0.0001 and p = 0.0003, respectively). An exponential model fitted well the PH and ETP decay after intoxication. In contrast to PT, the decreases were observed immediately following VKA intake and had comparable time to halving values: 10.5 h (95% CI [8.2; 13.6]) for ETP and 10.4 h (95% CI [7.8; 14.1]) for PH. The decrease of FVII and FX preceded that of PH, ETP and FII while FIX decreased later on, contributing to the severe hypo-coagulability. We demonstrated that TGT performed in samples of intoxicated rats complemented with normal plasma is a reliable tool for evaluation of VKA rodenticide PD in rats.

Management of Rodent Populations by Anticoagulant Rodenticides: Toward Third-Generation Anticoagulant Rodenticides.

Second-generation anticoagulant rodenticides (SGARs) have been used since the 1980s for pest management. They are highly efficient even in warfarin-resistant rodents. Nevertheless, because of their tissue persistence, nontarget poisoning by SGARs is commonly described in wildlife. Due to this major problem, a new generation of anticoagulants must be developed to limit this risk. This study proposes a method of developing a new generation of anticoagulant rodenticides by revisiting the old SGARs based on the concept of stereochemistry. Each current SGAR is a mixture of diastereomers. Diastereomers of each compound were purified, and their biologic properties were compared by determining their ability to inhibit vitamin K epoxide reductase (VKOR) activity involved in the activation of vitamin K-dependent clotting factors and their toxicokinetic properties. Systematically, for each SGAR, both diastereomers are as effective in inhibiting VKOR activity. However, their toxicokinetic properties are very different, with one of the two diastereomers always more rapidly cleared than the other one. For all SGARs except flocoumafen, the less persistent diastereomer is always the less predominant isomer present in the current mixture. Therefore, the development of baits containing only the less persistent diastereomer would avoid the ecotoxicological risk associated with their use without decreasing their efficacy.

Monitoring of antivitamin K-dependent anticoagulation in rodents - Towards an evolution of the methodology to detect resistance in rodents.

Vitamin K antagonists are used as rodenticides for pest control management. In rodents, prothrombin time is used to monitor their effect despite its limits and the emergence of many coagulation methods. The aim of this study is to explore different coagulation monitoring methods in order to propose the best method and the best parameter to monitor vitamin K antagonists effect in rodents. The coagulation function was thus monitored with global coagulation assays and specialty assays after difethialone administration in rats. Despite many parameters obtained by thromboelastometry, only clotting time and clot formation time obtained by ExTEM were modified. Their evolution was fast with doubling time respectively of 4.0h and 3.7h but their increases were delayed with a lag time higher than 8h. Conversely, prothrombin time evolution presented a lag time of only 2h, but a higher doubling time of 7.2h. The measurements of factor VII and X activities were the most sensitive assays to monitor vitamin K antagonists effect with almost no lag time and the fastest evolution. Nevertheless, factor X was shown to be the only key factor driving prothrombin time. Monitoring factor X activity enables to follow most effectively the anticoagulation status in rats after rodenticides administration.

False Negative Results in Clostridium difficile Testing.

BACKGROUND: Accurate diagnosis of Clostridium difficile infection (CDI) is paramount for patient management. The wrong diagnosis places patients at risk, delays treatment, and/ or contributes to transmission of infection in the healthcare setting. Although amplification of the toxin B gene by polymerase chain reaction (PCR) is a sensitive method for detecting toxigenic C. difficile, false negative results still occur and could impact the diagnosis and treatment of this infection. METHODS: This study investigated 48 patients that tested negative for toxigenic C. difficile via GeneXpert C. difficile epi test, while simultaneously testing positive for toxigenic C. difficile via stool culture. Fifty discrepant samples were collected over a 15-month period and all C. difficile isolates were characterized by ribotype. Patient charts were reviewed to assess whether discrepant results impacted the treatment course or clinical outcome of affected patients. RESULTS: Fifty samples of a total of 2308 samples tested in an acute healthcare facility over a 15-month period had negative PCR and positive stool culture for toxigenic C. difficile. C. difficile isolated from the discrepant samples resulted in diverse ribotyping patterns suggesting they were derived from different strains. The samples belonged to patients who were distributed evenly between age groups and wards in the hospital. In the majority of cases, the false negative C. difficile test results did not seem to impact the clinical outcome in these patients. CONCLUSIONS: The PCR limit of detection may impact the results of molecular methods for C. difficile detection. Both clinical and analytical sensitivity of C. difficile tests should be considered when deciding which diagnostic assay to use, and clinical correlates should be examined carefully before excluding CDI as a cause of disease.

