Evaluation of the impact of iPSC differentiation protocols on transcriptomic signatures.

Human induced pluripotent stem cells (iPSC) have the potential to produce desired target cell types in vitro and allow for the high-throughput screening of drugs/chemicals at population level thereby minimising the cost of drug discovery and drug withdrawals after clinical trials. There is a substantial need for the characterisation of the iPSC derived models to better understand and utilise them for toxicological relevant applications. In our study, iPSC (SBAD2 or SBAD3 lines obtained from StemBANCC project) were differentiated towards toxicologically relevant cell types: alveolar macrophages, brain capillary endothelial cells, brain cells, endothelial cells, hepatocytes, lung airway epithelium, monocytes, podocytes and renal proximal tubular cells. A targeted transcriptomic approach was employed to understand the effects of differentiation protocols on these cell types. Pearson correlation and principal component analysis (PCA) separated most of the intended target cell types and undifferentiated iPSC models as distinct groups with a high correlation among replicates from the same model. Based on PCA, the intended target cell types could also be separated into the three germ layer groups (ectoderm, endoderm and mesoderm). Differential expression analysis (DESeq2) presented the upregulated genes in each intended target cell types that allowed the evaluation of the differentiation to certain degree and the selection of key differentiation markers. In conclusion, these data confirm the versatile use of iPSC differentiated cell types as standardizable and relevant model systems for in vitro toxicology.

Avian Haemosporidian Infection in Wildlife Rehabilitation Centres of Portugal: Causes, Consequences, and Genetic Diversity.

In the last decade, over 40% of bird species in Europe have experienced poor and bad conservation status, with more than 30% of bird species in mainland Portugal threatened with extinction. Along with anthropogenic factors, parasites and pathogens such as avian haemosporidians have been suggested to be responsible for these avian population declines. Wildlife rehabilitation centres play an essential role in species conservation and preservation. Moreover, animals admitted for rehabilitation can provide valuable information regarding transmission and pathogenicity of many diseases that affect wild birds that are rarely sampled in nature. However, reports of haemosporidians in captive birds are still limited. Here, we explored the prevalence and genetic diversity of avian haemosporidians in 89 birds from 29 species admitted to rehabilitation centres in Portugal, showing an overall infection prevalence of 30.3%. The prevalence of infection was higher in Strigiformes and in birds admitted to rehabilitation centres due to debilitating diseases. Remarkably, 30% of the infected bird species have not been found to harbour malaria parasites in preceding studies. We detected 15 different haemosporidian lineages infecting a third of bird species sampled. Notably, 2 out of these 15 detected haemosporidian lineages have not been obtained previously in other studies. Furthermore, we also identified nine new host-parasite interactions representing new host records for these haemosporidian parasites. Finally, our results revealed that birds infected with haemosporidians require longer rehabilitation treatments, which increase the economic costs for rehabilitation and may impair their survival prospects. These findings emphasise the importance of integrating haemosporidian infection considerations into rehabilitation protocols, highlighting the challenges posed by these infections in avian conservation and rehabilitation, including economic and logistical demands.

HTSplotter: An end-to-end data processing, analysis and visualisation tool for chemical and genetic in vitro perturbation screening.

In biomedical research, high-throughput screening is often applied as it comes with automatization, higher-efficiency, and more and faster results. High-throughput screening experiments encompass drug, drug combination, genetic perturbagen or a combination of genetic and chemical perturbagen screens. These experiments are conducted in real-time assays over time or in an endpoint assay. The data analysis consists of data cleaning and structuring, as well as further data processing and visualisation, which, due to the amount of data, can easily become laborious, time-consuming and error-prone. Therefore, several tools have been developed to aid researchers in this process, but these typically focus on specific experimental set-ups and are unable to process data of several time points and genetic-chemical perturbagen screens. To meet these needs, we developed HTSplotter, a web tool and Python module that performs automatic data analysis and visualization of visualization of eitherendpoint or real-time assays from different high-throughput screening experiments: drug, drug combination, genetic perturbagen and genetic-chemical perturbagen screens. HTSplotter implements an algorithm based on conditional statements to identify experiment types and controls. After appropriate data normalization, including growth rate normalization, HTSplotter executes downstream analyses such as dose-response relationship and drug synergism assessment by the Bliss independence (BI), Zero Interaction Potency (ZIP) and Highest Single Agent (HSA) methods. All results are exported as a text file and plots are saved in a PDF file. The main advantage of HTSplotter over other available tools is the automatic analysis of genetic-chemical perturbagen screens and real-time assays where growth rate and perturbagen effect results are plotted over time. In conclusion, HTSplotter allows for the automatic end-to-end data processing, analysis and visualisation of various high-throughput in vitro cell culture screens, offering major improvements in terms of versatility, efficiency and time over existing tools.

