Retrospective Detection of Ophidiomyces ophidiicola from Snake Moults Collected in Bieszczady Mountains, Poland.

Ophidiomyces ophidiicola, the causative agent of ophidiomycosis, poses a potential threat to wild snakes worldwide. This study aimed to retrospectively investigate the prevalence of O. ophidiicola in archived snake moults collected from the San River Valley in the Bieszczady Mountains, Poland, from 2010 to 2012. Using qPCR for O. ophidiicola detection and conventional PCR for clade characterisation, we analysed 58 moults and one road-killed specimen of Zamenis longissimus and Natrix natrix. A novel combination of primers (ITS2L) was used to simultaneously confirm SYBR Green-based qPCR results and perform genotyping. O. ophidiicola has been detected from two Z. longissimus and one N. natrix specimens. The identified clade (I-B) is consistent with those found in wild snakes of eastern Europe and San River Valley, indicating that O. ophidiicola has been present in this region for at least a decade. This study underscores the value of historical samples in understanding the long-term presence of pathogens and highlights the potential role of environmental reservoirs in the persistence of O. ophidiicola. Our findings are crucial for informing conservation strategies for the endangered Aesculapian snake populations in Poland, emphasising the need for ongoing monitoring and habitat management to mitigate the potential impact of ophidiomycosis.

Temporal variation characteristics of microbial aerosols in the goose house environment.

1. Preventing disease is important in poultry production systems, but this has mainly been studied in chickens. The aim of this study is to explore the impact of microbial aerosols in intensive goose house environments.2. To evaluate the environmental quality of geese housing, fine particulate matter (PM2.5) was collected using an ambient air particulate matter sampler. High-throughput sequencing was used to analyse bacterial diversity and relative abundance. Results showed that the number of general and operational taxonomic units (OTUs) were 1,578 and 19 112 in all PM2.5 samples. Firmicutes, Bacteroidota, Proteobacteria, Acidobacterota were the four most abundant phyla in PM2.5.3. Compared with bacterial phyla in the PM2.5 from chicken houses, those in the genus Acidobacterota were increased in goose housing. There are various genera of bacteria present in PM2.5, and their composition was similar across different samples. No significant change was observed in the diversity of microbiota in the PM2.5, although multiple pathogenic bacteria were detected.4. A prediction function showed that a variety of bacterial phyla correlated positively with the human diseases.5. In summary, the microbial aerosols in the goose shed pose significant risks to the health of the geese. Regular monitoring of the composition of microbial aerosols is important for the healthy growth of geese and disease prevention and control.

A Guide to the Clinical Management of Vipera Snakebite in Italy.

The genus Vipera encompasses most species of medically significant venomous snakes of Europe, with Italy harbouring four of them. Envenomation by European vipers can result in severe consequences, but underreporting and the absence of standardised clinical protocols hinder effective snakebite management. This study provides an updated, detailed set of guidelines for the management and treatment of Vipera snakebite tailored for Italian clinicians. It includes taxonomic keys for snake identification, insights into viper venom composition, and recommendations for clinical management. Emphasis is placed on quick and reliable identification of medically relevant snake species, along with appropriate first aid measures. Criteria for antivenom administration are outlined, as well as indications on managing potential side effects. While the protocol is specific to Italy, its methodology can potentially be adapted for other European countries, depending on local resources. The promotion of comprehensive data collection and collaboration among Poison Control Centres is advocated to optimise envenomation management protocols and improve the reporting of epidemiological data concerning snakebite at the country level.

Evaluation of the ecological risk of pesticide residues from the European LUCAS Soil monitoring 2018 survey.

