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- Voices of youth: youth participation in the CO-CREATE ProjectPublication . Meshkovska, Biljana; Moerman, Gerben; Baillergeau, Evelyne; Bröer, Christian; Lien, Nanna; Luszczynska, Aleksandra; Rito, Ana Isabel; Knai, Cécile; Helleve, ArnfinnIntroduction: Applying youth participatory action research (YPAR) has become common practice in public health. However, there are challenges in applying YPAR and there is a need for improving the process by learning from youth that has participated in such projects. This study explores youth involvement in the CO-CREATE project which focused on developing overweight and obesity prevention policies. Methods: This is a qualitative study, based on three data sources: 1) fieldnotes taken by project staff during observation of youth (approximately 150 observations) 2) semi-structured interviews with project staff (n = 12) 3) direct feedback from participating youth through feedback forms (n = 51). Data was analyzed in NVivo, following the principles of reflexive thematic analysis. Results: Five themes were generated which showcase youth involvement in CO-CREATE: 1) an inspired and engaged youth; 2) insights through respectful interactions; 3) a strength of voice and call to action; 4) challenges in understanding project and disengagement; and 5) interpersonal conflict and pressure to participate. Conclusion: Taking the time to build trust and create a feeling of equality is important. It is also important to allow youth to take the lead when they wish so and have clear ideas of how to do so. Voices of rebelliousness can be a show of strength and help reach the goals of the project. Participation in CO-CREATE may have contributed toward individual empowerment of youth.
- Influenza vaccine effectiveness in Europe and the birth cohort effect against influenza A(H1N1)pdm09: VEBIS primary care multicentre study, 2023/24Publication . Kissling, Esther; Maurel, Marine; Pozo, Francisco; Pérez-Gimeno, Gloria; Buda, Silke; Sève, Noémie; Domegan, Lisa; Hooiveld, Mariëtte; Oroszi, Beatrix; Martínez-Baz, Iván; Guiomar, Raquel; Latorre-Margalef, Neus; Mlinarić, Ivan; Lazar, Mihaela; Giménez Duran, Jaume; Dürrwald, Ralf; Enouf, Vincent; McKenna, Adele; de Lange, Marit; Túri, Gergő; Trobajo-Sanmartín, Camino; GOMEZ TEIXEIRA PINTO, VERÓNICA DEL PILAR; Samuelsson Hagey, Tove; Višekruna Vučina, Vesna; Cherciu, Maria Carmen; García Vazquez, Miriam; Erdwiens, Annika; Masse, Shirley; Bennett, Charlene; Meijer, Adam; Kristóf, Katalin; Castilla, Jesús; Rodrigues, Ana Paula; Kurečić Filipović, Sanja; Ivanciuc, Alina Elena; Bacci, Sabrina; Kaczmarek, MarlenaIntroduction: Influenza A(H1N1)pdm09, A(H3N2) and B/Victoria viruses circulated in Europe in 2023/24, with A(H1N1)pdm09 dominance. First influenza infections in childhood may lead to different vaccine effectiveness (VE) in subsequent years. Aim: The VEBIS primary care network estimated influenza VE in Europe using a multicentre test-negative study. Methods: Primary care practitioners collected information and specimens from patients consulting with acute respiratory infection. We estimated VE against influenza (sub)type and clade, by age group and by year of age for A(H1N1)pdm09, using logistic regression. Results: We included 29,958 patients, with 3,054, 1,053 and 311 influenza A(H1N1)pdm09, A(H3N2) and B cases, respectively. All-age VE against influenza A(H1N1)pdm09 was 52% (95% CI: 44-59). By year of age, VE was 27% (95% CI: -2 to 47) at 44 years with peaks at 72% (95% CI: 52-84) and 54% (95% CI: 41-64) among children and those 65 years and older, respectively. All-age A(H1N1)pdm09 VE against clade 5a.2a was 41% (95% CI: 24-54) and -11% (95% CI: -69 to 26) against clade 5a.2a.1. The A(H3N2) VE was 35% (95% CI: 20-48) among all ages and ranged between 34% and 40% by age group. All-age VE against clade 2a.3a.1 was 38% (95% CI: 1-62). All-age VE against B/Victoria was 83% (95% CI: 65-94), ranging between 70 and 92% by age group. Discussion: The 2023/24 VEBIS primary care VE against medically attended symptomatic influenza infection was high against influenza B/Victoria, but lower against influenza A(H1N1)pdm09 and A(H3N2). Clade- and age-specific effects may have played a role in the lower A(H1N1)pdm09 VE.