Insights into planktonic food-web dynamics through the lens of size and season.

Knowledge of the trophic structure and variability of planktonic communities is a key factor in understanding food-web dynamics and energy transfer from zooplankton to higher trophic levels. In this study, we investigated how stable isotopes of mesozooplankton species varied seasonally (winter, spring, autumn) in relation to environmental factors and plankton size classes in a temperate coastal ecosystem. Our results showed that spring is characterized by the strongest vertical and size-structured plankton food-web, mainly fueled by the phytoplankton bloom. As a result, spring displayed the largest isotopic niche space and trophic divergence among species. On the contrary, both pelagic and benthic-derived carbon influenced low productive seasons (winter and autumn), resulting in more generalist strategies (trophic redundancy). Stable isotope mixing models were used to explore how different seasonal structures influenced the overall food web up to predatory plankton (i.e., mysids, chaetognaths, and fish larvae). Different feeding strategies were found in spring, with predators having either a clear preference for larger prey items (> 1 mm, for herring and dab larvae) or a more generalist diet (sprat and dragonets larvae). During low productive seasons, predators seemed to be more opportunistic, feeding on a wide range of size classes but focusing on smaller prey. Overall, the food-web architecture of plankton displayed different seasonal patterns linked to components at the base of the food web that shaped the main energy fluxes, either from phytoplankton or recycled material. Additionally, these patterns extended to carnivorous plankton, such as fish larvae, emphasizing the importance of bottom-up processes.

Antifouling action of polyisoprene-based coatings by inhibition of photosynthesis in microalgae.

Previous studies have demonstrated that ionic and non-ionic natural rubber-based coatings inhibit adhesion and growth of marine bacteria, fungi, microalgae, and spores of macroalgae. Nevertheless, the mechanism of action of these coatings on the different micro-organisms is not known. In the current study, antifouling activity of a series of these rubber-based coatings (one ionic and two non-ionic) was studied with respect to impacts on marine microalgal photosynthesis using pulse-amplitude-modulation (PAM) fluorescence. When grown in contact with the three different coatings, an inhibition of photosynthetic rate (relative electron transport rate, rETR) was observed in all of the four species of pennate diatoms involved in microfouling, Cocconeis scutellum, Amphora coffeaeformis, Cylindrotheca closterium, and Navicula jeffreyi. The percentage of inhibition ranged from 44% to 100% of the controls, depending on the species and the coating. The ionic coating was the most efficient antifouling (AF) treatment, and C. scutellum and A. coffeaeformis are the most sensitive and tolerant diatoms tested, respectively. Photosynthetic inhibition was reversible, as almost complete recovery of rETR was observed 48 h post exposure, after detachment of cells from the coatings. Thus, the antifouling activity seemed mostly due to an effect of contact with materials. It is hypothesized that photosynthetic activity was suppressed by coatings due to interference in calcium availability to the microalgal cells; Ca(2+) has been shown to be an essential micro/macro nutrient for photosynthesis, as well as being involved in cell adhesion and motility in pennate diatoms.

Interest of the faecal and plasma matrix for monitoring the exposure of wildlife or domestic animals to anticoagulant rodenticides.