The 2018 LUCAS (Land Use and Coverage Area frame Survey) Soil Pesticides survey provides a European Union (EU)-scale assessment of 118 pesticide residues in more than 3473 soil sites. This study responds to the policy need to develop risk-based indicators for pesticides in the environment. Two mixture risk indicators are presented for soil based, respectively, on the lowest and the median of available No Observed Effect Concentration (NOECsoil,min and NOECsoil,50) from publicly available toxicity datasets. Two further indicators were developed based on the corresponding equilibrium concentration in the aqueous phase and aquatic toxicity data, which are available as species sensitivity distributions. Pesticides were quantified in 74.5% of the sites. The mixture risk indicator based on the NOECsoil,min exceeds 1 in 14% of the sites and 0.1 in 23%. The insecticides imidacloprid and chlorpyrifos and the fungicide epoxiconazole are the largest contributors to the overall risk. At each site, one or a few substances drive mixture risk. Modes of actions most likely associated with mixture effects include modulation of acetylcholine metabolism (neonicotinoids and organophosphate substances) and sterol biosynthesis inhibition (triazole fungicides). Several pesticides driving the risk have been phased out since 2018. Following LUCAS surveys will determine the effectiveness of substance-specific risk management and the overall progress toward risk reduction targets established by EU and UN policies. Newly generated data and knowledge will stimulate needed future research on pesticides, soil health, and biodiversity protection. Integr Environ Assess Manag 2024;00:1-15. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Targeting WIP1 phosphatase promotes partial remission in experimental collapsing glomerulopathy.

Collapsing focal segmental glomerulosclerosis (FSGS), also known as collapsing glomerulopathy (CG), is the most aggressive variant of FSGS and is characterized by a rapid progression to kidney failure. Understanding CG pathogenesis represents a key step for the development of targeted therapies. Previous work implicated the telomerase protein component TERT in CG pathogenesis, as transgenic TERT expression in adult mice resulted in a CG resembling that seen in human primary CG and HIV-associated nephropathy (HIVAN). Here, we used the telomerase-induced mouse model of CG (i-TERTci mice) to identify mechanisms to inhibit CG pathogenesis. Inactivation of WIP1 phosphatase, a p53 target acting in a negative feedback loop, blocked disease initiation in i-TERTci mice. Repression of disease initiation upon WIP1 deficiency was associated with senescence enhancement and required transforming growth factor-ß functions. The efficacy of a pharmacologic treatment to reduce disease severity in both i-TERTci mice and in a mouse model of HIVAN (Tg26 mice) was then assessed. Pharmacologic inhibition of WIP1 enzymatic activity in either the telomerase mice with CG or in the Tg26 mice promoted partial remission of proteinuria and ameliorated kidney histopathologic features. Histological as well as high-throughput sequencing methods further showed that selective inhibition of WIP1 does not promote kidney fibrosis or inflammation. Thus, our findings suggest that targeting WIP1 may be an effective therapeutic strategy for patients with CG.

Codon optimality modulates protein output by tuning translation initiation.

The impact of synonymous codon choice on protein output has important implications for understanding endogenous gene expression and design of synthetic mRNAs. Previously, we used a neural network model to design a series of synonymous fluorescent reporters whose protein output in yeast spanned a seven-fold range corresponding to their predicted translation speed. Here, we show that this effect is not due primarily to the established impact of slow elongation on mRNA stability, but rather, that an active mechanism further decreases the number of proteins made per mRNA. We combine simulations and careful experiments on fluorescent reporters to argue that translation initiation is limited on non-optimally encoded transcripts. Using a genome-wide CRISPRi screen to discover factors modulating the output from non-optimal transcripts, we identify a set of translation initiation factors including multiple subunits of eIF3 whose depletion restored protein output of a non-optimal reporter. Our results show that codon usage can directly limit protein production, across the full range of endogenous variability in codon usage, by limiting translation initiation.

TKPlate 1.0: An Open-access platform for toxicokinetic and toxicodynamic modelling of chemicals to implement new approach methodologies in chemical risk assessment.

This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8441/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8440/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8437/full.

Circadian clock disruption promotes the degeneration of dopaminergic neurons in male Drosophila.

Sleep and circadian rhythm disruptions are frequent comorbidities of Parkinson's disease (PD), a disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. However, the causal role of circadian clocks in the degenerative process remains uncertain. We demonstrated here that circadian clocks regulate the rhythmicity and magnitude of the vulnerability of DA neurons to oxidative stress in male Drosophila. Circadian pacemaker neurons are presynaptic to a subset of DA neurons and rhythmically modulate their susceptibility to degeneration. The arrhythmic period (per) gene null mutation exacerbates the age-dependent loss of DA neurons and, in combination with brief oxidative stress, causes premature animal death. These findings suggest that circadian clock disruption promotes dopaminergic neurodegeneration.

