National Institute of Health Dr. Ricardo Jorge
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Modeling Lysosomal Storage Disorders in an Innovative Way: Establishment and Characterization of Stem Cell Lines from Human Exfoliated Deciduous Teeth of Mucopolysaccharidosis Type II Patients
No full textThis article belongs to the Special Issue The Lysosome in Human Health and Diseases.Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.This work was financed by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., within the scope of the project EXPL/BTM-SAL/0659/2021 (http://doi.org/10.54499/EXPL/BTM-SAL/0659/2021). This work was also partially supported by Sanfilippo Children’s Foundation (2019DGH1656/SCF2019I&D) and the Portuguese Society for Metabolic Disorders, SPDM (Bolsa SPDM de apoio à investigação Dr. Aguinaldo Cabral 2018; 2019DGH1629/SPDM2018I&D). Some specific tasks also benefited from additional funds: LAMP1 staining was performed according to the method implemented under the scope of the ongoing FCT project EXPL/BTM-TEC/1477/2021 and using its associated reagents; similarly, the differentiation into the three-germ-layer protocol was performed using a kit acquired and implemented under the scope of another FCT project, previously conducted in the lab (PTDC/BIM-MEC/4762/2014)
Effectiveness of XBB.1.5 Vaccines Against Symptomatic SARS‐CoV‐2 Infection in Older Adults During the JN.1 Lineage‐Predominant Period, European VEBIS Primary Care Multicentre Study, 20 November 2023–1 March 2024
No full textMapear com DDIWe estimated XBB.1.5 vaccine effectiveness (VE) against symptomatic SARS-CoV-2 infection among adults aged ≥65 years during the 2023/2024 JN.1 lineage-predominant period in a European multi-country test-negative case–control study at primary care level. We estimated VE adjusted by study site, age, sex, chronic conditions and onset date. We included 220 cases and 1733 controls. The VE was 48% (95% CI: 12–71), 23% (95% CI: −11–48) and 5% (95% CI: −92–56) among those with symptom onset 1–5, 6–11, and ≥12weeks after vaccination, respectively. XBB.1.5 vaccine provided short and moderate protection against JN.1 symptomatic infection.Funding for this project was received from the European Centre for Disease Prevention and Control (ECDC) implementing Framework Contract ECDC/2021/019 ‘Vaccine effectiveness, burden and impact of COVID-19 and influenza’.info:eu-repo/semantics/publishedVersio
Exploring Cynara cardunculus L. by-products potential: Antioxidant and antimicrobial properties
No full textCynara cardunculus L. (cardoon), a perennial crop indigenous to the Mediterranean region, has gained recognition for its remarkable resilience to diverse weather conditions and its multifaceted applications across various industries, which includes the use of the flower as a vegetable rennet to produce some cheeses, as a source of biomass for energy, or its seed oil for human consumption, biodiesel, and animal feed. In some applications (e.g. biomass or seed production), when crop is harvested at the end of the growth cycle, the leaves remain as the main by-products, along with the flowers. In the context of a circular economy, the aim of this work was to undergone studies to determinate their biological properties (antioxidant and antimicrobial). Methanolic and ethanolic extracts of C. cardunculus L. (globe artichoke var. scolymus (L.) Fiori) and cultivated cardoon (var. altilis DC.)) leaves and flowers were characterised in terms of their polyphenol profile (total phenolic content (TPC), total flavonoid compounds (TFC), and ultra-high performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-ToF-MS)), antioxidant capacity (free radical DPPH inhibition system, β-carotene bleaching assay), and antimicrobial capacity (minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antifungal). In addition, the cultivated cardoon leaves extracts were assessed before and after they were dried in an oven with forced air circulation to evaluate if this treatment affected their bioactive profile. Chlorogenic acid, apigenin, and luteolin were the most quantified of a total of sixteen compounds identified by UHPLC-ToF-MS. Cultivated cardoon dry leaf extract presented the best antioxidant capacity for both methanolic (EC50 = 0.8 mg/mL, antioxidant activity coefficient (AAC) = 279.67) and ethanolic (EC50 =2.1 mg/mL, AAC = 448.06) extracts, compared