Anticoagulant rodenticides (ARs), particularly second-generation compounds (SGAR), are known to be a potential threat to unintended species due to their tissue persistence. The liver is the storage tissue of ARs and is a matrix of choice in diagnosing exposure and intoxication of non-target fauna. However, it is only available on dead animals. Blood and faeces can be used on living animals. These two biological matrices were compared in terms of their relevance to exposure to ARs. In addressing this question, we compared the faecal, plasma and liver concentrations of bromadiolone, one of the SGAR frequently implicated in wildlife exposure. We studied this comparison at the individual level and at the population level, considering three influencing factors: dose, sex and time. Our findings demonstrate that faecal analyses are more valuable than plasma analyses for monitoring AR exposure of domestic and wild animals, even if faecal concentrations cannot be correlated with liver concentrations.

The effect of interspecific and intraspecific diversity on microplastic ingestion in two co-occurring mussel species in South Africa.

Interspecific and intraspecific diversity are essential components of biodiversity with far-reaching implications for ecosystem function and service provision. Importantly, genotypic and phenotypic variation within a species can affect responses to anthropogenic pressures more than interspecific diversity. We investigated the effects of interspecific and intraspecific diversity on microplastic ingestion by two coexisting mussel species in South Africa, Mytilus galloprovincialis and Perna perna, the latter occurring as two genetic lineages. We found significantly higher microplastic abundance in M. galloprovincialis (0.54 ± 0.56 MP items g-1WW) than P. perna (0.16 ± 0.21 MP items g-1WW), but no difference between P. perna lineages. Microbeads were the predominant microplastic (76 % in P. perna, 99 % in M. galloprovincialis) and polyethylene the prevalent polymer. Interspecific differences in microplastic abundance varied across locations, suggesting diverse sources of contamination. We suggest that microplastic ingestion can be species-specific even in organisms that coexist and play similar functional roles within ecosystems.

Larval growth of the polychaete Arenicola marina under different temperature and food conditions: consequences on bioenergetic models.

Arenicola marina, a marine benthic polychaete, is widespread on sandy beaches in Europe and considered as an ecosystem engineer despite commonly used as bait by fishermen. Data regarding the bioenergetics of the lugworm larval stages are still incomplete. Trochophore is initially lecithotroph and then becomes planktotroph while growing as metatrochophore on subtidal area, a quite stable daily temperature environment compared with the foreshore, where juveniles and adult live, with daily temperature fluctuating up to 15°C. These discrepancies in temperature ranges may influence the temperature corrections (TCs) that control metabolic rates during the life cycle of A. marina. We carried out laboratory experiments in microcosms by inducing artificial spawning of lugworms, and then undertaken in vitro fertilization to obtain embryos and, finally, to follow, the larval development up to 10 segments with chaetae for 50 days under three temperature conditions (13°C, 15°C and 17°C) and two food conditions ('fed' and 'non-fed'). The first feeding ('birth') of A. marina larvae was deciphered anatomically for a size between 450 and 500 µm and described at 17 days post-fertilization for larvae reared at 15°C and 17°C. Using a biphasic model with a von Bertalanffy growth before 'birth' and an exponential growth after 'birth', among the three temperature treatments, the 15°C condition exhibited the best larval performance. TC based on embryonic and larval metabolic rates gave an Arrhenius temperature of ~6661 K and a higher boundary temperature tolerance range of ~294.5 K. Both temperature values differ from those calculated from TC based mostly on juvenile and adult metabolic rates. We claim to use two sets of Arrhenius temperatures according to the life history stages of A. marina while using Dynamic Energy Budget model. This model was developed initially in order to manage the conservation of the lugworm species.

The MANA (MANagement of Atolls, 2017-2022) project for pearl oyster aquaculture management in the Central Pacific Ocean using modelling approaches: Overview of first results.

This editorial presents results of the MANA (MANagement of Atolls) project compiled in the form of a Marine Pollution Bulletin collection of 14 articles. MANA is a project funded by the French Agence National pour la Recherche that specifically addresses the development of knowledge and management tools for pearl farming atolls, with a focus on the spat collecting activity in French Polynesia. The 14 papers cover the range of thematic tasks described in the initial project, including atoll geomorphology and bathymetry, climate forcing, atoll lagoon and rim hydrodynamics, typology of atolls, evaluation of remote sensing data for monitoring atoll lagoons, and development of numerical models and spatially-explicit tools that altogether have contributed to the applied objectives. In addition, this editorial draws an update on the pearl farming industry in French Polynesia with the latest statistics, and discusses the next targeted priorities for research programs focusing on pearl farming atolls.