An in silico insight on the mechanistic aspects of gelsenicine toxicity: A reverse screening study pointing to the possible involvement of acetylcholine binding receptor.

Gelsedine-type alkaloids are highly toxic plant secondary metabolites produced by shrubs belonging to the Gelsemium genus. Gelsenicine is one of the most concerning gelsedine-type alkaloids with a lethal dose lower than 1 mg/Kg in mice. Several reported episodes of poisoning in livestock and fatality cases in humans due to the usage of Gelsemium plants extracts were reported. Also, gelsedine-type alkaloids were found in honey constituting a potential food safety issue. However, their toxicological understanding is scarce and the molecular mechanism underpinning their toxicity needs further investigations. In this context, an in silico approach based on reverse screening, docking and molecular dynamics successfully identified a possible gelsenicine biological target shedding light on its toxicodynamics. In line with the available crystallographic data, it emerged gelsenicine could target the acetylcholine binding protein possibly acting as a partial agonist against α7 nicotinic acetylcholine receptor (AChR). Overall, these results agreed with evidence previously reported and prioritized AChR for further dedicated analysis.

Exploring Dimensionality Reduction Techniques for Deep Learning Driven QSAR Models of Mutagenicity.

Dimensionality reduction techniques are crucial for enabling deep learning driven quantitative structure-activity relationship (QSAR) models to navigate higher dimensional toxicological spaces, however the use of specific techniques is often arbitrary and poorly explored. Six dimensionality techniques (both linear and non-linear) were hence applied to a higher dimensionality mutagenicity dataset and compared in their ability to power a simple deep learning driven QSAR model, following grid searches for optimal hyperparameter values. It was found that comparatively simpler linear techniques, such as principal component analysis (PCA), were sufficient for enabling optimal QSAR model performances, which indicated that the original dataset was at least approximately linearly separable (in accordance with Cover's theorem). However certain non-linear techniques such as kernel PCA and autoencoders performed at closely comparable levels, while (especially in the case of autoencoders) being more widely applicable to potentially non-linearly separable datasets. Analysis of the chemical space, in terms of XLogP and molecular weight, uncovered that the vast majority of testing data occurred within the defined applicability domain, as well as that certain regions were measurably more problematic and antagonised performances. It was however indicated that certain dimensionality reduction techniques were able to facilitate uniquely beneficial navigations of the chemical space.

The VEGA Tool to Check the Applicability Domain Gives Greater Confidence in the Prediction of In Silico Models.

A sound assessment of in silico models and their applicability domain can support the use of new approach methodologies (NAMs) in chemical risk assessment and requires increasing the users' confidence in this approach. Several approaches have been proposed to evaluate the applicability domain of such models, but their prediction power still needs a thorough assessment. In this context, the VEGA tool capable of assessing the applicability domain of in silico models is examined for a range of toxicological endpoints. The VEGA tool evaluates chemical structures and other features related to the predicted endpoints and is efficient in measuring applicability domain, enabling the user to identify less accurate predictions. This is demonstrated with many models addressing different endpoints, towards toxicity of relevance to human health, ecotoxicological endpoints, environmental fate, physicochemical and toxicokinetic properties, for both regression models and classifiers.

Pilot survey reveals ophidiomycosis in dice snakes Natrix tessellata from Lake Garda, Italy.

