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Articles publicats en revistes (Ciències Fisiològiques)

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    Development of a microRNA-based prognostic model for accurate prediction of distant metastasis in breast cancer patients
    (BioMed Central, 2025-09-29) Fontana, Andrea; Barbano, Raffaela; Pasculli, Barbara; Mazza, Tommaso; Palumbo, Orazio; Binda, Elena; Trivieri, Nadia; Mencarelli, Gandino; Laurenzana, Ilaria; Lamorte, Daniela; De Luca, Luciana; Caivano, Antonella; Biagini, Tommaso; Rendina, Michelina; Lo Mele, Antonio; Prencipe, Giuseppene; Bravaccini, Sara; Murgo, Roberto; Ciuffreda, Luigi; Morritti, Maria; Valori, Vanna Maria; Di Lisa, Francesca Sofia; Vici, Patrizia; Castelvetere, Marina; Carella, Massimo; Graziano, Paolo; Maiello, Evaristo; Copetti, Massimiliano; Esteller, Manel; Parrella, Paolo
    Background: The attempt to exploit molecular subtyping for risk stratification in breast cancer patients has been only partially successful with a limited application in the clinical practice. In the BREMIR study, we aimed to identify a panel of miRNAs as prognostic biomarkers for breast cancer. We first confirmed the association of previously linked miRNAs with critical clinical parameters, then adopted a discovery-driven approach to identify novel biomarkers. Methods: miRNA expression was analyzed using the Affymetrix Gene Chip 4.0 array in a discovery cohort of 34 patients (3 with synchronous metastases, 14 who developed metastases after 10 years, and 17 who remained metastasis-free) and 6 controls. RT-qPCR validated selected miRNAs in an extended cohort (n = 223) with a median follow up of 6.6 years. A stepwise logistic regression model incorporating miRNA levels and clinicopathological features was developed to predict metastasis risk. Additionally, miRNA expression was assessed in plasma extracellular vesicles (EVs) using digital PCR in an independent cohort (n = 39). In silico enrichment analyses explored the functional role of relevant miRNAs in metastasis development. Results: Eight differentially expressed miRNAs were identified in the discovery cohort. In the extended cohort, miR-3916 and miR-3613-5p were the most effective in distinguishing patients who developed metastases. Higher miR-3916 expression was associated with reduced metastasis risk (OR = 0.42, 95%CI 0.23-0.70, p = 0.002), while higher miR-3613-5p expression was linked to increased risk (OR = 2.06, 95%CI 1.27-3.50, p = 0.005). Adding these miRNAs to a model with clinicopathological features improved discrimination (AUC = 0.85 vs. AUC = 0.76, p = 0.001). The model was effective across all breast cancer subtypes. In extracellular vesicles, miR-3613-5p was more abundant in tumors than benign lesions (p = 0.039), while miR-3916 was lower in metastatic samples than in non-metastatic tumors (p = 0.020). In-silico pathway enrichment analyses indicates their involvement in critical steps of the metastatic process including EMT plasticity, DNA damage response and metastatic niche formation. Conclusions: This is the first study integrating miRNA expression with clinicopathological features in a logistic model for breast cancer prognosis. While further validation is needed, our model shows promise as a prognostic tool across all breast cancer subtypes. In silico pathway enrichment analysis highlights miR-3613-5p and miR-3916 as critical regulators of metastasis development, underscoring the need for further investigation.
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    Pipeline Olympics: continuable benchmarking of computational workflows for DNA methylation sequencing data against an experimental gold standard
    (Oxford University Press, 2025-10-21) Lin, Yu-Yu; Breuer, Kersten; Weichenhan, Dieter; Lafrenz, Pascal; Sarnataro, Antonella; Wilk, Agata; Chepeleva, Maryna; Mücke, Oliver; Schönung, Maximilian; Petermann, Franziska; Kensche, Philip Reiner; Weiser, Lena; Thommen, Frank; Giacomelli, Gideon; Nordstroem, Karl; Gonzalez-Avalos, Edahi; Merkel, Angelika; Kretzmer, Helene; Fischer, Jonas; Krämer, Stephen; Iskar, Murat; Wolf, Stephan; Buchhalter, Ivo; Esteller, Manel; Lawerenz, Christian; Twardziok, Sven; Zapatka, Marc; Hovestadt, Volker; Schlesner, Matthias; Schulz, Marcel H.; Hoffmann, Steve; Gerhauser, Clarissa; Walter, Jörn; Hartmann, Mark; Lipka, Daniel B.; Assenov, Yassen; Bock, Christoph; Plass, Christoph; Toth, Reka; Lutsik, Pavlo
    DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five whole-genome profiling protocols. As an evaluation reference, we employed accurate locus-specific measurements from our previous benchmark of targeted DNA methylation assays. Based on this experimental gold-standard assessment and multiple performance metrics, we identified workflows that consistently demonstrated superior performance and revealed major workflow development trends. To ensure the long-term utility of our benchmark, we implemented an interactive workflow execution and data presentation platform, adaptable to user-defined criteria and readily expandable to future software.
