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- European pilot interlaboratory comparison study on Mpox virus whole genome sequencingPublication . Fuchs, Jonas; Bertelli, Claire; Pillonel, Trestan; Cordeiro, Rita; Izopet, Jacques; Pasquier, Christophe; Lewandowski, Kuiama; Maks, Anastasija; Michel, Janine; Rodriguez-Sanchez, Belen; Sanches-Seco, Maria Paz; Ledesma, Juan; Sobral, Daniel; Vercauteren, Koen; de Block, Tessa; Rezende, Antonio Mauro; Brinkmann, Annika; Nitsche, Andreas; Greub, Gilbert; Panning, MarcusObjectives: Since 2022, distinct Mpox virus (MPXV) clades have been spreading across different geographic regions, causing a challenging epidemiological situation. Whole genome sequencing (WGS) proved to be instrumental for patient management and global public health. We report a pilot interlaboratory comparison study for MPXV WGS. Methods: We distributed noninfectious DNA samples, including the main MPXV clades I and II, to eight European laboratories. We included one cowpox (CPXV) sample as a specificity control. Participants were free to choose their WGS pipeline of choice to mimic a real-world scenario and were asked to report on the sequencing pipeline used, average genome coverage, and MPXV species, clade, and subclade assignments. Results: Seven of the eight invited laboratories reported results back. All participants largely identified the MPXV clades and reported high-quality genomes with minimal variations, specifically for MPXV clade IIb 2022 outbreak strains. However, reconstructed genomes showed high variability for nonclade IIb MPXV strains. The CPXV sample was correctly identified by three laboratories. Conclusions: Although results for MPXV clade IIb 2022 outbreak strains are reassuring, the inclusion of MPXV clade I and IIa strains highlights pitfalls for targeted sequencing approaches and subsequent bioinformatic analyses. Our findings underscore the need for standardized external quality assessment studies.
- Assessing exposure and early biological effects in waste management workers using a harmonized occupational biomonitoring studyPublication . Tavares, Ana; Rosário, Rita; Aimonen, K.; Louro, Henriqueta; Martins, Carla; Viegas, Susana; Santonen, Tiina; Silva, Maria JoãoObjective: Human biomonitoring provides measurements of internal exposure to all chemicals by all routes of exposures and also addresses associated early biological effects. An occupational biomonitoring study requires a comprehensive and harmonised planning, including participants’ recruitment, sampling strategy, biomarkers definition and analysis, data collection, management, and reporting, in order to generate comparable and replicable data. In the framework of the European Partnership for the Assessment of Risks from Chemicals (PARC), a multi-centric biomonitoring campaign was planned to assess occupational exposure to hazardous chemicals from electronic and plastic waste management industries. We here present its design, comprising the assessment of external and internal exposure to several substances and characterization of early biological effects. Methods: Target participants are workers involved in e-waste and plastic recycling activities, and non-exposed individuals as controls. Besides industrial hygiene samples (e.g., air, settle dust) also biological samples (hair, blood, epithelial buccal cells and urine) are collected after informed consent, and a questionnaire is applied to all participants. Exposure biomarkers comprise measurements of heavy metals, flame retardants, per- and polyfluoroalkyl substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), bisphenols, polychlorobiphenyls (PCBs), and other plasticizers in hair, blood, or urine samples by qualified laboratories. Effect biomarkers are assessed in blood (genotoxicity – micronucleus and comet assay - inflammation, epigenetic and oxidative stress markers), buccal cells (micronucleus) and urine samples (metabolic changes). Occupational hygiene measurements and contextual information will facilitate the interpretation of biomarkers data and the identification of potential confounding variables. Expected Results: The findings are expected to provide valuable information on exposure and associated early (preclinical) effects, and also to uncover groups at increased risk, supporting the promotion of good work practices and/or the implementation of efficient risk management strategies further improving worker’s health protection.
- Discriminative Protein Markers of Second-Hand Smoke Exposure Identified by Shotgun ProteomicsPublication . Neves, Sofia; Pacheco, Solange A.; Vaz, Fátima; Saraiva, Joana; James, Peter; Simões, Tânia; Penque, DeborahObjective: Chronic exposure to second-hand smoke (SHS) increases the risk of developing tobacco-related pathologies such as lung cancer and cardiovascular diseases. This study aimed to identify potential protein biomarkers for response and risk assessment of SHS exposure. Methods: A shotgun proteomics approach was employed to analyse protein expression profiles in nasal epithelium and plasma samples from healthy, non-smoking restaurant workers who were either exposed or not exposed to SHS in the workplace. A label-free quantification strategy was used to measure differential protein expression between the two groups. Logistic regression modelling was applied to identify the proteins that best discriminated exposed individuals from non-exposed controls, with the goal of establishing an expression profile indicative of SHS-related response. Results: In the nasal epithelium, SHS exposure was associated with modulation of proteins involved in HIF1α-regulated glycolytic pathways, xenobiotic metabolism, cell proliferation, and differentiation. In plasma, differentially expressed proteins were related to systemic inflammation and atherosclerosis. Among these, three plasma proteins—Histidine-rich glycoprotein (HRG), Vitamin D-binding protein (GC), and Leucine-rich alpha-2-glycoprotein (LRG1)—showed a significant discriminatory potential between SHS-exposed and non-exposed individuals. Conclusions: The identified proteins, particularly HRG, GC, and LRG1, are promising response biomarkers for SHS exposure. Their expression profiles may support the development of molecular tools for individual response assessment associated with environmental tobacco smoke.
