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- Evaluating the Impact of Newborn Screening for Cystic Fibrosis in Portugal: A Decade of Insights and OutcomesPublication . Camacho, Bernardo; Pereira, Luísa; Bragança, Raquel; Castanhinha, Susana; Penteado, Raquel; Silva, Teresa Reis; Miragaia, Pedro; Silva, Sónia; Cardoso, Ana L.; Barbosa, Telma; Freitas, Cristina; Gonçalves, Juan; Marcão, Ana; Vilarinho, Laura; Barreto, Celeste; Constant, CarolinaThe implementation of newborn screening (NBS) has revolutionized the diagnostic landscape of cystic fibrosis (CF). In Portugal, NBS was initiated in October 2013 through a pilot study and was subsequently fully integrated into a nationwide program by December 2018. Infants with positive screening results are referred to a specialized CF reference center for diagnostic confirmation, employing Sweat Chloride Testing (SCT) and genetic testing for CFTR variants. We aimed to analyze infants with a positive CF screening and determine the false positive and false negative rates, as well as to calculate the positive predictive value and sensitivity of our NBS program. A retrospective nationwide analysis was conducted on infants with a positive NBS for CF between October 2013 and February 2023. Two hundred and forty infants were referred from the NBS program; 74 (30.8%) were confirmed to have CF through SCT and genetic testing. Sensitivity was 93.2%, and the positive predictive value (PPV) was 30.8%. In addition, 48.5% were homozygous for F508del variants, and 87.8% had at least one F508del variant. Guidelines set forth by the European Cystic Fibrosis Society advise NBS programs to achieve a minimum PPV of 30% and a minimum sensitivity of 95%. Our report demonstrated good compliance with these recommendations.
- Impact of PPP1R1A Knockdown on the Proteomic Landscape of INS-1 Cells: A Focus on Significant Modulated PathwaysPublication . Taneera, Jalal; Giddey, Alexander D.; Soares, Nelson C.; Khalique, Anila; Mohammed, Abdul Khader; Mahgoub, Mohamed Omer; Mahgoub, EglalPPP1R1A (protein phosphatase 1 regulatory inhibitor subunit 1A) is a cAMP/PKA-responsive inhibitor of protein phosphatase 1 (PP1) with a pivotal role in pancreatic β-cell physiology. To investigate its functional impact, Ppp1r1a was silenced in INS-1 (832/13) rat β-cells, and proteomic alterations were profiled using label-free DIA mass spectrometry (Orbitrap Exploris 480) with a rat spectral library. Quantitative analysis (n = 4/group) identified ∼2846 proteins with >2-fold change, revealing extensive proteome reprogramming. Key biological processes affected included vesicle trafficking and exocytosis, insulin biosynthesis and processing, organelle organization, mRNA processing, and autophagy. Pathway enrichment highlighted disruptions in insulin secretion, insulin resistance, and mTOR signaling. Crucial β-cell proteins, including INS2, Cacna1a, CPEB2, PCSK2, SNAP25, SYT5, and VAMP7, were significantly downregulated. Validation confirmed reduced phosphorylated AKT levels and p-AKT/T-AKT ratio, consistent with impaired mTOR signaling. Collectively, these findings demonstrate that PPP1R1A is essential for maintaining β-cell function and insulin secretion, and its depletion triggers broad proteomic and signaling alterations. Thus, PPP1R1A emerges as a regulatory node with potential therapeutic relevance in modulating β-cell activity and insulin dynamics in diabetes.
- A regulatory perspective on the applicability of NAMs in genotoxicity and carcinogenicity assessment in EU: current practices and future directionsPublication . Bossa, Cecilia; Alivernini, Silvia; Andreoli, Cristina; Aquilina, Gabriele; Attias, Leonello; Benfenati, Emilio; Dusinska, Maria; El Yamani, Naouale; Louro, Henriqueta; Marcon, Francesca; Raitano, Giuseppa; Rundén-Pran, Elise; Russo, Maria Teresa; Silva, Maria João; Battistelli, Chiara LauraNew Approach Methodologies (NAMs) are gaining significant momentum globally to reduce animal testing and enhance the efficiency and human relevance of chemical safety assessment. Even with substantial EU commitment from regulatory agencies and the academic community, the full regulatory adoption of NAMs remains a distant prospect. This challenge is further complicated by the fact that the academic world, oriented toward NAMs development, and regulatory agencies, focused on practical application, frequently operate in separate spheres. Addressing this disconnect, the present paper, developed within the European Partnership for the Assessment of Risks from Chemicals (PARC), provides a clear overview of both the available non-animal tests and current evaluation practices for genotoxic and carcinogenic hazard assessment, while simultaneously highlighting existing regulatory needs, gaps, and challenges toward greater human health protection and the replacement of animal testing through NAMs adoption. The analysis reveals a complex landscape: while the EU is deeply committed to developing and adopting NAMs, as outlined in its Chemical Strategy for Sustainability and supported by initiatives like PARC, prescriptive regulations such as Classification, Labelling and Packaging (CLP) and Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) still heavily mandate in vivo animal data for hazard classification, particularly for germ cell mutagenicity and carcinogenicity. This reliance creates a "too-short-blanket-problem," where efforts to reduce animal testing may impact human health protection because of the current in vivo-based classification criteria. In contrast, sectors such as cosmetics and certain European Food Safety Authority (EFSA)-regulated products demonstrate greater flexibility toward progressive integration of NAMs. While the deep mechanistic understanding of genotoxicity and carcinogenicity has significantly advanced the integration of alternatives to animal tests into regulatory chemical hazard assessment, their broader and full implementation faces considerable challenges due to both scientific complexities (i.e., the development and validation of fit-for-purpose NAMs) and existing legislative provisions.