- Trans-ethnic GWAS meta-analysis of idiopathic spermatogenic failure highlights the immune-mediated nature of Sertoli cell-only syndromePublication . González-Muñoz, Sara; Long, Yichen; Guzmán-Jiménez, Andrea; Cerván-Martín, Miriam; Higueras-Serrano, Inmaculada; Castilla, José A.; Clavero, Ana; Garrido, Nicolás; Luján, Saturnino; Yang, Xiaoyu; Guo, Xuejiang; Liu, Jiayin; Bassas, Lluís; Seixas, Susana; Gonçalves, João; Lopes, Alexandra M.; Larriba, Sara; Bossini-Castillo, Lara; Palomino-Morales, Rogelio J.; Wang, Cheng; Hu, Zhibin; Carmona, F. DavidNon-obstructive azoospermia, a severe form of male infertility caused by spermatogenic failure (SPGF), has a largely unknown genetic basis across ancestries. To our knowledge, this is the first trans-ethnic meta-analysis of genome-wide association studies on SPGF, involving 2255 men with idiopathic SPGF and 3608 controls from European and Asian populations. Using logistic regression and inverse variance methods, we identify two significant genetic associations with Sertoli cell-only (SCO) syndrome, the most extreme SPGF phenotype. The G allele of rs34915133, in the major histocompatibility complex class II region, significantly increases SCO risk (P = 5.25E-10, OR = 1.57), supporting a potential immune-related cause. Additionally, the rs10842262 variant in the SOX5 gene region is also a genetic marker of SCO (P = 5.29E-09, OR = 0.72), highlighting the key role of this gene in the male reproductive function. Our findings reveal shared genetic factors in male infertility across ancestries and provide insights into the molecular mechanisms underlying SCO.
- Diminished DNA binding affinity of DMRT1 caused by heterozygous DM domain mutations is a cause of male infertilityPublication . Marić, Tihana; Castillo-Madeen, Helen; Klarić, Monika Logara; Barišić, Antun; Trgovec-Greif, Lovro; Murphy, Mark W.; Juchnewitsch, Anna-Grete; Lillepea, Kristiina; Dutta, Avirup; Žunić, Lucija; Stendahl, Alexandra M.; Punab, Margus; Pomm, Kristjan; Mendoza, Daniel M.; Lopes, Alexandra M.; Šorgić, Ana Merkler; Vugrek, Oliver; Gonçalves, João; Almstrup, Kristian; Aston, Kenneth I.; Belužić, Robert; Ježek, Davor; Bertoša, Branimir; Laan, Maris; Bojanac, Ana Katušić; Conrad, Donald F.; Barbalić, MajaThe most severe form of male infertility is idiopathic non-obstructive azoospermia (NOA), a complete sperm absence in the ejaculate. We performed exome sequencing in the Croatian infertile brothers with NOA and found a variant in DMRT1 (Doublesex and mab-3 related transcription factor 1) gene that was further assessed by the EMSA assay and molecular dynamic simulations. We additionally screened for DMRT1 mutations in 1940 infertile men diagnosed with spermatogenic failure, 644 normozoospermic controls, and 105 females with primary ovarian insufficiency (POI) recruited to the GEnetics of Male INfertility Initiative (GEMINI) or Estonian Andrology (ESTAND) cohorts. DMRT1 p.Pro74Leu (chr9:g.842059C > T) variant was detected in infertile brothers in the highly conserved position within the DNA binding DM domain of the protein. EMSA assay showed reduced DNA binding of DMRT1P74L and molecular dynamic simulations showed differences in structural and dynamical properties between the wild type protein and DMRT1P74L. Plausible disease-causing DMRT1 variants were only identified in infertile men (13/1940; 0.67%), and none in 639 fertile controls. Burden testing showed an excess of rare deleterious DM domain mutations in the infertility cohort compared to gnomAD v.4.0 population-based controls (Fisher’s exact test, p = 1.44 x 10−5). Three rare deleterious variants in DMRT1 were found in 104 cases of POI. The findings of this study strengthen the evidence of DMRT1 variants being a causal factor for male infertility and provide the distribution of likely pathogenic variants across the gene. This is also the first study to suggest that DMRT1 variants may also be linked to POI.