- Impact of nanocelluloses on genome-wide DNA methylation pattern of human pulmonary and intestinal cellsPublication . Ventura, Célia; Vital, Nádia; Valente, Ana; Vieira, Luís; Louro, Henriqueta; Silva, Maria JoãoObjective: Nanocellulose is an innovative nanomaterial with interesting physicochemical properties for several industrial and biomedical applications and its safety for human health must be ensured. This study aimed to identify DNA methylation changes in human pulmonary and intestinal cells after exposure to two fibrillar celluloses with different physicochemical properties, both derived from Eucaliptus globulus. Their cellular effects were investigated in silico by functional pathway and gene ontology (GO) analysis. Methods: We applied Reduced Representation Bisulfite Sequencing to analyze the methylation differences in DNA CpG-rich regions from human bronchial (BEAS-2B) and intestinal (Caco-2) cells exposed for 24h to 14.3 µg/mL of cellulose nanofibrils (CNF) or microfibrils (CMF) versus non-exposed ones. A bioinformatics pipeline was implemented for identifying differentially methylated genes (DMGs), functional pathways, and GO associations. Results: CNF and CMF exposure resulted in 11 and 14 DMGs, respectively, in BEAS-2B cells, 6 being common to both nanocelluloses. In Caco-2 cells, 36 and 31 DMGs were identified, sharing 12 DMGs. No DMGs were shared between these cell lines. Hypomethylation predominated in BEAS-2B cells, and hypermethylation in Caco-2 cells. In BEAS-2B cells, both nanocelluloses affected similar pathways and GO terms (e.g., regulation of DNA replication, damage repair and senescence, telomere maintenance, and D-glucose transport). In Caco-2 cells, both CNF and CMF enriched, for instance, signal transduction, glycosylation, and cytoskeletal dynamics. Each nanocellulose type also affected other different pathways and terms. Conclusions: Nanocellulose may have a wide impact on the metabolism and survival of pulmonary and intestinal cells through several regulatory pathways, which depend on nanocellulose physicochemical properties. Cell type also influences the outcome, suggesting tissue-specific effects. These findings highlight the relevance of DNA methylation in nanotoxicology, providing insights into underlying molecular mechanisms of action. Keywords: gene ontology; nanomaterial; pathway analysis; RRBS
- Genotoxic effects of Alternaria mycotoxins in human liver HepG2 cellsPublication . Ventura, Célia; Vilela, Rita Sofia; Guerreiro, Beatriz; Louro, Henriqueta; Silva, Maria JoãoObjective: Mycotoxins are natural toxic compounds produced by filamentous fungi as secondary metabolites. Human exposure to mycotoxins occurs predominantly through ingestion of contaminated food, and have been associated with nephrotoxicity, hepatotoxicity, immunotoxicity, carcinogenicity, and teratogenicity. Alternaria mycotoxins are produced by black moulds of the genus Alternaria, which are common plant pathogens and saprophytes widely distributed in the environment. However, limited toxicological data exists on Alternaria mycotoxins. Within the scope of the European Partnership for the Assessment of Risks from Chemicals (PARC; https://www.eu-parc.eu/) these toxins were considered as priority substances, and several studies are underway with the aim of filling knowledge gaps regarding their genotoxicity, among other toxic effects. Methods: Genotoxicity was evaluated using the In Vitro Mammalian Cell Micronucleus Assay with cytokinesis block (CBMN) according to the OECD TG 487. HepG2 liver cells were exposed for 48 h to a concentration-range of each Alternaria mycotoxin following dose-range finding based on cytotoxicity testing (MTT assay). Vinblastine was used as a positive control. Results: All tested mycotoxins were cytotoxic in the MTT assay and genotoxic in the CBMN assay. In addition to the significant increase in micronucleated binucleated cells, some mycotoxins also induced a significant increase in other nuclear anomalies in HepG2 cells, showing a dose-response relationship. Conclusions: These results indicate that the studied Alternaria mycotoxins induce chromosomal damage, which can lead to genomic instability, a key driver in cancer. Therefore, this study contributes to PARC objectives, providing critical toxicological data for their hazard assessment following human oral exposure. The data will contribute to support their risk assessment and management by regulators and policy makers in order to protect human health from these emerging food contaminants.