Browsing by Issue Date, starting with "2022-02"
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- Multiparametric In Vitro Toxicity Assessment of Consumer-relevant Nanomaterials in a Human Placental ModelPublication . Pires, Joana; Moreira, Luciana; Teixeira, João Paulo; Fraga, SóniaThe increasing number of consumer products containing nanomaterials (NM) has raised serious concerns regarding their environmental and human safety. Among the categories of NM of large market volume are metal nanoparticles (M-NP), inorganic non-metallic and carbon-based NM [1]. So far, little is known about the impact of NM exposure on placental development and function. Thus, our study aimed at evaluating the in vitro toxicity of NM of consumer relevance in human trophoblastic BeWo clone b30 epithelial cells, a widely used human placental model. BeWo b30 cells were exposed for 24 h to varied concentrations (0.8-48 µg/cm2) of NM of different chemical composition [Au, Ag, TiO2, SiO2 and graphene oxide (nano_GO)], primary size (10, 30 and 60 nm Au- and AgNP), capping (citrate- and PEGylated AuNP) and crystal structure (TiO2 NP rutile and anatase forms). In vitro cytotoxicity was assessed by determining changes in cell morphology (optical microscopy), metabolic activity (AlamarBlue assay), plasma membrane integrity (LDH release) and intracellular reactive oxygen species (ROS; DCFH-DA assay) and ATP levels (luminescent assay). In vitro genotoxicity (DNA strand breaks and oxidative damage) was assessed by the comet assay. No evident changes in cell morphology were observed after exposure to any tested NM. Overall, NM can be ranked for cytotoxicity as AgNP > nano_GO > AuNP ~TiO2 NP ~ SiO2 NP, being the effects more visible at higher concentrations. Regarding M-NP, the influence of the size in the cytotoxic-induced effects was more evident for AgNP than for AuNP, with the smaller NP causing more cytotoxicity in BeWo b30 cells. PEG capping was an effective protection, preventing the cytotoxic effects that were visible in cells exposed to the tested citrate-capped AuNP. No significant differences between rutile-anatase and anatase TiO2 NP-induced cytotoxicity were observed. Exposure to AgNP and nano_GO significantly increased ROS levels of the exposed cells suggesting that oxidative stress is a possible mechanism underlying their cytotoxicity in BeWo b30 cells. All tested NM significantly increased intracellular ATP levels compared to control cells, except for 10 nm AuNP. No significant changes in both DNA strand-breaks levels and DNA oxidative damage were detected for all tested NM. Our findings highlight the potential hazard associated with human placental exposure to NM, where the physicochemical properties are important determinants of their toxicity. Additional research is needed for a deeper understanding of NM impact on human placental barrier, to unravel their mechanisms of action and the properties responsible for NM-mediated toxicity, and thus support regulatory decisions that protect consumers and ultimately assist in the development of safer NM.
- Epilepsy progression is associated with cumulative DNA methylation changes in inflammatory genesPublication . Martins-Ferreira, Ricardo; Leal, Bárbara; Chaves, João; Li, Tianlu; Ciudad, Laura; Rangel, Rui; Santos, Agostinho; Martins da Silva, António; Pinho Costa, Paulo; Ballestar, EstebanMesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) is the most common focal epilepsy in adults. It is characterized by alarming rates of pharmacoresistance. Epileptogenesis is associated with the occurrence of epigenetic alterations, and the few epigenetic studies carried out in MTLE-HS have mainly focused on the hippocampus. In this study, we obtained the DNA methylation profiles from both the hippocampus and anterior temporal neocortex of MTLE-HS patients subjected to resective epilepsy surgery and autopsied non-epileptic controls. We assessed the progressive nature of DNA methylation changes in relation to epilepsy duration. We identified significantly altered hippocampal DNA methylation patterns encompassing multiple pathways known to be involved in epileptogenesis. DNA methylation changes were even more striking in the neocortex, wherein pathogenic pathways and genes were common to both tissues. Most importantly, DNA methylation changes at many genomic sites varied significantly with epilepsy duration. Such progressive changes were associated with inflammation-related genes in the hippocampus. Our results suggest that the neocortex, relatively spared of extensive histopathological damage, may also be involved in epilepsy development. These results also open the possibility that the observed neocortical impairment could represent a preliminary stage of epileptogenesis before the establishment of chronic lesions or a consequence of prolonged seizure exposure. Our two-tissue multi-level characterization of the MTLE-HS DNA methylome suggests the occurrence of a self-propagating inflammatory wave of epigenetic dysregulation.