- Translon: a single term for translated regionsPublication . Świrski, Michał I.; Tierney, Jack A. S.; Albà, M. Mar; Andreev, Dmitry E.; Aspden, Julie L.; Atkins, John F.; Bassani-Sternberg, Michal; Berry, Marla J.; Biffo, Stefano; Boris-Lawrie, Kathleen; Borodovsky, Mark; Brierley, Ian; Brook, Matthew; Brunet, Marie A.; Janusz M. Bujnicki; Caliskan, Neva; Calviello, Lorenzo; Carvunis, Anne-Ruxandra; Cate, Jamie H. D.; Cenik, Can; Chang, Kung Yao; Chen, Yiwen; Chothani, Sonia; Choudhary, Jyoti S.; Clark, Patricia L.; Clauwaert, Jim; Cooley, Lynn; Dassi, Erik; Dean, Kellie; Diaz, Jean-Jacques; Dieterich, Christoph; Dikstein, Rivka; Dinman, Jonathan D.; Dmitriev, Sergey E.; Dontsova, Olga A.; Dunham, Christine M.; Eswarappa, Sandeep M.; Farabaugh, Philip J.; Faridi, Pouya; Fierro-Monti, Ivo; Firth, Andrew E.; Gatfield, David; Gebauer, Fátima; Gelfand, Mikhail S.; Gray, Nicola K.; Green, Rachel; Hill, Chris H.; Hou, Ya-Ming; Hübner, Norbert; Ignatova, Zoya; Ivanov, Pavel; Iwasaki, Shintaro; Johnson, Rory; Jomaa, Ahmad; Jovanovic, Marko; Jungreis, Irwin; Kellis, Manolis; Kieft, Jeffrey S.; Kochetov, Alex V.; Koonin, Eugene V.; Korostelev, Andrei A.; Kufel, Joanna; Kulakovskiy, Ivan V.; Kurian, Leo; Lafontaine, Denis L. J.; Larsson, Ola; Loughran, Gary; Lukeš, Julius; Mariotti, Marco; Martens-Uzunova, Elena S.; Martinez, Thomas F.; Matsumoto, Akinobu; McManus, Joel; Medenbach, Jan; Melnikov, Sergey V.; Menschaert, Gerben; Merchante, Catharina; Mikl, Martin; Miller, W. Allen; Mühlemann, Oliver; Namy, Olivier; Nedialkova, Danny D.; Nosek, Jozef; Orchard, Sandra; Ozretić, Petar; Pertea, Mihaela; Pervouchine, Dmitri D.; Romão, Luísa; Ron, David; Roucou, Xavier; Rubtsova, Maria P.; Ruiz-Orera, Jorge; Saghatelian, Alan; Salzberg, Steven L.; Seale, Lucia A.; Seoighe, Cathal; Sergiev, Petr V.; Shah, Premal; Shirokikh, Nikolay; Slavoff, Sarah A.; Sonenberg, Nahum; Stasevich, Timothy J.; Szczesny, Roman J.; Tamm, Tiina; Tchórzewski, Marek; Topisirovic, Ivan; Tremblay, Michel L.; Tuller, Tamir; Ulitsky, Igor; Valášek, Leoš Shivaya; Van Damme, Petra; Viero, Gabriella; Vizcaino, Juan Antonio; Vogel, Christine; Wallace, Edward W. J.; Weissman, Jonathan S.; Westhof, Eric; Whiffin, Nicola; Wilson, Daniel N.; Xie, Zhi; Yewdell, Jonathan W.; Yordanova, Martina M.; Yu, Chien-Hung; Yurchenko, Vyacheslav; Zagrovic, Bojan; TRANSLACORE; Baranov, Pavel V.; Valen, EivindNo abstract available
- Metabolomics insights into doxorubicin and 5-fluorouracil combination therapy in triple-negative breast cancer: a xenograft mouse model studyPublication . Hassanein, Mai M.; Hagyousif, Yousra A.; Zenati, Ruba A.; Al-Hroub, Hamza M.; Khan, Farman Matloob; Abuhelwa, Ahmad Y.; Alzoubi, Karem H.; Soares, Nelson C.; El-Huneidi, Waseem; Abu-Gharbieh, Eman; Omar, Hany; Zaher, Dana M.; Bustanji, Yasser; Semreen, Mohammad H.Background: Breast cancer is one of the most prevalent malignancies and a leading cause of death among women worldwide. Among its subtypes, triple-negative breast cancer (TNBC) poses significant clinical challenges due to its aggressive behavior and limited treatment options. This study aimed to investigate the effects of doxorubicin (DOX) and 5-fluorouracil (5-FU) as monotherapies and in combination using an established MDA-MB-231 xenograft model in female BALB/C nude mice employing advanced metabolomics analysis to identify molecular alterations induced by these treatments. Methods: We conducted comprehensive plasma and tumor tissue sample profiling