to the cardoon flower extracts and the globe artichoke leaf ex tracts. Dried cultivated cardoon leaf extracts presented higher antioxidant capacity than fresh cultivated cardoon leaf extracts, but a greater number of polyphenolic compounds were identified in fresh cultivated cardoon leaf extract. The Gram-positive bacteria were more sensitive to the activity of both ethanolic and methanolic extracts than the Gram-negative and cultivated cardoon dry leaf ethanolic extract presented lower MIC and MBC values (125–2000 µg/mL) for most of the tested microorganisms, thus showing higher antimicrobial activity. As for the cultivated cardoon leaf extracts, the dried leaf extracts exhibited better antimicrobial activity, with lower MIC values, than the fresh leaf extracts. The extracts only demonstrated a slight inhibition against the fungi Aspergillus fumigatus. In conclusion, studies performed indicate that dried leaves maintain their biological activities compared to fresh leaves, and that flowers present significant biological activity which suggests the great.Highlights: - The antioxidant activity of different Cynara cardunculus extracts was tested; - Chlorogenic acid, luteolin and apigenin were the main phenolic compounds quantified; - The antimicrobial activity of different C. cardunculus extracts was tested; - The extracts were tested against different Aspergillus species; - Cardoon dried leaf extract had greater antioxidant and antimicrobial activities.This work was supported by the Mechanical Engineering and Resource Sustainability Center—MEtRICs, financed by national funds from the FCT/MCTES (https://doi.org/10.54499/UIDB/04077/2020,
https://doi.org/10.54499/UIDP/04077/2020). This research was funded by PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior)
through the grant UIDB/00211/2020. Cassia H. Barbosa would like to thank for the Ph.D. scholarship from Fundaçao para a Ciência e Tecnologia (FCT) — 2021.08154.BD (https://doi.org/10.54499/2021.08154.BD).info:eu-repo/semantics/publishedVersio
Strengthening the genomic surveillance of Francisella tularensis by using culture-free whole-genome sequencing from biological samples
No full textIntroduction: Francisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited.
Methods: To surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity.
Results: We obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries.
Discussion: Overall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis, being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen.This work was supported by funding from the European Union EU4Health Programme under grant agreement No 101113460 (GENEO) and by the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). The acquisition of WGS-related reagents was supported by the Ministerio de Economía y Competitividad, Government of Spain/FEDER, RESERTULA project (CLG2015-66962-C2-2-R). Fieldwork for sample collection was funded by the Ministerio de Economía y Competitividad, Government of Spain/FEDER ECOTULA project (CGL2015-66962-C2-1-R) and by the Ministerio de Ciencia, Innovación y Universidades BOOMRAT project (PID2019-109327RB-I00) from Spain.info:eu-repo/semantics/publishedVersio
Biomonitoring of firefighters’ exposure to priority pollutant metal(loid)s during wildland fire combat missions: Impact on urinary levels and health risks
No full textWildland firefighters are exposed to metal(loid)s released during wildfires through vegetation combustion, which also promotes remobilization of accumulated anthropogenic metal(loid)s. Studies biomonitoring metal(loid)s exposure promoted exclusively by wildfire suppression activities are lacking. This work aimed to characterize, for the first time, the impact of real-life wildland firefighting operations on urinary levels of priority pollutant metal(loid)s [14 included in ATSDR, 11 in USEPA, and 4 in Human Biomonitoring for Europe Initiative priority lists] in firefighters. Spot urines were sampled pre-exposure (105 non-smokers, 76 smokers) and post-exposure to firefighting activities (20 non-smokers, 25 smokers); among those, paired samples were collected from 14 non-smoking and 24 smoking firefighters. Smokers displayed significantly higher baseline levels of zinc (28 %), lithium (29 %), cadmium (55 %), rubidium (13 %), and copper (20 %) than non-smokers. Following wildfire suppression, the concentration of