- The Clinical Genome Resource (ClinGen) Familial Hypercholesterolemia Variant Curation Expert Panel consensus guidelines for LDLR variant classificationPublication . Chora, Joana R.; Iacocca, Michael A.; Tichý, Lukáš; Wand, Hannah; Kurtz, C. Lisa; Zimmermann, Heather; Leon, Annette; Williams, Maggie; Humphries, Steve E.; Hooper, Amanda J.; Trinder, Mark; Brunham, Liam R.; Costa Pereira, Alexandre; Jannes, Cinthia E.; Chen, Margaret; Chonis, Jessica; Wang, Jian; Kim, Serra; Johnston, Tami; Soucek, Premysl; Kramarek, Michal; Leigh, Sarah E.; Carrié, Alain; Sijbrands, Eric J.; Hegele, Robert A.; Freiberger, Tomáš; Knowles, Joshua W.; Bourbon, Mafalda; ClinGen Familial Hypercholesterolemia Expert PanelPurpose: In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published consensus standardized guidelines for sequence-level variant classification in Mendelian disorders. To increase accuracy and consistency, the Clinical Genome Resource Familial Hypercholesterolemia (FH) Variant Curation Expert Panel was tasked with optimizing the existing ACMG/AMP framework for disease-specific classification in FH. In this study, we provide consensus recommendations for the most common FH-associated gene, LDLR, where >2300 unique FH-associated variants have been identified. Methods: The multidisciplinary FH Variant Curation Expert Panel met in person and through frequent emails and conference calls to develop LDLR-specific modifications of ACMG/AMP guidelines. Through iteration, pilot testing, debate, and commentary, consensus among experts was reached. Results: The consensus LDLR variant modifications to existing ACMG/AMP guidelines include (1) alteration of population frequency thresholds, (2) delineation of loss-of-function variant types, (3) functional study criteria specifications, (4) cosegregation criteria specifications, and (5) specific use and thresholds for in silico prediction tools, among others. Conclusion: Establishment of these guidelines as the new standard in the clinical laboratory setting will result in a more evidence-based, harmonized method for LDLR variant classification worldwide, thereby improving the care of patients with FH.
- Genomic surveillance of Neisseria meningitidis serogroup W in Portugal from 2003 to 2019Publication . Bettencourt, Célia; Nunes, Alexandra; Gomes, João Paulo; Simões, Maria JoãoIn recent years, a change in the epidemiology of meningococcal disease caused by Neisseria meningitidis serogroup W (MenW) has been observed worldwide, with the emergence of new sublineages associated with a higher rate of fatal cases. The present study intends to describe the epidemiology of invasive meningococcal disease (IMD) due to MenW in Portugal between 2003 and 2019, and to genetically characterize population structure. Despite MenW has a low incidence in Portugal, having almost disappeared from 2008 to 2015, since 2016, the number of MenW cases has been steadily increasing at a rate of ~ twofold per year, with more than 80% of the characterized isolates belonging to clonal complex 11 (cc11). Core-genome phylogeny of 25 Portuguese (PT) MenW isolates showed a strain clustering mainly either with the Original UK or the UK 2013 sublineages. Our study also reported for the first time the presence of distinct prophages with a notable overrepresentation of an ~ 32-35-kb PS_1-like prophage found in MenW cc11 genomes. The presence of the PS_1-like prophage in almost all 4723 cc11 genomes selected from Neisseria PubMLST database regardless of the capsular group they belong to suggests an ancestral acquisition of this mobile element prior to capsular switching events. Overall, by mimicking the scenario observed worldwide, this study reinforces the importance of a close monitoring of MenW disease, especially from cc11, in order to promptly adapt the vaccination plan for IMD control in Portugal. Moreover, future studies are needed to understand the putative contribution of prophages to fitness and virulence of PT MenW strains.
- A Multidisciplinary Approach to the First Autochthonous Case of Tularemia Reported in PortugalPublication . Cunha, F.; Lopes de Carvalho, I.; Torres, C.; Gonçalves, R.Francisella tularensis, a Gram-negative coccobacillus, is a highly virulent pathogen responsible for several zoonotic outbreaks in Europe in the last few decades. The authors report the case of a 46-year-old male who developed fever, myalgias and headache a week after having contact with animal feed contaminated by rodents. Serological tests were positive for Francisella tularensis. This first case of autochthonous tularemia in Portugal led to an intensive investigation involving several healthcare services and national governmental authorities. The authors address the possible underdiagnosis of this infection in the country.