- A genetic variant in the 3′-UTR of PIWIL4 confers risk for extreme phenotypes of male infertility by altering miR-215 and miR-136 binding affinityPublication . González-Muñoz, Sara; Cerván-Martín, Miriam; Guzmán-Jiménez, Andrea; Rodríguez-Martín, Ana Isabel; Garrido, Nicolás; Castilla, José A.; Gonzalvo, M. Carmen; Clavero, Ana; Molina, Marta; Vilches, Miguel Ángel; Espuch-Oliver, Andrea; Maldonado, Vicente; García-Peña, María Luisa; Galiano-Gutiérrez, Noelia; Santamaría, Esther; González, Cristina; Quintana-Ferraz, Fernando; Gómez, Susana; Amorós, David; Martínez-Granados, Luis; Ortega-González, Yanira; Burgos, Miguel; Pereira-Caetano, Iris; Pinto, Graça S.; Aguiar, Ana; Pereira, Isabel S.; López-Rodrigo, Olga; Bassas, Lluís; Seixas, Susana; Gonçalves, João; Lopes, Alexandra M.; Larriba, Sara; Bossini-Castillo, Lara; Carmona, F. David; Palomino-Morales, Rogelio J.Study question: What is the functional impact of the rs508485 genetic polymorphism, located in the 3'-untranslated region (UTR) region of the PIWIL4 gene, on non-obstructive azoospermia (NOA)? Summary answer: The rs508485 genetic variant contributes to the pathogenesis of extreme patterns of NOA by modulating PIWIL4 expression through microRNA (miRNA) interactions. What is known already: Male infertility represents a significant global health challenge with profound societal and economic consequences. One of the most severe forms of male infertility is NOA, which is characterized by severe spermatogenic failure (SPGF) of idiopathic origin in most cases. Cumulating knowledge increasingly suggests that this idiopathic form of NOA may represent a multifactorial condition involving complex interactions between genetic and environmental factors. The PIWI protein subfamily, particularly PIWIL4, plays a pivotal role in spermatogenesis by processing PIWI-interacting RNAs, which silence retrotransposons to protect genomic integrity. Genetic variations in this gene have been found to be associated with susceptibility to NOA. Study design, size, duration: A case-control study was conducted in a European cohort including 1516 infertile men with SPGF and 2451 fertile controls. Logistic regression and functional assays were employed to investigate the functional role of the rs508485 polymorphism in PIWIL4. Participants/materials, setting, methods: Participants were genotyped for the rs508485 polymorphism. Associations between the polymorphism and NOA phenotypes, including Sertoli cell-only (SCO) syndrome and testicular sperm extraction (TESE) outcomes, were assessed. In silico tools predicted miRNA binding effects, which were subsequently validated using luciferase reporter assays. Main results and the role of chance: The T allele of rs508485 was significantly associated with the SCO phenotype (P = 2.69E-03, OR = 1.34) and unfavourable TESE outcomes (P = 1.09E-03, OR = 1.54). In silico analyses predicted that the rs508485 variant might alter binding sites in the 3'-UTR region of PIWIL4 for different miRNAs, such as hsa-miR-215-3p and hsa-miR-136-3p. Functional validation using luciferase assays confirmed that these miRNAs differentially bind to the T and C alleles of this polymorphism, influencing PIWIL4 regulation. Large scale data: N/A. Limitations, reasons for caution: The study is limited to a single genetic polymorphism and functional assays were performed in vitro. Additional studies are required to validate these findings across diverse populations and explore additional genetic interactions. Wider implications of the findings: These findings highlight the critical role of miRNA regulation in extreme forms of male infertility by influencing the expression of essential spermatogenesis genes, such as PIWIL4. Our study sheds light on the genetic mechanisms underlying spermatogenesis and suggests potential therapeutic targets for NOA.