the WHO potentially toxic metal(loid)s rose from 2 % to 3 % in smokers and 2 % to 5 % in non-smokers (up to 4 % for all firefighters and up to 5 % in paired samples). Levels of nickel (33-53 %), antimony (45-56 %), and cesium (40-47 %) increased significantly post-exposure in non-smokers (in all firefighters and in paired samples), whose urinary concentrations were generally more impacted by wildfire emissions than those of smokers. Arsenic (80 %) displayed the only significant increase post-exposure in smokers, being the best discriminant of exposure to wildfire emissions in these subjects. Significant positive correlations were found for age and/or career length with cadmium, lead, barium, strontium, and mercury, and for body mass index with arsenic. The reference/guidance values were exceeded for arsenic, zinc, cesium, nickel, antimony, cadmium, lead, thallium, mercury, copper, and cobalt in 1-90 % of firefighters suggesting augmented health risks due to wildfire combating and emphasizing the need of mitigation strategies. This study also provides biomonitoring data to help setting reference values for the occupationally exposed part of population.Highlights: - 18 urinary metal(loid)s were biomonitored pre- and post-wildfire combating; - Smokers presented elevated baseline levels of Zn, Li, Cd, Rb, and Cu; - The best discriminant of wildfire emissions exposure was As in smokers; - Cd, Pb, Ba, Sr, and Hg were associated with career length and/or age; - Urinary As, Zn, Cs, Ni, Sb, Cd, Pb, Tl, Hg, Cu, Co guidance values were exceeded.This work was financially supported by the project PCIF/SSO/0017/2018 (http://doi.org/10.54499/PCIF/SSO/0017/2018) by the Fundaçao para a Ciencia e a Tecnologia (FCT), Ministerio da Ciencia, Tecnologia e Ensino Superior through national funds
Psychological impact of life events in systemic lupus erythematosus patients - Differences between flares and remission.
No full textBackground: Stress has been linked to worsening symptoms and increased disease activity in patients with Systemic lupus erythematosus (SLE). Life-events are individual stress points, and there is conflicting evidence regarding their role in SLE activity and disease perception.
Methods: Adult SLE patients were recruited for the study. Clinical and laboratory features of SLE were recorded, and previous diagnosis of anxiety or depression were retrieved from patients' electronic charts. Flares were defined by the Systemic Lupus Erythematosus Disease Activity (SLEDAI) flare Index, and flares during the previous year were documented. During a routine visit, they completed validated Portuguese translations of the 10-item Perceived Stress Scale (PSS-10), Hospital Anxiety and Depression Scale (HADS) and Life Experience Survey (LES) for the previous year.
Results: A total of 47 female SLE patients were recruited. Ten patients (21.3%) had experienced recent flares. Patients with recent flares reported fewer life events, with lower positive, negative, and total weightings sums compared to those without recent flares. Although 42.2% of patients perceived pathological levels of stress in the previous month, 48.9% had anxiety symptoms, and 34% were at high risk for an anxiety disorder, these psychometric measures did not differ significantly between the recent flare and no-flare groups.
Conclusions: There is a high prevalence of pathological levels of stress among SLE patients. SLE patients with recent flares report less psychological impact from life events, both positive and negative, independent of other psychological or pharmacological factors.DGO work is funded by a doctoral research grant from FCT – Foundation for Science and Technology (UI/BD/150742/2020); UMIB (UIDB/00215/2020; UIDP/00215/2020); ITR (LA/P/0064/2020)
Raw milk cheeses from Beira Baixa, Portugal - A contributive study for the microbiological hygiene and safety assessment