Rosin from Pinus pinaster Portuguese forests shows a regular profile of resin acids.

Pinus pinaster forestry occupies >20% of the forest ecosystem area in the continental territory of Portugal with a high impact on the national economy. This species' major derived non-wood product is oleoresin, the raw material for rosin production. Rosin comprises mainly a blend of resin acids and has broad industrial and pharmaceutical applications. Oleoresin production in Portugal has been progressively reduced due to low-cost producers in other countries; currently, it reaches only 2% of the existing P. pinaster trees. To support this value chain, the chemical fingerprint of rosin derived from the national forest requires focused analysis. In the present study, we collected oleoresin within seven geographically distinct pure P. pinaster forests in two consecutive collection years. A high-resolution nuclear magnetic resonance (NMR) method was used to quantify the diversity of resin acids in the corresponding rosin samples. Overall, the acquired data highlighted that the profile of resin acids in P. pinaster rosin produced in Portugal is highly regular, regardless of the forest location, having as the major constituents abietic acid and dehydroabietic acid. The diversity of resin acids is possibly influenced, to a minor extent, by some edaphoclimatic factors.

'From the sky to the ground': fishers' knowledge, landscape analysis and hydrological data indicate long-term environmental changes in Amazonian clear water rivers.

Fishers possess detailed local ecological knowledge (LEK) which can be a valuable resource for tracking long-term environmental changes in less studied tropical rivers. Our goal was to investigate such changes in three clear water rivers in the Brazilian Amazon, focusing on hydrology, water quality and land cover. Additionally, we aimed to compare these changes among three rivers (Trombetas, Tapajós and Tocantins) representing a potential gradient of environmental changes. We interviewed 129 fishers (67 in Tapajós, 33 in Tocantins and 29 in Trombetas), and analyzed temporal series on land cover and hydrology respectively through maps produced by the project MapBiomas, and data from the Brazilian National Water Agency across the last 34 years (from 1985 to 2019). The complementary analyses of these three databases (mapping, hydrological data and fishers' knowledge) revealed environmental changes in the studied rivers. The maps showed a gradient of anthropic changes on land cover, from the less altered Trombetas river, the moderately altered Tapajós and the more intensely changed landscape in the Tocantins River. Fishers from the Tocantins River reported a greater variety of negative changes in water quality related to anthropic actions, such as dams, deforestation, and pollution. Additionally, most fishers indicated hydrological changes making the Tocantins River drier in more recent years, which would cause negative effects on fish populations. In the Tapajós River, fishers mentioned more varied hydrological patterns and negative effects on water quality linked to mining activities, whereas in Trombetas fishers perceived increased floods. The changes mentioned by the interviewed fishers matched observed trends from hydrological data indicating a trend of increasing droughts in the more impacted Tocantins River. Fishers' knowledge provided exclusive 'on the ground' data to track long-term changes on local hydrology and water quality, as well as inform the effects of these changes on fish and fisheries.

Integrating distribution kinetics and toxicodynamics to assess repeat dose neurotoxicity in vitro using human BrainSpheres: a case study on amiodarone.