- Assessment of occupational exposure to hexavalent chromium – recommendations from HBM4EU chromate studyPublication . Santonen, Tiina; Bocca, Beatrice; Bousoumah, Radia; Duca, Radu Corneliu; Galea, Karen S.; Godderis, Lode; Göen, Thomas; Hardy, Emilie; Iavicoli, Ivo; Janasik, Beata; Jones, Kate; Leese, Elizabeth; Leso, Veruscka; Louro, Henriqueta; Majery, Nicole; Ndaw, Sophie; Pinhal, Hermínia; Porras, Simo P.; Scheepers, Paul T.J.; Sepai, Ovnair; Silva, Maria João; van Nieuwenhuyse, An; Verdonck, Jelle; Viegas, Susana; Wasowicz, WojciechIntroduction: Hexavalent chromium (Cr(VI)) is an important occupational carcinogen. In addition to air monitoring biomonitoring is commonly applied to monitor exposure to Cr(VI). Within the EU biomonitoring initiative, HBM4EU, we explored the applicability of different biomonitoring methods in the assessment of occupational exposure to Cr(VI) in welding and surface treatment activities. Materials and Methods: A multi-center cross-sectional study was performed in Belgium, Finland, France, Italy, Poland, Portugal, the Netherlands, Luxembourg and United Kingdom. Harmonized procedures were used to collect biological and industrial hygiene samples. Contextual information was collected using questionnaires. Altogether 602 exposed workers and controls were included in the study. Exposure biomarkers studied included urinary, red blood cell (RBC) and plasma Cr, and exhaled breath condensate (EBC) Cr(VI)/Cr(III). In addition, number of effect biomarkers were studied. Results: All exposure markers showed highest exposure levels among chrome plating workers. U-Cr showed a good correlation with air Cr(VI) in bath platers and welders. Observed low correlations between different exposure biomarkers suggest that these approaches are not interchangeable but rather complementary. Conclusions: U-Cr showed its value as the first approach for the assessment of internal exposure to Cr(VI). We recommend pre- and post-shift samples for low exposure levels. RBC/P-Cr and EBC-Cr(VI)/Cr(III) provide additional information when more specific information on exposure is needed. The current exposure levels require analytical methods with high sensitivity.
- Molecular Identification and Ecology of Portuguese Wild-Caught Phlebotomine Sandfly SpecimensPublication . Zé-Zé, L.; Amaro, F.; Osório, H.; Giovanetti, M.; Lourenço, J.; Alves, M.J.Phlebotomine sandflies are important vectors of Leishmania spp. and phleboviruses causing disease in animals and humans. Morphological identification of phlebotomine sandflies to the species level is challenging, requiring microscopical examination of the genitalia, which is demanding and time consuming. Molecular sandfly species identification can be a practical solution to save resources since it enables further molecular studies capable of generating data, such as biting preferences by blood meal analysis. In this study, resorting to a sandfly dataset collected between 2014 and 2018 across Portuguese territory under active mosquito surveillance and sandfly specific surveys, we used molecular methods to explore the genetic diversity and spatial distribution, further exploring ecological co-variants of four sandfly species—Phlebotomus ariasi, P. perniciosus, P. sergenti, and Sergentomyia minuta—all of which are of public health importance. Sandflies were collected from Spring to Autumn (May–November) following local temperature patterns. P. perniciosus was the most widespread detected species, with a nationwide distribution. All studied species clustered together with known samples from the Iberian Peninsula. Further monitoring studies of sandfly species diversity, distribution, and seasonality are essential for surveillance and control of sandfly-borne pathogens both nationally and globally
- Nocturnal Birds of Prey as Carriers of Staphylococcus aureus and Other Staphylococci: Diversity, Antimicrobial Resistance and Clonal LineagesPublication . Silva, Vanessa; Lopes, Ana Filipa; Soeiro, Vanessa; Caniça, Manuela; Manageiro, Vera; Pereira, José Eduardo; Maltez, Luís; Capelo, José Luis; Igrejas, Gilberto; Poeta, PatríciaOwls are nocturnal predators that inhabit urbanized and farmlands. They are in direct contact with other animals, both livestock and small wild rodents that they mostly feed on. Staphylococci can be both commensal and pathogenic bacteria that are widespread across the various ecological niches. We aimed to isolate staphylococci from owls and to characterize their antimicrobial resistance, virulence factors and genetic lineages. Swab samples were collected from the throat and cloaca of 114 owls admitted to two rehabilitation centers in Portugal. The identification of staphylococci species was performed by MALDI-TOF. Staphylococci antimicrobial resistance and virulence genes were investigated by means of the disk diffusion method and PCR. Staphylococcus aureus isolates were characterized by MLST, agr and spa-typing. Of the tested animals, 66 isolates were recovered, including 10 different species of staphylococci, of which 25 were coagulase-positive (CoPS) and 41 were coagulase-negative (CoNS). Twenty-three S. aureus were isolated, of which one mecC-MRSA was identified. The isolates were mainly resistant to penicillin, aminoglycosides, clindamycin and tetracycline. mecC-MRSA belonged to ST1245 and spa-type t843 and the remaining S. aureus were ascribed to 12 STs and 15 spa types. A high diversity of clonal lineages was identified among the S. aureus isolated from wild owls. Owls feed mainly on small rodents often exposed to waste and anthropogenic sources, which may explain the moderate prevalence of S. aureus in these animals.