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    Interrelation of adipose tissue macrophages and fibrosis in obesity.
    (Elsevier B.V., 2024-05-28) Dahdah, N.; Tercero Alcázar, Carmen; Malagón, María M.; García-Roves, Pablo M. (Pablo Miguel); Guzmán Ruiz, Rocío
    Obesity is characterized by adipose tissue expansion, extracellular matrix remodelling and unresolved inflammation that contribute to insulin resistance and fibrosis. Adipose tissue macrophages represent the most abundant class of immune cells in adipose tissue inflammation and could be key mediators of adipocyte dysfunction and fibrosis in obesity. Although macrophage activation states are classically defined by the M1/M2 polarization nomenclature, novel studies have revealed a more complex range of macrophage phenotypes in response to external condition or the surrounding microenvironment. Here, we discuss the plasticity of adipose tissue macrophages (ATMs) in response to their microenvironment in obesity, with special focus on macrophage infiltration and polarization, and their contribution to adipose tissue fibrosis. A better understanding of the role of ATMs as regulators of adipose tissue remodelling may provide novel therapeutic strategies against obesity and associated metabolic diseases.
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    Epigenetic inactivation of the 5-methylcytosine RNA methyltransferase NSUN7 is associated with clinical outcome and therapeutic vulnerability in liver cancer
    (BioMed Central, 2023-05-12) Ortiz Barahona, Vanessa; Soler, Marta; Davalos, Veronica; García-Prieto, Carlos A.; Janin, Maxime; Setién, Fernando; Fernández-Rebollo, Irene; Bech-Serra, Joan J.; De La Torre, Carolina; Guil, Sonia; Villanueva Garatachea, Alberto; Zhang, Pei-Hong; Yang, Li; Guarnacci, Marco; Schumann, Ulrike; Preiss, Thomas; Balaseviciute, Ugne; Montal, Robert; Llovet i Bayer, Josep Maria; Esteller, Manel
    Background: RNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggests that the epitranscriptome and its associated enzymes are altered in human tumors. Methods: Combining data mining and conventional experimental procedures, NSUN7 methylation and expression status was assessed in liver cancer cell lines and primary tumors. Loss-of-function and transfection-mediated recovery experiments coupled with RNA bisulfite sequencing and proteomics determined the activity of NSUN7 in downstream targets and drug sensitivity. Results: In this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in transformed cell lines, identified that the NOL1/NOP2/Sun domain family member 7 (NSUN7) undergoes promoter CpG island hypermethylation-associated with transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to find the RNA targets of this poorly characterized putative RNA methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript stability. Most importantly, proteomic analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival. Interestingly, NSUN7 unmethylated status was enriched in the immune active subclass of liver tumors. Conclusion: The 5-methylcytosine RNA methyltransferase NSUN7 undergoes epigenetic inactivation in liver cancer that prevents correct mRNA methylation. Furthermore, NSUN7 DNA methylation-associated silencing is associated with clinical outcome and distinct therapeutic vulnerability.
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    Neural cell diversity in the light of single-cell transcriptomics
    (CRC Press, Taylor and Francis Group, 2024-01-17) Fernandez Moya, Sandra M.; Jaya Ganesh, Akshay; Plass, Mireya
    The development of highly parallel and affordable high-throughput single-cell transcriptomics technologies has revolutionized our understanding of brain complexity. These methods have been used to build cellular maps of the brain, its different regions, and catalog the diversity of cells in each of them during development, aging and even in disease. Now we know that cellular diversity is way beyond what was previously thought. Single-cell transcriptomics analyses have revealed that cell types previously considered homogeneous based on imaging techniques differ depending on several factors including sex, age and location within the brain. The expression profiles of these cells have also been exploited to understand which are the regulatory programs behind cellular diversity and decipher the transcriptional pathways driving them. In this review, we summarize how single-cell transcriptomics have changed our view on the cellular diversity in the human brain, and how it could impact the way we study neurodegenerative diseases. Moreover, we describe the new computational approaches that can be used to study cellular differentiation and gain insight into the functions of individual cell populations under different conditions and their alterations in disease.