Ophidiomycosis is an emerging infectious disease caused by the fungus Ophidiomyces ophidiicola (Oo). To date, Oo presence or associated disease condition has been recorded in wild and/or captive snakes from North America, Europe, Asia and Australia, but the data is still scarce outside the Nearctic. Although Italy is a country with a high snake biodiversity in the European panorama, and animals with clinical signs compatible with Oo infection have been documented, to date no investigations have reported the disease in the wild. Therefore, a pilot survey for the Italian territory was performed in conjunction with setting up a complete diagnostic workflow including SYBR Green-based real-time PCR assay for the detection of Oo genomic and mitochondrial DNA combined with histopathology of scale clips. Oo presence was investigated in 17 wild snake specimens from four different species. Four snakes were sampled in a targeted location where the mycosis was suspected via citizen science communications (i.e. North of the Lake Garda), whereas other ophidians were collected following opportunistic sampling. Oo genomic and mitochondrial DNA were detected and sequenced from all four Lake Garda Natrix tessellata, including three juveniles with macroscopic signs such as discolouration and skin crusts. From histopathological examination of scale clips, the three young positive individuals exhibited ulceration, inflammation and intralesional hyphae consistent with Oo infection, and two of them also showed the presence of arthroconidial tufts and solitary cylindrical arthrospores, allowing "Ophidiomycosis and Oo shedder" categorisation. For the remaining snake samples, the real-time PCR tested negative for Oo. This pilot survey permitted to localise for the first time Oo infection in free-ranging ophidians from Italy. Ophidiomycosis from Lake Garda highlights the need to increase sampling efforts in this area as well as in other northern Italian lakes to assess the occurrence of the pathogen, possible risk factors of the infection, its impact on host population fitness and the disease ecology of Oo in European snakes.

A computational study on the biotransformation of alkenylbenzenes by a selection of CYPs: Reflections on their possible bioactivation.

Alkenylbenzenes are aromatic compounds found in several vegetable foods that can cause genotoxicity upon bioactivation by members of the cytochrome P450 (CYP) family, forming 1'-hydroxy metabolites. These intermediates act as proximate carcinogens and can be further converted into reactive 1'-sulfooxy metabolites, which are the ultimate carcinogens responsible for genotoxicity. Safrole, a member of this class, has been banned as a food or feed additive in many countries based on its genotoxicity and carcinogenicity. However, it can still enter the food and feed chain. There is limited information about the toxicity of other alkenylbenzenes that may be present in safrole-containing foods, such as myristicin, apiole, and dillapiole. In vitro studies showed safrole as mainly bioactivated by CYP2A6 to form its proximate carcinogen, while for myristicin this is mainly done by CYP1A1. However, it is not known whether CYP1A1 and CYP2A6 can activate apiole and dillapiole. The present study uses an in silico pipeline to investigate this knowledge gap and determine whether CYP1A1 and CYP2A6 may play a role in the bioactivation of these alkenylbenzenes. The study found that the bioactivation of apiole and dillapiole by CYP1A1 and CYP2A6 is limited, possibly indicating that these compounds may have limited toxicity, while describing a possible role of CYP1A1 in the bioactivation of safrole. The study expands the current understanding of safrole toxicity and bioactivation and helps understand the mechanisms of CYPs involved in the bioactivation of alkenylbenzenes. This information is essential for a more informed analysis of alkenylbenzenes toxicity and risk assessment.

The use of new approach methodologies for the environmental risk assessment of food and feed chemicals.

New Approach Methodologies (NAMs) provide tools for supporting both human and environmental risk assessment (HRA and ERA). This short review provides recent insights regarding the use of NAMs in ERA of food and feed chemicals. We highlight the usefulness of tiered methods supporting weight-of-evidence approaches in relation to problem formulation (i.e., data availability, time, and resource availability). In silico models, including quantitative structure activity relationship models, support filling data gaps when no chemical property or ecotoxicological data are available, and biologically-based models (e.g., toxicokinetic-toxicodynamic models, dynamic energy models, physiologically-based models and species sensitivity distributions) are applicable in more data rich situations, including landscape-based modelling approaches. Particular attention is given to provide practical examples to apply the approaches described in real-world settings. We conclude with future perspectives, with regards to the need for addressing complex challenges such as chemical mixtures and multiple stressors in a wide range of organisms and ecosystems.

A Computational Inter-Species Study on Safrole Phase I Metabolism-Dependent Bioactivation: A Mechanistic Insight into the Study of Possible Differences among Species.