For ethical, economical, and scientific reasons, animal experimentation, used to evaluate the potential neurotoxicity of chemicals before their release in the market, needs to be replaced by new approach methodologies. To illustrate the use of new approach methodologies, the human induced pluripotent stem cell-derived 3D model BrainSpheres was acutely (48 h) or repeatedly (7 days) exposed to amiodarone (0.625-15 µM), a lipophilic antiarrhythmic drug reported to have deleterious effects on the nervous system. Neurotoxicity was assessed using transcriptomics, the immunohistochemistry of cell type-specific markers, and real-time reverse transcription-polymerase chain reaction for various genes involved in the lipid metabolism. By integrating distribution kinetics modeling with neurotoxicity readouts, we show that the observed time- and concentration-dependent increase in the neurotoxic effects of amiodarone is driven by the cellular accumulation of amiodarone after repeated dosing. The development of a compartmental in vitro distribution kinetics model allowed us to predict the change in cell-associated concentrations in BrainSpheres with time and for different exposure scenarios. The results suggest that human cells are intrinsically more sensitive to amiodarone than rodent cells. Amiodarone-induced regulation of lipid metabolism genes was observed in brain cells for the first time. Astrocytes appeared to be the most sensitive human brain cell type in vitro. In conclusion, assessing readouts at different molecular levels after the repeat dosing of human induced pluripotent stem cell-derived BrainSpheres in combination with the compartmental modeling of in vitro kinetics provides a mechanistic means to assess neurotoxicity pathways and refine chemical safety assessment for humans.

Morphologic and Genetic Analysis of Synhimantus (Synhimantus) laticeps from a Long-Eared Owl (Asio otus).

The long-eared owl (Asio otus) is a medium-sized owl species that is well-distributed in almost all of the territories in Portugal. Nematodes were found in the oral cavity of a long-eared owl (A. otus) admitted to CRASSA (Wildlife Rehabilitation Centre of Santo André). During a physical exam and stabilization of the bird, five nematodes were collected. The worms were examined and measured under light microscopy, and photos were taken. After a morphological analysis was conducted, all the nematodes (five females) were identified as Synhimantus (Synhimantus) laticeps. Two specimens were subjected to molecular analysis, which confirmed the result. This study provides a combined morphological and genetic approach to S. laticeps. To the authors' best knowledge, this is the first report including genetic sequencing of S. laticeps in a long-eared owl (A. otus) from Portugal.

Anamnesis ambiental como parte de la consulta pediátrica: estudio piloto / Environmental history-taking as part of pediatric consultations: a pilot study

Introducción. La salud ambiental infantil es la rama de la pediatría que estudia la influencia del medioambiente en la salud y la enfermedad de los niños. Las exposiciones ambientales globales representan una seria amenaza para la salud, lo que justifica una mayor investigación y acción. Objetivo. Evaluar la salud ambiental de una muestra de niños que viven en áreas urbanas y rurales de la ciudad de Uruguaiana, Brasil. Población y métodos. Se incluyeron padres/tutores (n = 714) de niños atendidos en el Policlínico Infantil de la Ciudad de Uruguaiana de enero a octubre de 2021, que respondieron la anamnesis ambiental en pediatría (Sociedad Brasileña de Pediatría). Los datos obtenidos se analizaron según la residencia en zona urbana o rural, o el ingreso familiar. Resultados. Al comparar los habitantes de la zona urbana (n = 660) con los de la zona rural (n = 54), verificamos que entre los de la zona rural fue significativamente mayor la actividad con productos químicos (15 % vs. 32,7 %; p = 0,004), vivir cerca de plantación (7,5 % vs. 74,5 %; p <0,001) o con fuente de contaminación (4,8 % vs. 32,7 %; p <0,001), tener perro (62 % vs. 87,3 %; p <0,001), usar plaguicidas (0,6 % vs. 32,7 %; p <0,001) y exposición a contaminación química (2,6 % vs. 18,2 %; p <0,001). En el área urbana predominó la exposición al tránsito de vehículos cerca de la vivienda (85 % vs. 48,1 %; p <0,001), renta media inferior a 3 salarios mínimos (90 %) y baja escolaridad. Conclusión. Realizar la anamnesis ambiental es fundamental para la detección de amenazas ambientales presentes en los lugares donde los niños y adolescentes viven, aprenden, juegan y estudian.