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    Energy substrate metabolism, mitochondrial structure and oxidative stress after cardiac ischemia-reperfusion in mice lacking UCP3.
    (Elsevier B.V., 2023-08-20) Sánchez-Pérez, Patricia; Mata, Ana; Torp, May-Kristin; López Bernardo, Elia; Heiestad, Christina M.; Aronsen, Jan Magnus; Molina-Iracheta, Antonio; Jiménez-Borreguero, Luis J.; García-Roves, Pablo M. (Pablo Miguel); Costa, Ana S.H.; Frezza, Christian; Murphy, Michael P.; Stenslokken, Kåre-Olav; Cadenas, Susana
    Myocardial ischemia-reperfusion (IR) injury may result in cardiomyocyte dysfunction. Mitochondria play a critical role in cardiomyocyte recovery after IR injury. The mitochondrial uncoupling protein 3 (UCP3) has been proposed to reduce mitochondrial reactive oxygen species (ROS) production and to facilitate fatty acid oxidation. As both mechanisms might be protective following IR injury, we investigated functional, mitochondrial structural, and metabolic cardiac remodeling in wild-type mice and in mice lacking UCP3 (UCP3-KO) after IR. Results showed that infarct size in isolated perfused hearts subjected to IR ex vivo was larger in adult and old UCP3-KO mice than in equivalent wild-type mice, and was accompanied by higher levels of creatine kinase in the effluent and by more pronounced mitochondrial structural changes. The greater myocardial damage in UCP3-KO hearts was confirmed in vivo after coronary artery occlusion followed by reperfusion. S1QEL, a suppressor of superoxide generation from site IQ in complex I, limited infarct size in UCP3-KO hearts, pointing to exacerbated superoxide production as a possible cause of the damage. Metabolomics analysis of isolated perfused hearts confirmed the reported accumulation of succinate, xanthine and hypoxanthine during ischemia, and a shift to anaerobic glucose utilization, which all recovered upon reoxygenation. The metabolic response to ischemia and IR was similar in UCP3-KO and wild-type hearts, being lipid and energy metabolism the most affected pathways. Fatty acid oxidation and complex I (but not complex II) activity were equally impaired after IR. Overall, our results indicate that UCP3 deficiency promotes enhanced superoxide generation and mitochondrial structural changes that increase the vulnerability of the myocardium to IR injury.
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    A review of vancomycin, gentamicin and amikacin population pharmacokientic models in neonates and infants.
    (Springer Nature Switzerland, 2025-01-15) Albanell Fernández, Marta; Bastida Fernández, Carla; Rodríguez Reyes, Montse; Soy Muner, Dolors
    Abstract Population pharmacokinetic (popPK) models are an essential tool when implementing therapeutic drug monitoring (TDM) and to overcome dosing challenges in neonates in clinical practice. Since vancomycin, gentamicin, and amikacin are among the most prescribed antibiotics for the neonatal population, we aimed to characterize the popPK models of these antibiotics and the covariates that may influence the pharmacokinetic parameters in neonates and infants with no previous pathologies. We searched the PubMed, Embase, Web of Science, and Scopus databases and the bibliographies of relevant articles from inception to the beginning of February 2024. The search identified 2064 articles, of which 68 met the inclusion criteria (34 for vancomycin, 21 for gentamicin, 13 for amikacin). A one-compartment popPK model was more frequently used to describe the pharmacokinetics of the three antibiotics (91.2% vancomycin, 76.9% gentamicin, 57.1% amikacin). Pharmacokinetic parameter (mean ± standard deviation) values calculated for a “typical” neonate weighing 3 kg were as follows: clearance (CL) 0.34 ± 0.80 L/h for vancomycin, 0.27 ± 0.49 L/h for gentamicin, and 0.19 ± 0.07 L/h for amikacin; volume of distribution (V d): 1.75 ± 0.65 L for vancomycin, 1.54 ± 0.53 L for gentamicin, and 1.67 ± 0.27 L for amikacin for one compartment models. Total body weight, postmenstrual age, and serum creatinine were common predictors (covariates) for describing the variability in CL, whereas only total body weight predominated for V d. A single universal popPK model for each of the antibiotics reviewed cannot be implemented in the neonatal population because of the significant variability between them. Body weight, renal function, and postmenstrual age are important predictors of CL in the three antibiotics, and total body weight for V d. TDM represents an essential tool in this population, not only to avoid toxicity but to attain the desired pharmacokinetic/pharmacodynamic index. The characteristics of the neonatal population, coupled with the lack of prospective studies and external validation of most models, indicate a need to continue investigating the pharmacokinetics of these antibiotics in neonates.