Safrole, a 162.2 Da natural compound belonging to the alkenylbenzenes class, is classified as a possible carcinogen to humans by IARC (group IIB) and has proven to be genotoxic and carcinogenic to rodents. Despite its use as a food or feed additive, it is forbidden in many countries due to its documented toxicity; yet, it is still broadly present within food and feed and is particularly abundant in spices, herbs and essential oils. Specifically, safrole may exert its toxicity upon bioactivation to its proximate carcinogen 1'-hydroxy-safrole via specific members of the cytochrome P450 protein family with a certain inter/intra-species variability. To investigate this variability, an in-silico workflow based on molecular modelling, docking and molecular dynamics has been successfully applied. This work highlighted the mechanistic basis underpinning differences among humans, cats, chickens, goats, sheep, dogs, mice, pigs, rats and rabbits. The chosen metric to estimate the likeliness of formation of 1'-hydroxy-safrole by the species-specific cytochrome P450 under investigation allowed for the provision of a knowledge-based ground to rationally design and prioritise further experiments and deepen the current understanding of alkenylbenzenes bioactivation and CYPs mechanics. Both are crucial for a more informed framework of analysis for safrole toxicity.

Risk Assessment of Combined Exposure to Multiple Chemicals at the European Food Safety Authority: Principles, Guidance Documents, Applications and Future Challenges.

Human health and animal health risk assessment of combined exposure to multiple chemicals use the same steps as single-substance risk assessment, namely problem formulation, exposure assessment, hazard assessment and risk characterisation. The main unique feature of combined RA is the assessment of combined exposure, toxicity and risk. Recently, the Scientific Committee of the European Food Safety Authority (EFSA) published two relevant guidance documents. The first one "Harmonised methodologies for the human health, animal health and ecological risk assessment of combined exposure to multiple chemicals" provides principles and explores methodologies for all steps of risk assessment together with a reporting table. This guidance supports also the default assumption that dose addition is applied for combined toxicity of the chemicals unless evidence for response addition or interactions (antagonism or synergism) is available. The second guidance document provides an account of the scientific criteria to group chemicals in assessment groups using hazard-driven criteria and prioritisation methods, i.e., exposure-driven and risk-based approaches. This manuscript describes such principles, provides a brief description of EFSA's guidance documents, examples of applications in the human health and animal health area and concludes with a discussion on future challenges in this field.

Perfluoroalkyl substances (PFASs) are substrates of the renal human organic anion transporter 4 (OAT4).

Poly- and perfluoroalkyl substances (PFASs) are omnipresent in the environment and have been shown to accumulate in humans. Most PFASs are not biotransformed in animals and humans, so that elimination is largely dependent on non-metabolic clearance via bile and urine. Accumulation of certain PFASs in humans may relate to their reabsorption from the pre-urine by transporter proteins in the proximal tubules of the kidney, such as URAT1 and OAT4. The present study assessed the in vitro transport of 7 PFASs (PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS and PFOS) applying URAT1- or OAT4-transfected human embryonic kidney (HEK) cells. Virtually no transport of PFASs could be measured in URAT1-transfected HEK cells. All PFASs, except PFBS, showed clear uptake in OAT4-transfected HEK cells. In addition, these in vitro results were further supported by in silico docking and molecular dynamic simulation studies assessing transporter-ligand interactions. Information on OAT4-mediated transport may provide insight into the accumulation potential of PFASs in humans, but other kinetic aspects may play a role and should also be taken into account. Quantitative information on all relevant kinetic processes should be integrated in physiologically based kinetic (PBK) models, to predict congener-specific accumulation of PFASs in humans in a more accurate manner.

Congeners-Specific Intestinal Absorption Of Microcystins In An In Vitro 3D Human Intestinal Epithelium: The Role Of Influx/Efflux Transporters.

Microcystins constitute a group of over 200 variants and are increasingly considered as emerging toxins in food and feed safety, particularly with regards to sea-food and fish consumption. Toxicity of MCs is congener-specific, being characterised by different acute potencies, likely related to the differential activity of metabolic enzymes and transporters proteins involved in their cellular uptake. However, the active transport of MCs across intestinal membranes has not been fully elucidated. Our results, obtained using a fit for purpose 3D human reconstructed intestinal epithelium, provide new information on the complex mechanisms involved in the absorption of 5 MC variants': it is indeed characterised by the equilibrium between uptake and extrusion, since the selected congeners are substrates of both influx and efflux proteins. In the range of tested nominal concentrations (10-40 µM) fully representative of relevant exposure scenarios, none of the active tested transporters were saturated. The comparison of permeability (Papp) values of MCs variants highlighted a dose independent relationship for MC-LR, -YR and -RR (Papp x 10-7 ranged from 2.95 to 3.54 cm/s), whereas -LW and-LF showed a dose dependent increase in permeability reaching Papp values which were similar to the other congeners at 40 µM. MC-RR, -LR, -YR show absorption values around 5% of the administered dose. Due to their lipophilicity, MC-LW and -LF were also detected within the cellular compartment. The intestinal uptake was only partially attributable to OATPs, suggesting the involvement of additional transporters. Regarding the efflux proteins, MCs are not P-gp substrates whereas MRP2 and to a lesser extent Breast cancer resistance protein are active in their extrusion. Despite the presence of GST proteins, as an indication of metabolic competence, in the intestinal tissue, MC-conjugates were never detected in our experimental settings.