using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). Results: Each treatment group exhibited unique metabolic profiles in plasma and tumor analysis. Univariate and enrichment analyses identified alterations in metabolic pathways. The combination treatment of DOX + 5-FU induced the most extensive metabolic alterations disrupting key pathways including purine, pyrimidine, beta-alanine, and sphingolipid metabolism. It significantly reduced critical metabolites such as guanine, xanthine, inosine, L-fucose, and sphinganine, demonstrating enhanced cytotoxic effects compared to individual treatments. The DOX treatment uniquely increased ornithine levels, while 5-FU altered sphingolipid metabolism, promoting apoptosis. Significance: This in vivo study highlights TNBC's metabolic alterations to chemotherapeutics, identifying potential biomarkers like L-fucose and beta-alanine, and provides insights for improving treatment strategies.
- Shaping the future of human biomonitoring (HBM): progress, strategy, and global vision from ISES Europe and the HBM Global NetworkPublication . Zare Jeddi, Maryam; Hopf, Nancy B.; Galea, Karen S.; Jones, Kate; Louro, Henriqueta; Silva, Maria João; Covaci, Adrian; Santonen, Tiin; Scheepers, Paul T.J.; Viegas, Susana; Quirós-Alcalá, Lesliam; Qureshi, Asif; Marder, M. Elizabeth; von Goetz, Natalie; Kasiotis, Konstantinos M.; Machera, Kyriaki; Sepai, Ovnair; Duca, Radu-Corneliu; Ghosh, Manosij; van Nieuwenhuyse, An; Kei Chung, Ming; Kil, Jihyon; Nakayama, Shoji F.; Menouni, Aziza; Chbihi, Kaoutar; Vekic, Ana Maria; Souza, Gustavo; Waras, Maisarah Nasution; Ali, Imran; Bader, Michael; Kumar, Eva; Makris, Konstantinos C.; Ziying Lin, Elizabeth; Haynes, Erin N.; Bamai, Yu Ait; Kwon, Jung-Hwan; Huang, Po-Chin; Pasanen-Kase, RobertHuman biomonitoring (HBM) continues to play an indispensable role within exposure science, offering insights into aggregate chemical exposures across populations and life stages. Since 2018, the European chapter of the International Society of Exposure Science Human Biomonitoring Working Group (ISES Europe HBM WG) has aimed to facilitate generation of more and high-quality HBM data. The working group aims to strengthen integration of HBM data into regulatory frameworks through improved study design, harmonized methodologies, and enhanced reporting practices. Key achievements in the past seven years include the harmonization of HBM metadata through development of minimum information requirements for HBM (MIR-HBM), development of chemical-specific BASIC Guides for occupational health and hygiene professionals, and establishment of the FAIR (Findable, Accessible, Interoperable, and Reusable) Environmental and Health Registry (FAIREHR) to enhance data transparency and reusability. Recognizing the need for broader impact, the HBM Global Network was launched in 2025 to promote worldwide collaboration, capacity building, and policy integration. Together, ISES Europe HBM WG and the HBM Global Network form a coordinated platform with shared governance, strategic priorities, and digital infrastructure. This short communication outlines the progress to date, strategic pillars guiding our work, and ongoing initiatives linking science, policy, and practice. We call on researchers, regulators, and stakeholders worldwide to join these networks, strengthen harmonized approaches, and ensure that HBM becomes a cornerstone of 21st-century chemical risk governance.
- 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.