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    Chromatin activity of IκBα mediates the exit from naïve pluripotency
    (eLife Sciences, 2025-10-22) Palma, Luis G.; Alvarez-Villanueva, Daniel; Maqueda, Maria; Barrero, Mercedes; Iglesias, Arnau; Bertran, Joan, 1964-; Alvarez, Damiana; García Prieto, Carlos Antonio; Ballare, Cecilia; Esteller, Manel; Rodríguez Cortez, Virginia Carolina; Bueno, Clara; Vidal-Bel, August; Villanueva Garatachea, Alberto; Menéndez, Pablo; Stik, Gregoire; Croce, Luciano Di; Payer, Bernhard; Espinosa, Lluís; Bigas Salvans, Anna
    Maintenance of pluripotency is a multifactorial process in which NF-κB is a negative regulator. Our previous work identified a chromatin role for IκBα, the master regulator of NF-κB signaling, that is critical for the proper regulation of various tissue stem cells. Here, we found that IκBα accumulates specifically in the chromatin fraction of mouse pluripotent stem cells. IκBα depletion does not affect NF-kB-dependent transcription, but causes a profound epigenetic rewiring in pluripotent stem cells, including alterations in H3K27me3, a histone mark catalyzed by Polycomb repression complex 2. Chromatin changes induced by IκBα depletion affect a subset of pluripotency genes and are associated with altered gene transcription. At the cellular level, IκBα-deficient embryonic stem cells are arrested in a naive pluripotency state when cultured in serum/LIF conditions and fail to exit pluripotency under differentiation conditions. By constructing separation-of-function mutants, we show that the effects of IκBα in regulating stem cell pluripotency are NF-κB-independent, but mainly rely on its chromatin-related function. Taken together, our results reveal a novel mechanism by which IκBα participates in the regulation of the pluripotent state of mouse embryonic stem cells and shed light on the interplay between inflammatory signals and the regulation of pluripotency.
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    DNA Methylation: A profile of methods and applications
    (Eaton Pub. Co.], 2018-09-24) Esteller, Manel; Fraga, Mario F.
    Ever since methylcytosine was found in genomic DNA, this epigenetic alteration has become a center of scientific attraction, especially because of its relation to gene silencing in disease. There is currently a wide range of methods designed to yield quantitative and qualitative information on genomic DNA methylation. The earliest approaches were concentrated on the study of overall levels of methylcytosine, but more recent efforts havefocused on the study ofthe methylation status of specific DNA sequences. Particularly, optimization of the methods based on bisulfite modification of DNA permits the analysis of limited CpGs in restriction enzyme sites (e.g., combined bisulfite restriction analyses and methylation-sensitive single nucleotide primer extension) and the overall characterization based on differential methylation states (e.g., methylation-specific PCR, MethyLight, and methylation-sensitive single-stranded conformational polymorphism) and allows very specific patterns of methylation to be revealed (bisulfite DNA sequencing). In addition, novel methods designed to search for new methylcytosine hot spots have yielded further data without requiring prior knowledge of the DNA sequence. We hope this review will be a valuable tool in selecting the best techniques to address particular questions concerning the cytosine methylation status of genomic DNA.