No full textDue to specific bacterial microbiota, raw milk cheeses have appreciated sensory properties. However, they may pose a threat to consumer safety due to potential pathogens presence. This study evaluated the microbiological contamination of 98 raw milk cheeses from Beira Baixa, Portugal. Presence and enumeration of Coagulase Positive Staphylococci (CPS), Listeria monocytogenes, Salmonella spp., pathogenic Escherichia coli, and indicator microorganisms (non-pathogenic E. coli and Listeria spp.) was attained. E. coli antimicrobial resistance (AMR) was also evaluated. PCR and/or Whole genome sequencing (WGS) was used to characterize E. coli, Salmonella spp. and L. monocytogenes isolates. Sixteen cheeses (16.3%) were classified as Satisfactory, 59 (60.2%) as Borderline and 23 (23.5%) as Unsatisfactory/Potential Injurious to Health. L. monocytogenes, CPS > 104 cfu g−1, Extraintestinal pathogenic E. coli (ExPEC) and Salmonella spp. were detected in 4.1%, 6.1%, 3.1% and 1.0% of the samples, respectively. Listeria innocua (4.1%) and E. coli > 104 cfu g−1 (16.3%) were also detected. AMR E. coli was detected in 23/98 (23.5%) of the cheese samples, of which two were multidrug resistant. WGS identified genotypes already associated to human disease and Listeria spp. cluster analysis indicated that cheese contamination might be related with noncompliance with Good Hygiene Practices during cheese production
Knowledge and regulation on fungal contamination of sand and water: progress report and perspectives
No full textECMM/ISHAM MYCOSANDS study group: Sevtap Arikan-Akdagli, Aleksandra Barac, Sébastien Bertout, Andra-Cristina Bostanaru, Sara Brito, Michelle Bull, Nilgün Çerikçioğlu, Belinda Chapman, Laurence Delhaes, Maria Efstratiou, Çagri Ergin, Michael Frenkel, Alexis Danielle Guerra, Aurora Gitto, Cláudia Isabel Gonçalves, Hélène Guegan, Nina Gunde-Cimerman, Mümtaz Güran, Laszlo Irinyi, Sunny Jiang, Egle Jonikaitė, Slaven Jozić, Marija Kataržytė, Lena Klingspor, Mihai Mares, Wim Meijer, Willem Melchers, Joseph Meletiadis, Wieland Meyer, Valentin Nastasa, Monika Novak-Babič, Dilara Ogunc, Betil Ozhak, Anna Prigitano, Stéphane Ranque, Malcolm Richardson, Frédéric Roger, Raluca-Oana Rusu, Raquel Sabino, Ana Sampaio, Susana Silva, Helena Solo-Gabriele, Jayne Stephens, Marlou Tehupeiory-Kooreman, Anna-Maria Tortorano, Aristea Velegraki, Cristina Veríssimo, Darija Vukić Lušić, Georgoa Wunderlich.ReviewFungal flora in coastal/inland beach sand and recreational water is a neglected field of study, despite its potential impact on human health. A joint International Society for Human and Animal Mycology/European Confederation for Medical Mycology (ISHAM/ECMM) working group was formed in 2019 with the task to set up a vast international initiative aimed at studying the fungal contamination of beaches and bathing waters. Here we review the importance of the topic, and list the main results and achievements from 12 scientific publications. Fungal contamination exists at different levels, and the genera most frequently found were Aspergillus spp., Candida spp., Fusarium spp., and Cryptococcus spp., both in sand and in water. A site-blind median was found to be 89 colony-forming units of fungi per gram of sand in coastal/inland freshwaters. This threshold has been used for the sand quality criterion of the blue flag in Portugal. Additionally, our data were considered pivotal and therefore used for the first inclusion of fungi as a biological taxon of interest in water quality and sand monitoring recommendations of the World Health Organization's new guidelines on recreational water quality (Vol.1-Chap7). The findings of the consortium also suggest how environmental conditions (climate, salinity, soil pH, nitrogen, etc.) influence microbial communities in different regions, and that yeast species like Candida glabrata, Clavispora lusitaniae, and Meyerozyma guilliermondii have been identified as potential fungal indicators of fecal contamination. Climate change and natural disasters may affect fungal populations in different environments, and because this is still a field of study under exploration, we also propose to depict the future challenges of research and unmet needs.info:eu-repo/semantics/publishedVersio
Genomic study of European Clostridioides difficile ribotype 002/ sequence type 8
No full textClostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.This work was supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement no. 773830: One Health European Joint Programme
COVID-19 Vaccine Effectiveness in Autumn and Winter 2022 to 2023 Among Older Europeans
No full textKey Points: - Question: What was the effectiveness of COVID-19 vaccines administered in autumn and winter 2022 to 2023 against symptomatic SARS-CoV-2 infection among people aged 60 years or older in Europe, and how did different exposed or reference groups affect effectiveness?