Remote sensing provides new insights on phytoplankton biomass dynamics and black pearl oyster life-history traits in a Pacific Ocean deep atoll.

Thus far, no long-term in situ observation of planktonic biomass have been undertaken to optimize the black-lip pearl oyster aquaculture in the remote Tuamotu atolls. The feasibility of using data from the OLI sensor onboard Landsat-8 satellite to determine chlorophyll a concentrations (Chla) in a deep atoll, Ahe, was then assessed over the 2013-2021 period using 153 images. Validations with in situ observations were satisfactory, while seasonal and spatial patterns in Chla were evidenced within the lagoon. Then, a bioenergetic modelling exercise was undertaken to estimate oyster life-history traits when exposed to the retrieved Chla. The outputs provide spatio-temporal variations in pelagic larval duration (11.1 to 30.6 days), time to reach commercial size (18.8 to 45.3 months) and reproductive outputs (0.5 to 1.7 event year-1). This first study shows the potential of using remote sensing to monitor the trophic status of deep pearl farming lagoons and help aquaculture management.

Asymptomatic Anticoagulant Rodenticide Exposure in Dogs and Cats-A French and Belgian Rural and Urban Areas Study.

Anticoagulant rodenticides (ARs) are important tools for controlling rodent pests, but they also pose a health threat to non-target species. ARs are one of the most common causes of pet poisoning. However, exposure of domestic animals to subclinical doses of ARs is poorly documented. To study the random exposure of dogs and cats to ARs, feces from animals showing no clinical signs of rodenticide poisoning were collected from a network of French and Belgian veterinarians. We analyzed fresh feces from 304 dogs and 289 cats by liquid chromatography-tandem mass spectrometry. This study showed a limited prevalence of AR exposure in dogs and cats of 2.6 and 4.5% respectively. In both species, access to the outdoors is a risk factor for ARs exposure. In contrast, the sex of the animals did not affect the ARs exposure status. The observation of the ratio of cis and trans isomers suggested primary exposure in dogs, but also in some cats. While primary exposure in dogs appears to be related to the use of ARs as plant protection products, primary exposure in cats may be malicious, as warfarin, an anticoagulant formerly used as a rodenticide and now used only in humans, was found in 4 of 13 exposed cats. Secondary exposure may also occur in cats.Our study showed reduced exposure in dogs and cats, compared to wildlife, which often has high exposure, especially in areas where rodent control is important.

Inferences to estimate consumer's diet using stable isotopes: Insights from a dynamic mixing model.

Stable isotope ratios are used to reconstruct animal diet in trophic ecology via mixing models. Several assumptions of stable isotope mixing models are critical, i.e., constant trophic discrimination factor and isotopic equilibrium between the consumer and its diet. The isotopic turnover rate (λ and its counterpart the half-life) affects the dynamics of isotopic incorporation for an organism and the isotopic equilibrium assumption: λ involves a time lag between the real assimilated diet and the diet estimated by mixing models at the individual scale. Current stable isotope mixing model studies consider neither this time lag nor even the dynamics of isotopic ratios in general. We developed a mechanistic framework using a dynamic mixing model (DMM) to assess the contribution of λ to the dynamics of isotopic incorporation and to estimate the bias induced by neglecting the time lag in diet reconstruction in conventional static mixing models (SMMs). The DMM includes isotope dynamics of sources (denoted δs), λ and frequency of diet-switch (ω). The results showed a significant bias generated by the SMM compared to the DMM (up to 50% of differences). This bias can be strongly reduced in SMMs by averaging the isotopic variations of the food sources over a time window equal to twice the isotopic half-life. However, the bias will persist (∼15%) for intermediate values of the ω/λ ratio. The inferences generated using a case study highlighted that DMM enhanced estimates of consumer's diet, and this could avoid misinterpretation in ecosystem functioning, food-web structure analysis and underlying biological processes.