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    Resistance to PI3κδ inhibitors in marginal zone lymphoma can be reverted by targeting the IL-6/PDGFRA axis
    (Ferrata Storti Foundation, 2022-04-28) Arribas, Alberto J.; Napoli, Sara; Cascione, Luciano; Sartori, Giulio; Barnabei, Laura; Gaudio, Eugenio; Tarantelli, Chiara; Mensah, Afua Adjeiwaa; Spriano, Filippo; Zucchetto, Antonella; Rossi, Francesca M.; Rinaldi, Andrea; Castro de Moura, Manuel; Jovic, Sandra; Bordone-Pittau, Roberta; Di Veroli, Alessandra; Stathis, Anastasios; Cruciani, Gabriele; Stussi, Georg; Gattei, Valter; Brown, Jennifer R.; Esteller, Manel; Zucca, Emanuel; Rossi, Davide; Bertoni, Francesco
    PI3KPPinhibitors are active in patients with lymphoid neoplasms and a first series of them have been approved for the treatment of multiple types of B-cell lymphoid tumors, including marginal zone lymphoma (MZL). The identification of the mechanisms underlying either primary or secondary resistance is fundamental to optimize the use of novel drugs. Here, we present a model of secondary resistance to PI3Kffinhibitors obtained by prolonged exposure of a splenic MZL cell line to idelalisib. The VL51 cell line was kept under continuous exposure to idelalisib. The study included detailed characterization of the model, pharmacological screens, silencing experiments, validation experiments on multiple cell lines and on clinical specimens. VL51 developed resistance to idelalisib, copanlisib, duvelisib, and umbralisib. An integrative analysis of transcriptome and methylation underlined an enrichment of up-regulated transcripts and lowmethylated promoters in resistant cells, including IL-6/STAT3 and PDGFRA related genes and surface CD19 expression, alongside the repression of the let-7 family miRNAs, of miR-125, miR-130, miR-193 and miR-20. The use of the IL-6R blocking antibody tocilizumab, the STAT3 inhibitor stattic, the LIN28 inhibitor LIN1632, the PDGFR inhibitor masitinib and the anti-CD19 antibody drug conjugate loncastuximab tesirine were active compounds in the resistant cells as single agents and/or in combination with PI3K//inhibition. Findings were validated on additional in vitro lymphoma models and on clinical specimens. A novel model of resistance obtained from splenic MZL allowed the identification of therapeutic approaches able to improve the anti-tumor activity of PI3Kttinhibitors in B-cell lymphoid tumors.
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    Editorial: Mitochondrial proteomics: Understanding mitochondria function and dysfunction through the characterization of their proteome
    (Frontiers Media, 2020-12-10) Ferri, Alberto; García-Roves, Pablo M. (Pablo Miguel); Pieroni, Luisa
    The development of OMICS sciences, and in particular Proteomics, has provided new tools to investigate the mitochondrial role in health and disease on a system biology scale. This Research Topic aims to collect papers describing how evidence based on proteomics data can contribute to gaining a deep understanding of mitochondrial structure and function and to unveil unknown or multifactorial features of mitochondrial physiological function and dysfunction. This exciting editorial initiative has allowed us to collect seven interesting contributions. Three original research articles investigate mitochondrial proteome changes in response to non-physiological stimuli, such as micro-environmental modifications or neurological diseases. The paper by Maffioli et al. sheds light on mechanotransduction, a particular ability of cells to respond and adapt their structure and function to biophysical changes in their microenvironment. The authors studied this phenomenon in cellular models of rodent pancreatic β cells cultured on artificial microenvironment, a nanostructured surface assembled by Supersonic Cluster Beam Deposition of zirconia nanoparticles. Following their previous study, they used mass spectrometry-based proteomics to investigate the involvement of mitochondria. They showed that mitochondria morphology and proteome can be regulated by nanotopography leading to a metabolic switch in these cells.
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    State of art of dose individualization to support tacrolimus drug monitoring: What’s next?
    (Frontiers Media, 2025-12-01) Lloberas Blanch, Núria; Fernández-Alarcón, Beatriz; Vidal Alabró, Anna; Colom Codina, Helena
    Tacrolimus is an immunosuppressant with a narrow therapeutic index and a high intra- and inter-patient variability showing significant challenges in optimal dosing and monitoring. Historically, pre-dose concentration monitoring and simplified area under the curve measurements have been the standard approach. However, recent advances in pharmacokinetic modeling have improved individualized dosing strategies, moving beyond empirical methods. This review explores the evolving landscape of Tacrolimus therapeutic drug monitoring, focusing on advanced modeling techniques that support personalized dosing. Key methodological approaches include Population Pharmacokinetic (PopPK) modeling, Bayesian prediction, Physiologically-Based Pharmacokinetic (PBPK) modeling, and emerging machine learning and artificial intelligence technologies. While no single method provides a perfect solution, these approaches are complementary and offer increasingly sophisticated tools for dose individualization. The review critically examines the potential and limitations of current modeling strategies, highlighting the complexity of translating advanced statistical and mathematical techniques into clinically accessible tools. A significant challenge remains the gap between sophisticated modeling techniques and the practical usability for healthcare professionals. The need for user-friendly platforms is emphasized, with recognition of existing commercial solutions while also noting their inherent limitations. Future directions point towards more integrated, intelligent systems that can bridge the current technological and practical gaps in personalized immunosuppressant therapy.