Introduction. Children's environmental health studies the influence of the environment on health and disease in children. Global environmental exposures pose a serious threat to health, warranting further research and action. Objective. To assess the environmental health of a sample of children living in urban and rural areas in Uruguaiana, Brazil. Population and methods. We included parents/legal guardians (n = 714) of children seen at Policlinica Infantil de Uruguaiana between January and October 2021, who completed the environmental history- taking in pediatrics (Brazilian Society of Pediatrics). Collected data were analyzed based on place of residence (urban or rural) or household income. Results. The comparison between inhabitants of the urban area (n = 660) and the rural area (n = 54) established that, among those living in the rural area, activity with chemical substances (15% versus 32.7%; p = 0.004), living near a plantation (7.5% versus 74.5%; p < 0.001) or near a source of contamination (4.8% versus 32.7%; p < 0.001), having a dog (62% versus 87.3%; p < 0.001), using pesticides (0.6% versus 32.7%; p < 0.001), and exposure to chemical contamination (2.6% versus 18.2%; p < 0.001) were significantly higher. In the urban area, exposure to vehicle traffic near the house (85% versus 48.1%; p < 0.001), an average income below 3 minimum wages (90%), and a low level of education predominated. Conclusion. Environmental history-taking is critical for the detection of environmental threats present in the areas where children and adolescents live, learn, play, and study

Environmental history-taking as part of pediatric consultations: a pilot study. / Anamnesis ambiental como parte de la consulta pediátrica: estudio piloto.

Introduction. Children's environmental health studies the influence of the environment on health and disease in children. Global environmental exposures pose a serious threat to health, warranting further research and action. Objective. To assess the environmental health of a sample of children living in urban and rural areas in Uruguaiana, Brazil. Population and methods. We included parents/legal guardians (n = 714) of children seen at Policlinica Infantil de Uruguaiana between January and October 2021, who completed the environmental historytaking in pediatrics (Brazilian Society of Pediatrics). Collected data were analyzed based on place of residence (urban or rural) or household income. Results. The comparison between inhabitants of the urban area (n = 660) and the rural area (n = 54) established that, among those living in the rural area, activity with chemical substances (15% versus 32.7%; p = 0.004), living near a plantation (7.5% versus 74.5%; p < 0.001) or near a source of contamination (4.8% versus 32.7%; p < 0.001), having a dog (62% versus 87.3%; p < 0.001), using pesticides (0.6% versus 32.7%; p < 0.001), and exposure to chemical contamination (2.6% versus 18.2%; p < 0.001) were significantly higher. In the urban area, exposure to vehicle traffic near the house (85% versus 48.1%; p < 0.001), an average income below 3 minimum wages (90%), and a low level of education predominated. Conclusion. Environmental history-taking is critical for the detection of environmental threats present in the areas where children and adolescents live, learn, play, and study.

Introducción. La salud ambiental infantil es la rama de la pediatría que estudia la influencia del medioambiente en la salud y la enfermedad de los niños. Las exposiciones ambientales globales representan una seria amenaza para la salud, lo que justifica una mayor investigación y acción. Objetivo. Evaluar la salud ambiental de una muestra de niños que viven en áreas urbanas y rurales de la ciudad de Uruguaiana, Brasil. Población y métodos. Se incluyeron padres/tutores (n = 714) de niños atendidos en el Policlínico Infantil de la Ciudad de Uruguaiana de enero a octubre de 2021, que respondieron la anamnesis ambiental en pediatría (Sociedad Brasileña de Pediatría). Los datos obtenidos se analizaron según la residencia en zona urbana o rural, o el ingreso familiar. Resultados. Al comparar los habitantes de la zona urbana (n = 660) con los de la zona rural (n = 54), verificamos que entre los de la zona rural fue significativamente mayor la actividad con productos químicos (15 % vs. 32,7 %; p = 0,004), vivir cerca de plantación (7,5 % vs. 74,5 %; p <0,001) o con fuente de contaminación (4,8 % vs. 32,7 %; p <0,001), tener perro (62 % vs. 87,3 %; p <0,001), usar plaguicidas (0,6 % vs. 32,7 %; p <0,001) y exposición a contaminación química (2,6 % vs. 18,2 %; p <0,001). En el área urbana predominó la exposición al tránsito de vehículos cerca de la vivienda (85 % vs. 48,1 %; p <0,001), renta media inferior a 3 salarios mínimos (90 %) y baja escolaridad. Conclusión. Realizar la anamnesis ambiental es fundamental para la detección de amenazas ambientales presentes en los lugares donde los niños y adolescentes viven, aprenden, juegan y estudian.