- Findings: In this case-control study of 9308 primary care patients at 11 European sites, within 3 months of vaccination, all COVID-19 vaccine effectiveness (CVE) estimates were 29% to 39% against SARS-CoV-2 viruses and 44% to 52% against the XBB variants. All point estimates decreased by time after vaccination, with no vaccine protection after 6 months.
- Meaning: Findings of this study suggest that COVID-19 vaccination campaigns should precede peaks in SARS-CoV-2 incidence and that effectiveness of new vaccines against emerging variants should be continually monitored using seasonal CVE approaches.Importance: In the context of emerging SARS-CoV-2 variants or lineages and new vaccines, it is key to accurately monitor COVID-19 vaccine effectiveness (CVE) to inform vaccination campaigns.
Objective: To estimate the effectiveness of COVID-19 vaccines administered in autumn and winter 2022 to 2023 against symptomatic SARS-CoV-2 infection (with all circulating viruses and XBB lineage in particular) among people aged 60 years or older in Europe, and to compare different CVE approaches across the exposed and reference groups used.
Design, setting, and participants: This case-control study obtained data from VEBIS (Vaccine Effectiveness, Burden and Impact Studies), a multicenter study that collects COVID-19 and influenza data from 11 European sites: Croatia; France; Germany; Hungary; Ireland; Portugal; the Netherlands; Romania; Spain, national; Spain, Navarre region; and Sweden. Participants were primary care patients aged 60 years or older with acute respiratory infection symptoms who were recruited at the 11 sites after the start of the COVID-19 vaccination campaign from September 2022 to August 2023. Cases and controls were defined as patients with positive and negative, respectively, reverse transcription-polymerase chain reaction (RT-PCR) test results.
Exposures: The exposure was COVID-19 vaccination. The exposure group consisted of patients who received a COVID-19 vaccine during the autumn and winter 2022 to 2023 vaccination campaign and 14 days or more before symptom onset. Reference group included patients who were not vaccinated during or in the 6 months before the 2022 to 2023 campaign (seasonal CVE), those who were never vaccinated (absolute CVE), and those who were vaccinated with at least the primary series 6 months or more before the campaign (relative CVE). For relative CVE of second boosters, patients receiving their second booster during the campaign were compared with those receiving 1 booster 6 months or more before the campaign.
Main outcomes and measures: The outcome was RT-PCR-confirmed, medically attended, symptomatic SARS-CoV-2 infection. Four CVE estimates were generated: seasonal, absolute, relative, and relative of second boosters. CVE was estimated using logistic regression, adjusting for study site, symptom onset date, age, chronic condition, and sex.
Results: A total of 9308 primary care patients were included, with 1687 cases (1035 females; median [IQR] age, 71 [65-79] years) and 7621 controls (4619 females [61%]; median [IQR] age, 71 [65-78] years). Within 14 to 89 days after vaccination, seasonal CVE was 29% (95% CI, 14%-42%), absolute CVE was 39% (95% CI, 6%-60%), relative CVE was 31% (95% CI, 15% to 44%), and relative CVE of second boosters was 34% (95% CI, 18%-47%) against all SARS-CoV-2 variants. In the same interval, seasonal CVE was 44% (95% CI, -10% to 75%), absolute CVE was 52% (95% CI, -23% to 82%), relative CVE was 47% (95% CI, -8% to 77%), and relative CVE of second boosters was 46% (95% CI, -13% to 77%) during a period of high XBB circulation. Estimates decreased with time since vaccination, with no protection from 180 days after vaccination.
Conclusions and relevance: In this case-control study among older Europeans, all CVE approaches suggested that COVID-19 vaccines administered in autumn and winter 2022 to 2023 offered at least 3 months of protection against symptomatic, medically attended, laboratory-confirmed SARS-CoV-2 infection. The effectiveness of new COVID-19 vaccines against emerging SARS-CoV-2 variants should be continually monitored using CVE seasonal approaches.This study was funded by framework contract ECDC/2021/019 from the ECDC.info:eu-repo/semantics/publishedVersio