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    Increased intraocular insulin-like growth factor-I triggers blood-retinal barrier breakdown
    (American Society for Biochemistry and Molecular Biology, 2009-08-21) Haurigot, Virginia; Villacampa, Pilar; Ribera, Albert; Llombart, Cristina; Bosch, Assumpció; Nacher, Victor; Ramos, David; Ayuso, Eduard; Segovia, José C.; Bueren, Juan A.; Ruberte París, Jesús; Bosch i Tubert, Fàtima
    Blood-retinal barrier (BRB) breakdown is a key event in diabetic retinopathy and other ocular disorders that leads to increased retinal vascular permeability. This causes edema and tissue damage resulting in visual impairment. Insulin-like growth factor-I (IGF-I) is involved in these processes, although the relative contribution of increased systemic versus intraocular IGF-I remains controversial. Here, to elucidate the role of this factor in BRB breakdown, transgenic mice with either local or systemic elevations of IGF-I have been examined. High intraocular IGF-I, resulting from overexpression of IGF-I in the retina, increased IGF-I receptor content and signaling and led to accumulation of vascular endothelial growth factor. This was parallel to up-regulation of vascular Intercellular adhesion molecule I and retinal infiltration by bone marrow-derived microglial cells. These alterations resulted in increased vessel paracellular permeability to both low and high molecular weight compounds in IGF-I-overexpressing retinas and agreed with the loss of vascular tight junction integrity observed by electron microscopy and the altered junctional protein content. In contrast, mice with chronically elevated serum IGF-I did not show alterations in the retinal vasculature structure and permeability, indicating that circulating IGF-I cannot initiate BRB breakdown. Consistent with a key role of IGF-I signaling in retinal diseases, a strong up-regulation of the IGF-I receptor in human retinas with marked gliosis was also observed. Thus, this study demonstrates that intraocular IGF-I, but not systemic IGF-I, is sufficient to trigger processes leading to BRB breakdown and increased retinal vascular permeability. Therefore, therapeutic interventions designed to counteract local IGF-I effects may prove successful to prevent BRB disruption.
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    Diverse inhibitors of de novo purine synthesis promote AICAR-induced AMPK activation and glucose uptake in L6 myotubes
    (Wiley, 2025-07-01) Dolinar, Klemen; Miš, Katarina; Šopar, Katja; Šutar, Mateja; Božič, Meta; Kolar, Matic; Hropot, Tim; García-Roves, Pablo M. (Pablo Miguel); Chibalin, Alexander V.; Pirkmajer, Sergej
    Methotrexate, an immunosuppressant and anticancer drug, promotes glucose uptake and lipid oxidation in skeletal muscle via activation of AMP-activated protein kinase (AMPK). Methotrexate promotes AMPK activation by inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) formyltransferase/inosine monophosphate (IMP) cyclohydrolase (ATIC), which converts ZMP, an endogenous purine precursor and an active form of the pharmacological AMPK activator AICAR, to IMP during de novo purine synthesis. In addition to methotrexate, inhibition of purine synthesis underpins the therapeutic effects of a number of commonly used immunosuppressive, anticancer, and antimicrobial drugs, raising the question of whether activation of AMPK in skeletal muscle could be a recurrent feature of these drugs. Using L6 myotubes, we found that AICAR-induced AMPK activation and glucose uptake were enhanced by inhibitors of the conversion of IMP to GMP (mycophenolate mofetil) or of IMP to AMP (alanosine) as well as by indirect inhibitors of human (trimetrexate) and bacterial ATIC (sulfamethoxazole). 6-Mercaptopurine, which inhibits the conversion of IMP to GMP and AMP, activated AMPK, increased glucose uptake, and suppressed insulin signaling, but did not enhance the effect of AICAR. As determined by measuring oxygen consumption rate, none of these agents suppressed mitochondrial function. Overall, our results indicate that IMP metabolism is a gateway for the modulation of AMPK and its metabolic effects in skeletal muscle cells.