- The Prevalence of Arcobacteraceae in Aquatic Environments: A Systematic Review and Meta-AnalysisPublication . Venâncio, Igor; Luís, Ângelo; Domingues, Fernanda; Oleastro, Mónica; Pereira, Luísa; Ferreira, SusanaMembers of the family Arcobacteraceae are distributed widely in aquatic environments, and some of its species have been associated with human and animal illness. However, information about the diversity and distribution of Arcobacteraceae in different water bodies is still limited. In order to better characterize the health risk posed by members in the family Arcobacteraceae, a systematic review and meta-analysis-based method was used to investigate the prevalence of Arcobacteraceae species in aquatic environments based on available data published worldwide. The database search was performed using related keywords and considering studies up to February 2021. The pooled prevalence in aquatic environments was 69.2%, ranging from 0.6 to 99.9%. These bacteria have a wide geographical distribution, being found in diverse aquatic environments with the highest prevalence found in raw sewage and wastewater treatment plants (WWTP), followed by seawater, surface water, ground water, processing water from food processing plants and water for human consumption. Assessing the effectiveness of treatments in WWTP in eliminating this contamination, it was found that the wastewater treatment may not be efficient in the removal of Arcobacteraceae. Among the analyzed Arcobacteraceae species, Al. butzleri was the most frequently found species. These results highlight the high prevalence and distribution of Arcobacteraceae in different aquatic environments, suggesting a risk to human health. Further, it exposes the importance of identifying and managing the sources of contamination and taking preventive actions to reduce the burden of members of the Arcobacteraceae family.
- Avaliação das propriedades antibacterianas de cianobactérias de água doce para aplicação em antibióticos naturaisPublication . Dias, Iara; Dias, Elsa; Dias, Deodália Maria AntunesA descoberta dos antibióticos revolucionou a medicina, mas a sua eficácia está comprometida devido à disseminação de mecanismos de resistência a antibióticos, sendo urgente pesquisar novos compostos antibacterianos. As cianobactérias têm sido estudadas quanto à produção de metabolitos secundários com atividade antibacteriana. Este trabalho teve como objetivo avaliar a capacidade antibacteriana de vinte e cinco estirpes de cianobactérias pertencentes à coleção ESSACC. Para tal, foram produzidos extratos de hexano, diclorometano:metanol (2:1), metanol 70% e água destas estirpes, em duas extrações sequenciais com estes solventes: (I) do menos polar para o mais polar; (II) do mais polar para o menos polar. O fenótipo de suscetibilidade de nove bactérias patogénicas a esses extratos foi avaliado pelos métodos da Difusão em Disco e da Microdiluição. Pelo método da Difusão em Disco nenhum dos extratos inibiu totalmente o crescimento bacteriano. No entanto, houve um decréscimo do crescimento de Pseudomonas aeruginosa ATCC 27853 quando exposta aos extratos de hexano (I) de Aphanizomenon flos-aquae (LMECYA 088), metanol (II) de Oscillatoria limosa (SAG 42.87), diclorometano:metanol (I)/ metanol (I) de Arthrospira platensis (SAG 85.79) e diclorometano:metanol (I)/(II) de Nostoc sp. (LMECYA 330). Pelo teste da Microdiluição verificou-se que o extrato metanólico (I) de A. platensis (SAG 85.79), a 25% v/v, inibiu totalmente o crescimento de P. aeruginosa. Adicionalmente, analisou-se a atividade adjuvante destes extratos em antibióticos de referência para a P. aeruginosa e observou-se que todos potenciaram a atividade de aztreonam e piperaciclina/tazobactam, excetuando o extrato de diclorometano:metanol (I) de A. platensis (SAG 85.79) que apenas potenciou a atividade de aztreonam. O extrato de diclorometano:metanol (I) de Nostoc sp. (LMECYA 330) apresentou também atividade sinérgica com o ertapenem. Os resultados obtidos neste estudo permitiram identificar quatro estirpes cianobacterianas como potenciais fontes de compostos antibacterianos, o que pode contribuir para a mitigação do fenómeno da resistência a antibióticos.