Digoxin Induces Human Astrocyte Reaction In Vitro.

Astrocyte reaction is a complex cellular process involving astrocytes in response to various types of CNS injury and a marker of neurotoxicity. It has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes have been reported with age, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected. However, the relationship between energy metabolism and astrocyte reactivity in the context of neurotoxicity is not known. We hypothesized that changes in energy metabolism of astrocytes will be coupled to their activation by xenobiotics. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics, and extracellular flux analyses after 24 h of exposure of human ReN-derived astrocytes to digoxin (1-10 µM) or TNFα (30 ng/ml) used as a positive control. Strong astrocytic reaction was observed, accompanied by increased glycolysis at low concentrations of digoxin (0.1 and 0.5 µM) and after TNFα exposure, suggesting that increased glycolysis may be a common feature of reactive astrocytes, independent of the triggering molecule. In conclusion, whether astrocyte activation is triggered by cytokines or a xenobiotic, it is strongly tied to energy metabolism in human ReN-derived astrocytes. Increased glycolysis might be considered as an endpoint to detect astrocyte activation by potentially neurotoxic compounds in vitro. Finally, ReN-derived astrocytes may help to decipher mechanisms of neurotoxicity in ascertaining the ability of chemicals to directly target astrocytes.

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition.

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.

Glycidamide and cis-2-butene-1,4-dial (BDA) as potential carcinogens and promoters of liver cancer - An in vitro study.

Acrylamide and furan are environmental and food contaminants that are metabolized by cytochrome P450 2E1 (CYP2E1), giving rise to glycidamide and cis-2-butene-1,4-dial (BDA) metabolites, respectively. Both glycidamide and BDA are electrophilic species that react with nucleophilic groups, being able to introduce mutations in DNA and perform epigenetic remodeling. However, whereas these carcinogens are primarily metabolized in the liver, the carcinogenic potential of acrylamide and furan in this organ is still controversial, based on findings from experimental animal studies. With the ultimate goal of providing further insights into this issue, we explored in vitro, using a hepatocyte cell line and a hepatocellular carcinoma cell line, the putative effect of these metabolites as carcinogens and cancer promoters. Molecular alterations were investigated in cells that survive glycidamide and BDA toxicity. We observed that those cells express CD133 stemness marker, present a high proliferative capacity and display an adjusted expression profile of genes encoding enzymes involved in oxidative stress control, such as GCL-C, GSTP1, GSTA3 and CAT. These molecular changes seem to be underlined, at least in part, by epigenetic remodeling involving histone deacetylases (HDACs). Although more studies are needed, here we present more insights towards the carcinogenic capacity of glycidamide and BDA and also point out their effect in favoring hepatocellular carcinoma progression.

Upscaling biological complexity to boost neuronal and oligodendroglia maturation and improve in vitro developmental neurotoxicity (DNT) evaluation.