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    Lung diffusing capacity after different modalities of exercise at sea level and hypobaric simulated altitude of 4000 m
    (2023-09-30) García Alday, Iker; Drobnic, Franchek; Arrillaga, Beatriz; Cheng, Yinkiria; Javierre Garcés, Casimiro F.; Pons, Vicente; Viscor Carrasco, Ginés
    Introduction: Lung diffusion capacity of carbon monoxide (DLCO) provides a measure of gas transfer in the lungs, which increase in relation to exercise and decrease in the presence of lung interstitial disease. The aim of this study is to evaluate the changes in lung diffusion after anaerobic and aerobic exercise in a cycle ergometer. Material and method: The participants were 11 healthy active subjects, including 8 females and 3 males (age: 24.3 ± 3.1 years). Lung diffusion capacity for carbon monoxide (DLCO) was studied under two different protocols: In the first day, DLCO was measured at SL at rest (SL-R), after 30-s maximal exercise (SL-ANA), and after 15-min moderate continuous exercise (SL-AER). In the second day, DLCO was evaluated at rest at SL, and then at HA (4,000 m) at rest (HA-R) and after 30-min of moderate interval exercise (HA-AER). Results: There was an increase in DLCO from rest to after SL-ANA (32.5 ± 6.4 to 40.3 ± 11.6 mL·min-1·mmHg-1, P = 0.027). In the second day, DLCO was evaluated at rest at SL, and then at HA (4,000 m) at rest (HA-R) and after 30-min of moderate interval exercise (HA-AER). During HA exposure, there was no changes in DLCO, either at HA-R, or after HA-AER. Conclusions: Lung diffusion capacity largely increased after 30-s maximal exercise in a cycle ergometer, although the O2 -dependence is small during this type of anaerobic exercise. Thus, exercise intensity may be a key modulator of the changes in lung diffusing capacity in relation to exercise.
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    Early downregulation of hair cell (HC)-specific genes in the vestibular sensory epithelium during chronic ototoxicity
    (BioMed Central, 2025-12-01) Borrajo, Mireia; Greguske, Erin A.; Maroto, Alberto F.; Palou Miranda, Aida; Renner, Ana; Giménez-Esbrí, Víctor; Sedano, David; Gut, Marta; Esteve Codina, Anna; Martín Mur, Beatriz; Barrallo-Gimeno, Alejandro; Llorens i Baucells, Jordi
    Background: Exposure of mammals to ototoxic compounds causes hair cell (HC) loss in the vestibular sensory epithelia of the inner ear. In chronic exposure models, this loss often occurs by extrusion of the HC from the sensory epithelium towards the luminal cavity. HC extrusion is preceded by several steps that begin with detachment and synaptic uncoupling of the cells from the afferent terminals of their postsynaptic vestibular ganglion neurons. The purpose of this study was to identify gene expression mechanisms that drive these responses to chronic ototoxic stress. Methods: We conducted four RNA-seq experiments that generated five comparisons of control versus treated animals. These involved two species (rat and mouse), two compounds (streptomycin and 3,3'-iminodipropionitrile, IDPN), and three time points in our rat/IDPN model. We compared differentially expressed genes and their associated Gene Ontology terms, and several genes of interest were validated by in-situ hybridisation and immunofluorescence analyses. Results: Common and model-unique expression responses were identified. The earliest and most robust common response was downregulation of HC-specific genes, including stereocilium (Atp2b2, Xirp2), synaptic (Nsg2), and ion channel genes (Kcnab1, Kcna10), together with new potential biomarkers of HC stress (Vsig10l2). A second common response across species and compounds was the upregulation of the stress mediator Atf3. Model- or time-restricted responses included downregulation of cell-cell adhesion and mitochondrial ATP synthesis genes, and upregulation of the interferon response, unfolded protein response, and tRNA aminoacylation genes. Conclusions: The present results provide key information on the responses of the vestibular sensory epithelium to chronic ototoxic stress, potentially relevant to other types of chronic stress.
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    Biochemical titration of glycogen in vitro
    (JoVE, 2013-11-24) Pelletier, Joffrey; Bellot, Grégory; Pouysségur, Jacques; Mazure, Nathalie M.
    Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive(1).