- Morpholino Knockdown in Zebrafish: A Tool to Investigate the Functional Impact of Variants of Unknown Significance in Mitochondrial DiseasesPublication . Laranjeira, Mateus; Oliveira, Jorge Miguel; Santorelli, Filippo Maria; Marchese, Maria; Nogueira, CéliaMitochondrial diseases (MDs) are heterogeneous multisystemic disorders often caused by genetic defects in either nuclear or mitochondrial DNA. Although next-generation sequencing technologies have dramatically expanded the number of variants associated with these diseases, many remain variants of unknown significance (VUS). This review explores the utility of zebrafish (Danio rerio) as a vertebrate model system for studying mitochondrial dysfunction, with a focused analysis on the application of morpholino oligonucleotides (MOs) to functionally characterize and interpret VUS. MO-induced knockdown produces a transient suppression of target genes during zebrafish early development, recapitulating key MD phenotypes. Furthermore, rescue experiments involving co-injection of MO and either wild-type or mutant mRNA have proven useful to functionally assess the pathogenicity of specific variants. Specifically, while wild-type mRNA rescues the morphant phenotype, failure of mutant mRNA to do so confirms the variant’s pathogenic effect. This approach has successfully linked previously uncharacterized genes to MD and helped reclassify ambiguous variants. The use of MO-based strategies in zebrafish remains a valuable tool for variant interpretation and functional validation, bridging the gap between genomic data and clinical action, and ultimately reducing the diagnostic odyssey. Overall, this review places MO knockdown and rescue assays in zebrafish as a robust and versatile platform to address functional genomics in MD research.
- Measuring minerals in pseudocereals using inductively coupled plasma optical emission spectrometry: what is the optimal digestion method?Publication . Nascimento, Ana C.; Motta, Carla; Rego, Andreia; Delgado, Inês; Santiago, Susana; Assunção, Ricardo; Matos, Ana Sofia; Santos, Mariana; Castanheira, Isabel; .Pseudocereals have gained attention due to their adaptability to different climates, high nutritional value, and suitability for gluten-free and plant-based diets. However, a challenge lies in the necessary adaptations in the diet pathways, mainly due to the lack of matrix-matching metrological tools. To address this problem, we developed a classification system to support laboratory decisions without shaped Proficiency Testing (PT) or Certified/Standard References Material. This system evaluates method performance through limit of detection (LOD), maximum uncertainty, and statistical comparison. For that matter, the mineral contents (Cu, Mn, Fe, Zn, Mg, P, Ca, K, and Na) of quinoa (Chenopodium quinoa), amaranth (Amaranthus caudatus), and buckwheat (Fagopyrum esculentum) were determined, using three different digestion methods, including dry-ashing, microwave, and graphite block acid digestion. A decision was reached concerning the optimal digestion method to be employed, with the results classified into three categories: (i) “rejected if results failed in two categories; (ii) “use with caution” if results were not satisfactory in one category; or (iii) “accepted”, if the results passed in all the categories. The system efficacy was exemplified by the effectiveness of dry-ashing and graphite block acid digestion by comparison with microwave digestion. Neither dry-ashing nor graphite block acid digestion can be recommended as an alternative method to the microwave digestion method when all the prioritized nutrient minerals are understudied. Although the microwave method is preferable for multi-elemental analysis, it is possible to obtain, with caution, comparable results from all the digestion methods if a higher relative combined uncertainty is defined (target uncertainty < 11%) under the assumption that this is suitable for the study.
- Establishment of a Human iPSC Line from Mucolipidosis Type II That Expresses the Key Markers of the DiseasePublication . Moutinho, Maria Eduarda; Gonçalves, Mariana; Duarte, Ana J.; Encarnação, Marisa; Coutinho, Maria Francisca; Matos, Liliana; Santos, Juliana I.; Ribeiro, Diogo; Amaral, Olga; Gaspar, Paulo; Alves, Sandra; Moreira, Luciana V.Mucolipidosis type II (ML II) is a rare and fatal disease of acid hydrolase trafficking. It is caused by pathogenic variants in the GNPTAB gene, leading to the absence of GlcNAc-1-phosphotransferase activity, an enzyme that catalyzes the first step in the formation of the mannose 6-phosphate (M6P) tag, essential for the trafficking of most lysosomal hydrolases. Without M6P, these do not reach the lysosome, which accumulates undegraded substrates. The lack of samples and adequate disease models limits the investigation into the pathophysiological mechanisms of the disease and potential therapies. Here, we report the generation and characterization of an ML II induced pluripotent stem cell (iPSC) line carrying the most frequent ML II pathogenic variant [NM_024312.5(GNPTAB):c.3503_3504del (p.Leu1168fs)]. Skin fibroblasts were successfully reprogrammed into iPSCs that express pluripotency markers, maintain a normal karyotype, and can differentiate into the three germ layers. Furthermore, ML II iPSCs showed a phenotype comparable to that of the somatic cells that originated them in terms of key ML II hallmarks: lower enzymatic activity of M6P-dependent hydrolases inside the cells but higher in conditioned media, and no differences in an M6P-independent hydrolase and accumulation of free cholesterol. Thus, ML II iPSCs constitute a novel model for ML II disease, with the inherent iPSC potential to become a valuable model for future studies on the pathogenic mechanisms and testing potential therapeutic approaches.