Human induced pluripotent stem cell (iPSC)-derived neuronal and glial cell models are suitable to assess the effects of environmental chemicals on the developing brain. Such test systems can recapitulate several key neurodevelopmental features, such as neural stem cell formation and differentiation towards different neuronal subtypes and astrocytes, neurite outgrowth, synapse formation and neuronal network formation and function, which are crucial for brain development. While monolayer, two-dimensional (2D) cultures of human iPSC-neuronal or glial derivatives are generally suited for high-throughput testing, they also show some limitations. In particular, differentiation towards myelinating oligodendrocytes can only be achieved after extended periods in differentiation. In recent years, the implementation of three-dimensional (3D) neuronal and glial models obtained from human iPSCs has been shown to compensate for such limitations, enabling robust differentiation towards both neuronal and glial cell populations, myelination and formation of more mature neuronal network activity. Here we compared the differentiation capacity of human iPSC-derived neural stem cells cultured either as 2D monolayer or as 3D neurospheres, and assessed chlorpyrifos (CPF) effects. Data indicate that 3D neurospheres differentiate towards neurons and oligodendroglia more rapidly than 2D cultures; however, the 2D model is more suitable to assess neuronal functionality by analysis of spontaneous electrical activity using multielectrode array. Moreover, 2D and 3D test systems are diversely susceptible to CPF treatment. In conclusion, the selection of the most suitable in vitro test system (either 2D or 3D) should take into account the context of use and intended research goals ('fit for purpose' principle).

An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat.

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health.

Diversity of microorganisms in Hyalomma aegyptium collected from spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia.

Ticks carry a diverse community of microorganisms including non-pathogenic symbionts, commensals, and pathogens, such as viruses, bacteria, protozoans, and fungi. The assessment of tick-borne microorganisms (TBM) in tortoises and their ticks is essential to understand their eco-epidemiology, and to map and monitor potential pathogens to humans and other animals. The aim of this study was to characterize the diversity of microorganisms found in ticks collected from the spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia. Ticks feeding on wild T. graeca were collected, and pathogens were screened by polymerase chain reaction using group-specific primers. In total, 131 adult Hyalomma aegyptium ticks were collected from 92 T. graeca in Morocco (n = 48), Tunisia (n = 2), Algeria (n = 70), and Turkey (n = 11). Bacteria and protozoa detected included Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), relapsing-fever borreliae (8.4%), Ehrlichia spp. (7.6%), Rickettsia spp. (3.4%), Borrelia burgdorferi s.l. (0.9%), Francisella spp. (0.9%), and Wolbachia spp. (0.8%). The characterization of Rickettsia included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R.africae (Morocco). Hemolivia mauritanica and Ehrlichia spp. prevalence varied significantly with the sampling region/country. We did not detect significant associations in microorganism presence within ticks, nor between microorganism presence and tick mitochondrial DNA haplogroups. This is the first report of Francisella persica-like, relapsing fever borreliae, M. mitochondrii, and Wolbachia spp. in H. aegyptium ticks collected from wild hosts from the South and Eastern Mediterranean region, and of R. sibirica mongolitimonae and R. africae in H. aegyptium from Algeria and Morocco, respectively. Given that T. graeca is a common species in commercial and non-commercial pet trade, the evaluation of the role of this species and its ticks as hosts for TBM is particularly relevant for public health.

MEIS2 Is an Adrenergic Core Regulatory Transcription Factor Involved in Early Initiation of TH-MYCN-Driven Neuroblastoma Formation.

Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member (gm6890), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin (CLSPN) and three chromosome 17q loci CBX2, GJC1 and LIMD2. Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.

Prevalence of Antibodies to Toxoplasma gondii in Different Wild Bird Species Admitted to Rehabilitation Centres in Portugal.

Toxoplasma gondii is a worldwide zoonotic parasite. According to the "One Health" approach, studies on toxoplasmosis are essential since it affects humans and domestic and wild animals. In the present study, antibodies to T. gondii were determined in serum samples from 263 wild birds located in five wildlife rehabilitation centres in mainland Portugal by using the modified agglutination test (MAT) with a cut-off titre of 20. An overall seroprevalence of 36.5% (95% confidence interval [CI]: 30.7-42.6) was observed. For the first time, antibodies to T. gondii were detected in some avian species, including pallid swift (Apus pallidus) (33.3%), black-backed gull (Larus fuscus) (39.3%), European turtle-dove (Streptopelia turtur) (100%), bee-eater (Merops apiaster) (50.0%), carrion crow (Corvus corone) (33.3%), and Egyptian vulture (Neophron percnopterus) (100%), which expands the list of intermediate hosts of T. gondii. A lower seroprevalence was found in juvenile birds (31.9%) compared to adults (48.7%) (p = 0.016). The central region of Portugal was considered a risk factor for T. gondii infection in wild birds (odds ratio: 3.61; 95% CI: 1.09-11.91). This pioneer study calls attention to the need for further studies, to provide a clearer understanding of T. gondii epidemiology in Portugal, because it reflects wide dispersion of T. gondii oocysts in the environment.