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    A time-restricted feeding intervention in children and adolescents with obesity: The TRansForm study protocol
    (Frontiers Media, 2022-10-26) Molina-Giraldo, Paula; Murillo, Serafín; Meis, Laura; Sans, Oscar; Amat Bou, Montse; Llobet, Marina; Jiménez-Chillaron, Josep C.; Ramon-Krauel, Marta; Lerin, Carles
    Obesity during childhood is of special concern as adiposity is typically tracked into adult life and it constitutes a major risk factor for future obesity and associated metabolic disorders. Recent studies indicate that time-restricted feeding (TRF) interventions may provide a promising strategy for obesity treatment. However, TRF interventions have only been tested in adult subjects. This study aims to determine both short- and long-term effects of a TRF intervention in children and adolescents with obesity. We will also investigate potential mechanisms mediating the response to the intervention, including the circadian rhythm and the gut microbiota composition. We have designed a randomized-controlled parallel-group clinical study in which children and adolescents (age range 8-18 year-old) with obesity will be subjected to time-restricted eating or no time restrictions for 2 months. Follow-up visits will allow for long-term effect assessments. We will measure anthropometric (BMI, body composition) and metabolic parameters (glucose and lipid metabolism), indicators of the circadian rhythm, and gut microbiota composition will be analyzed. This study will (1) determine safety and effectiveness of the TRF intervention in children and adolescents; (2) assess its long-term effects; and (3) evaluate potential mechanisms involved in the response to the intervention.
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    Quantification of transcript isoforms at the single-cell level using SCALPEL
    (Nature Publishing Group, 2025-12-01) Ake, Franz; Schilling, Marcel; Fernandez Moya, Sandra M.; Jaya Ganesh, Akshay; Gutiérrez Franco, Ana; Li, Lei; Plass, Mireya
    Single-cell RNA sequencing (scRNA-seq) facilitates the study of transcriptome diversity in individual cells. Yet, many existing methods lack sensitivity and accuracy. Here we introduce SCALPEL, a Nextflow-based tool to quantify and characterize transcript isoforms from standard 3' scRNA-seq data. Using synthetic data, SCALPEL demonstrates higher sensitivity and specificity compared to other tools. In real datasets, SCALPEL predictions have a high agreement with other tools and can be experimentally validated. The use of SCALPEL on real datasets reveals novel cell populations undetectable using single-cell gene expression data, confirms known 3' UTR length changes during cell differentiation, and identifies cell-type specific miRNA signatures regulating isoform expression. Additionally, we show that SCALPEL improves isoform quantification using paired long- and short-read scRNA-seq data. Overall, SCALPEL expands the current scRNA-seq toolkit to explore post-transcriptional gene regulation across species, tissues, and technologies, advancing our understanding of gene regulatory mechanisms at the single-cell level.
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    Transgenerational inheritance of hepatic steatosis in mice: sperm methylome is largely reprogrammed and inherited but does not globally influence liver transcriptome
    (Oxford University Press, 2025-02-15) Ribó Gené, Sílvia; Ramon-Krauel, Marta; Marimon-Escudé, Josep M.; Busato, Florence; Palmieri, Flavio; Mourin-Fernandez, Marta; Palacios Marín, Ivonne; Diaz, Ruben; Lerin, Carles; Oliva Virgili, Rafael; Tost, Jorg; Jiménez-Chillaron, Josep C.
    Nutritional challenges and obesity can contribute to the transmission of metabolic diseases through epigenetic mechanisms. Among them, DNA methylation stands out as a potential carrier of information because germline cytosine methylation responds to environmental factors and can be transmitted across generations. Yet, it remains unclear whether inherited DNA methylation plays an active role in the inheritance of metabolic phenotypes or solely influences expression of a few genes that cannot recapitulate the whole metabolic spectrum in the next generation offspring. Previously, we established a mouse model of childhood obesity by reducing litter size at birth. Mice raised in small litters (SL) developed obesity, insulin resistance, and hepatic steatosis. The offspring (SL-F1) and grand-offspring (SL-F2) of SL males also exhibited hepatic steatosis. Here, we aimed to investigate whether germline DNA methylation could serve as a carrier of phenotypic information, hepatic steatosis, between generations. Litter size reduction significantly altered global DNA methylation profile in the sperm of SL-F0 males. Remarkably, 8% of these methylation marks remained altered in the sperm of SL-F1 mice and in the liver of SL-F2 mice. These data suggest that germline DNA methylation is sensitive to environmental challenges and holds significant heritability, either through direct germline transmission and/or through sequential erasure and reestablishment of the marks in the following generations. Yet, DNA methylation did not strongly correlate with the hepatic transcriptome in SL-F2 mice, suggesting that it does not directly drive phenotypes in the F2. As an alternative, germline DNA methylation could potentially influence the phenotype of the next generation by modulating the expression of a reduced number of key transcription factors that, through an amplification cascade, drive phenotypic outcomes in subsequent generations.