Cardiotoxicity of Chemical Substances: An Emerging Hazard Class.

(1) Background: Human health risks and hazards from chemical substances are well regulated internationally. However, cardiotoxicity, is not defined as a stand-alone hazard and therefore there are no defined criteria for the classification of substances as cardiotoxic. Identifying and regulating substances that cause cardiovascular adverse effects would undoubtedly strengthen the national health systems. (2) Methods: To overcome the aforementioned gap, a roadmap is proposed for identifying regulatory criteria from animal studies and endorse legislation in order to classify substances as cardiotoxic. The roadmap consists of: (i) the identification of the appropriate animal species and strains; (ii) the identification of the lines of scientific evidence (e.g., histopathological, biochemical and echocardiographic indices etc.) from animal studies with relevance to humans; (iii) the statistical analysis and meta-analysis for each line of scientific evidence after exposure to well-established cardiotoxicants to humans (e.g., anthracyclines) in order to identify threshold values or range of normal and/ or altered values due to exposure; (iv) validation of the above described lines of evidence in animals exposed to other alleged cardiotoxic substances (e.g., anabolic androgen steroids (AAS) and pesticides); (v) establishment of mechanisms of action based on information of either known or alleged cardiotoxicants; and (vi) introduction of novel indices and in silico methods. (3) Results: Preliminary results in rats indicate a clear distinction from normal values to values measured in rats exposed to anthracyclines regarding left ventricle (LV) fractional shortening (FS) and LV ejection fraction (EF). A distinctive pattern is similarly observed for Creatine Kinase-Myocardial Band isoenzyme (CK-MB) and cardiac tissue glutathione (GSH). These findings are encouraging and indicate that there is room for targeted research to this end, and that these specific indices and biochemical markers should be further investigated in order to be developed to regulatory criteria. (4) Conclusions: Further research should be conducted by both the scientific and regulatory community that aims to clearly define the cardiotoxicity hazard caused by chemicals and develop a full set of scientific criteria.

Risk assessment of uptake of trace elements through consumption of cereals: a pilot study in Yerevan, Armenia.

The study aimed to assess the dietary exposure and related human health risks associated with trace elements through the intake of staple cereals, including buckwheat, rice, and emmer. The contents of Lead (Pb), Cadmium (Cd), Mercury (Hg), Nickel (Ni), Molybdenum (Mo), Iron (Fe), and Copper (Cu) were determined using Atomic Absorption Spectrometry. Cereal consumption data were obtained through a Semi-Quantitative Food Frequency Questionnaire amongst the Yerevan adult population. Concentrations of Pb, Cd, Hg, Ni, Mo, Fe and Cu were 0.75-5.56 µg/kg, 1.21-6 µg/kg, 6.7-6.99 µg/kg, 50.6-111 µg/kg, 20-429 µg/kg, 2770-30500 µg/kg and 601-1720 µg/kg respectively. The estimated daily intakes (EDI) of all studied trace elements did not exceed the health-based guidance values. The margin of exposure (MOE) values of Pb, Cd, Hg, and Mo for all clusters were above the threshold (>10 for Pb, Cd, and >100 for Hg, Mo) and therefore did not indicate a health concern. Meanwhile, in the case of Ni exposure, the MOE values for the second and third clusters of buckwheat consumption and for the third cluster of emmer consumption were below the threshold (<10) indicating that a human health risk cannot be excluded. Future work is proposed.