Neuroinflammatory Response to TNFα and IL1ß Cytokines Is Accompanied by an Increase in Glycolysis in Human Astrocytes In Vitro.

Astrogliosis has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism, and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes as a function of age have been reported, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected in supporting a functional switch of astrocytes from neurotrophic to neurotoxic. This study aimed to explore the metabolic changes occurring in astrocytes during their activation. Astrocytes were derived from human ReN cell neural progenitors and characterized. They were activated by exposure to tumor necrosis factor alpha (TNFα) or interleukin 1ß (IL1ß) for 24 h. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics and extracellular flux analyses. ReN-derived astrocytes reactivity was observed by the modifications of genes and proteins linked to inflammation (cytokines, nuclear factor-kappa B (NFκB), signal transducers and activators of transcription (STATs)) and immune pathways (major histocompatibility complex (MHC) class I). Increased NFκB1, NFκB2 and STAT1 expression, together with decreased STAT3 expression, suggest an activation towards the detrimental pathway. Strong modifications of astrocyte cytoskeleton were observed, including a glial fibrillary acidic protein (GFAP) decrease. Astrogliosis was accompanied by changes in energy metabolism characterized by increased glycolysis and lactate release. Increased glycolysis is reported for the first time during human astrocyte activation. Astrocyte activation is strongly tied to energy metabolism, and a possible association between NFκB signaling and/or MHC class I pathway and glycolysis is suggested.

Interferon-gamma signaling is downregulated in peripheral blood mononuclear cells from asthma patients / A sinalização de interferon-gama é diminuída em células mononucleares do sangue periférico de pacientes asmáticos

Introduction: Interferon-gamma (IFN-g) signaling is mediated by crosstalk of receptors, such as IFN-g receptor 1 (IFN-g R1), transcription factors, such as signal transducer and activator of transcription 1 (STAT1) and suppressors of cytokine signaling 1 (SOCS1). Here, we evaluated the role of IFN-g signaling in peripheral blood mononuclear cells (PBMCs) from asthma patients and control individuals. Methods: PBMCs from adult healthy nonasthmatic controls (n = 12; male and female, 18-60 years old) and patients diagnosed with asthma (n = 18; male and female, 18-60 years old) were stimulated with IFN-g (0.25, 0.5 and/or 1.0 ng/mL) and, after 24h, the production of CXC motif chemokine 10 (CXCL10) was evaluated (by enzyme linked immunosorbent assay) as well as the expression of IFN-g R1, STAT1 (both by flow cytometry assay) and SOCS1 (by real-time qPCR assay). Results: CXCL10 production was reduced in a dose-dependent manner in PBMCs from asthma patients stimulated with IFN-g when compared to control individuals. While IFN-g induced an increase in IFN-g R1 expression and phosphorylated STAT1 (pSTAT1) activation in PBMCs from the control group, a reduction in both IFN-g R1 and pSTAT1 was observed in PBMCs from asthma patients. IFN-g increased SOCS1 mRNA expression in PBMCs from asthma patients when compared to IFN-g-stimulated cells from control individuals. Conclusion: Taken together, our results demonstrated that IFN-g signaling is downregulated in asthma patients.