- Systemic lupus erythematosus and the gut microbiome: To look forward is to look within – A systematic review and narrative synthesisPublication . Oliveira, Daniel Guimarães; Machado, Alexandra; Lacerda, Pedro Castro; Karakikla-Mitsakou, Zoe; Vasconcelos, CarlosBackground: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease shaped by complex interactions involving genetic and environmental factors. Among these, the gut microbiome is emerging as potentially modulating immune responses and influencing disease susceptibility, progression, and activity. Objectives: To synthesize current evidence on gut microbiome changes in adult SLE patients, framed along the clinical pathway – from diagnosis to treatment – to help bridge bench and bedside for microbiome-informed SLE care and research. Methods: A systematic search identified primary research studies examining gut microbiota in adult SLE patients. Studies were reviewed in a stepwise manner by independent investigators. Findings were synthesized narratively, emphasizing human data. Results: SLE patients exhibit gut microbiome dysbiosis, with reduced microbial richness and altered bacterial taxa. A lower Firmicutes/Bacteroidetes ratio is frequently observed. Enrichment of specific taxa, such as Enterococcus, Lactobacillus, and Ruminococcus gnavus, is reported. Dysbiosis correlates with increased gut permeability, immune activation, and autoreactivity. Clinical associations include disease activity, flares, nephritis, and other manifestations. SLE treatments, such as hydroxychloroquine and corticosteroids, influence the microbiome. Emerging interventions such as dietary modulation and fecal microbiota transplantation show promise in early studies. However, considerable heterogeneity exists across studies in terms of patient characteristics, methodology, and taxa-level findings. Conclusions: The gut microbiome has multifaceted associations with SLE pathogenesis, disease activity, and therapeutic response. Translation will require standardized methods, functional validation, longitudinal followup, and clinical integration. While uncertainties remain, the gut microbiome is increasingly relevant, and clinicians caring for patients with SLE should be aware of its emerging implications.
- Toward harmonizing protein data in food composition databases: evaluating perspectives, methods and implicationsPublication . Pferdmenges, Larissa E.; Colombani, Paolo C.; Carlsen, Monica Hauger; Pajari, Anne-Maria; Poulsen, Anders; Dias, Maria da Graça; Moller, Anders; Lisciani, Silvia; Wust, Matthias; Bonsmann, Stefan; Schweiggert-Weisz, UteProtein content in foods has historically been estimated by multiplying measured nitrogen content with a universal nitrogen-to-protein conversion factor (NCF) of 6.25. Despite scientific consensus that this approach leads to systematic overestimations due to variations in amino acid composition and non-protein nitrogen (NPN) content, no universally accepted revision has been implemented. This review critically examines diverse perspectives on protein quantification and their implications for Food Composition Databases (FCDBs). A structured definition of protein for FCDBs is proposed, including amino acid residues, free amino acids and small peptides, while explicitly excluding NPN and prosthetic groups. Furthermore, analytical methods and NCF calculations are evaluated in order to provide more accurate assessments of protein content across a range of food matrices. The review highlights the importance of selecting food-specific NCFs to reduce overestimations, ensuring both scientific accuracy and practical feasibility. By addressing methodological shortcomings and proposing a refined protein quantification framework, this work aims to facilitate the transition toward more precise and harmonized protein values in FCDBs, benefiting nutritional research, dietary guidelines, and food labeling regulations.