Introdução: A sinalização de interferon-gama (IFN-g) é mediada por receptores, como o receptor 1 de IFN-gama (IFN-gR1), fatores de transcrição, como o transdutor de sinal e o ativador de transcrição 1 (STAT1) e supressores de sinalização de citocina 1 (SOCS1). Neste trabalho, avaliamos o papel da sinalização de IFN-g em células mononucleares do sangue periférico (PBMCs) de indivíduos com asma e controle. Métodos: Células mononucleares do sangue periférico (PBMCs) de adultos saudáveis e não asmáticos (n = 12, homens e mulheres, 18-60 anos) e pacientes diagnosticados com asma (n = 18, homens e mulheres, 18-60 anos) foram estimuladas com IFN-g (0,25, 0,5 e/ou 1,0 ng/mL) e após 24 horas a produção de CXCL10 foi avaliada por ensaio de imunoabsorção enzimática (ELISA), bem como o receptor 1 de IFN-g (IFN-g R1). Também foram avaliadas as expressões do transdutor de sinal e ativador da transcrição 1 (STAT1) (por citometria de fluxo) e supressor de expressão de sinalização de citocinas 1 (SOCS1) (por ensaio qPCR em tempo real). Resultados: A produção de CXCL10, uma quimiocina induzida por IFNg, foi reduzida de maneira dependente da dose em PBMCs de pacientes com asma estimulados com IFN-g (0,25-1,0 ng/mL) quando comparado ao grupo controle. Enquanto IFN-g induziu um aumento da expressão de IFN-g R1 e ativação da fosforilação de STAT1 (pSTAT1) em PBMCs do grupo controle, uma redução de ambas (IFN-g R1 e pSTAT1) foi observada em PBMCs de pacientes com asma. O IFN-g aumentou as PBMCs de expressão do mRNA de SOCS1 de pacientes com asma quando comparado às células estimuladas por IFN-g do controle. Conclusão: Em conjunto, nossos resultados demonstraram que a sinalização de IFN-g é sub-regulada em pacientes com asma.

Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept.

BACKGROUND: In light of the vulnerability of the developing brain, mixture risk assessment (MRA) for the evaluation of developmental neurotoxicity (DNT) should be implemented, since infants and children are co-exposed to more than one chemical at a time. One possible approach to tackle MRA could be to cluster DNT chemicals in a mixture on the basis of their mode of action (MoA) into 'similar' and 'dissimilar', but still contributing to the same adverse outcome, and anchor DNT assays to common key events (CKEs) identified in DNT-specific adverse outcome pathways (AOPs). Moreover, the use of human in vitro models, such as induced pluripotent stem cell (hiPSC)-derived neuronal and glial cultures would enable mechanistic understanding of chemically-induced adverse effects, avoiding species extrapolation. METHODS: HiPSC-derived neural progenitors differentiated into mixed cultures of neurons and astrocytes were used to assess the effects of acute (3 days) and repeated dose (14 days) treatments with single chemicals and in mixtures belonging to different classes (i.e., lead(II) chloride and methylmercury chloride (heavy metals), chlorpyrifos (pesticide), bisphenol A (organic compound and endocrine disrupter), valproic acid (drug), and PCB138 (persistent organic pollutant and endocrine disrupter), which are associated with cognitive deficits, including learning and memory impairment in children. Selected chemicals were grouped based on their mode of action (MoA) into 'similar' and 'dissimilar' MoA compounds and their effects on synaptogenesis, neurite outgrowth, and brain derived neurotrophic factor (BDNF) protein levels, identified as CKEs in currently available AOPs relevant to DNT, were evaluated by immunocytochemistry and high content imaging analysis. RESULTS: Chemicals working through similar MoA (i.e., alterations of BDNF levels), at non-cytotoxic (IC20/100), very low toxic (IC5), or moderately toxic (IC20) concentrations, induce DNT effects in mixtures, as shown by increased number of neurons, impairment of neurite outgrowth and synaptogenesis (the most sensitive endpoint as confirmed by mathematical modelling) and increase of BDNF levels, to a certain extent reproducing autism-like cellular changes observed in the brain of autistic children. CONCLUSIONS: Our findings suggest that the use of human iPSC-derived mixed neuronal/glial cultures applied to a battery of assays anchored to key events of an AOP network represents a valuable approach to identify mixtures of chemicals with potential to cause learning and memory impairment in children.