CNIC - Artículos
http://hdl.handle.net/20.500.12105/2153
2024-03-29T09:23:11ZStem cell niche organization in the Drosophila ovary requires the ECM component Perlecan.
http://hdl.handle.net/20.500.12105/15268
Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.1,2 The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals.3-11 Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues.12 Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless.13-18 Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. We have used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs).19 We report that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, we provide evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche.
2021-04-26T00:00:00ZChromatin dynamics through mouse preimplantation development revealed by single molecule localisation microscopy.
http://hdl.handle.net/20.500.12105/15267
Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state. This analysis adds a new layer of information to previous biochemical studies and provides novel insight to current models of chromatin organisation during the first stages of development.
2022-08-15T00:00:00ZThe first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.
http://hdl.handle.net/20.500.12105/15129
The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.
2021-01-01T00:00:00ZNLRC4 -mediated activation of CD1c+ dendritic cells contributes to perpetuation of synovitis in rheumatoid arthritis.
http://hdl.handle.net/20.500.12105/15128
The individual contribution of specific myeloid subsets such as CD1c+ conventional dendritic cells (cDC) to perpetuation of Rheumatoid Arthritis (RA) pathology remains unclear. In addition, the specific innate sensors driving pathogenic activation of CD1c+ cDCs in RA patients and their functional implications have not been characterized. Here, we assessed phenotypical, transcriptional and functional characteristics of CD1c+ and CD141+ cDCs and monocytes from the blood and synovial fluid of RA patients. Increased levels of CCR2 and the IgG receptor CD64 on circulating CD1c+ cDC associated with the presence of this DC subset in the synovial membrane in RA patients. Moreover, synovial CD1c+ cDCs are characterized by increased expression of proinflammatory cytokines and high abilities to induce pathogenic IFNγ+IL-17+ CD4+ T cells in vitro. Finally, we identified the crosstalk between Fcγ Receptors and NLRC4 as a new potential molecular mechanism mediating pathogenic activation, CD64 upregulation and functional specialization of CD1c+ cDCs in response to dsDNA-IgG in RA patients.
2022-10-04T00:00:00ZSingle cell clonal analysis identifies an AID-dependent pathway of plasma cell differentiation.
http://hdl.handle.net/20.500.12105/15126
Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody-secreting plasma cells. Here, we have addressed the role of activation-induced deaminase (AID), which initiates somatic hypermutation and class switch recombination, in the terminal differentiation of GC B cells. By combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID-experienced cells, we have identified a novel subset of late-prePB cells (L-prePB), which shares the strongest clonal relationships with plasmablasts (PBs). Mice lacking AID have various alterations in the size and expression profiles of transcriptional clusters. We find that AID deficiency leads to a reduced proportion of L-prePB cells and severely impairs transitions between the L-prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.
2022-10-07T00:00:00ZRemote ischaemic preconditioning ameliorates anthracycline-induced cardiotoxicity and preserves mitochondrial integrity.
http://hdl.handle.net/20.500.12105/13456
Anthracycline-induced cardiotoxicity (AIC) is a serious adverse effect among cancer patients. A central mechanism of AIC is irreversible mitochondrial damage. Despite major efforts, there are currently no effective therapies able to prevent AIC.
Forty Large-White pigs were included. In Study 1, 20 pigs were randomized 1:1 to remote ischaemic preconditioning (RIPC, 3 cycles of 5 min leg ischaemia followed by 5 min reperfusion) or no pretreatment. RIPC was performed immediately before each intracoronary doxorubicin injections (0.45 mg/kg) given at Weeks 0, 2, 4, 6, and 8. A group of 10 pigs with no exposure to doxorubicin served as healthy controls. Pigs underwent serial cardiac magnetic resonance (CMR) exams at baseline and at Weeks 6, 8, 12, and 16, being sacrifice after that. In Study 2, 10 new pigs received 3 doxorubicin injections (with/out preceding RIPC) and were sacrificed at week 6. In Study 1, left ventricular ejection fraction (LVEF) depression was blunted animals receiving RIPC before doxorubicin (RIPC-Doxo), which had a significantly higher LVEF at Week 16 than doxorubicin treated pigs that received no pretreatment (Untreated-Doxo) (41.5 ± 9.1% vs. 32.5 ± 8.7%, P = 0.04). It was mainly due to conserved regional contractile function. In Study 2, transmission electron microscopy (TEM) at Week 6 showed fragmented mitochondria with severe morphological abnormalities in Untreated-Doxo pigs, together with upregulation of fission and autophagy proteins. At the end of the 16-week Study 1 protocol, TEM revealed overt mitochondrial fragmentation with structural fragmentation in Untreated-Doxo pigs, whereas interstitial fibrosis was less severe in RIPC+Doxo pigs.
In a translatable large-animal model of AIC, RIPC applied immediately before each doxorubicin injection resulted in preserved cardiac contractility with significantly higher long-term LVEF and less cardiac fibrosis. RIPC prevented mitochondrial fragmentation and dysregulated autophagy from AIC early stages. RIPC is a promising intervention for testing in clinical trials in AIC.
2021-04-01T00:00:00ZCross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.
http://hdl.handle.net/20.500.12105/13424
Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing.
All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden.
This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.
2021-04-01T00:00:00ZExploratory study of the long-term footprint of deep brain stimulation on brain metabolism and neuroplasticity in an animal model of obesity.
http://hdl.handle.net/20.500.12105/13389
Deep brain stimulation (DBS) is a powerful neurostimulation therapy proposed for the treatment of several neuropsychiatric disorders. However, DBS mechanism of action remains unclear, being its effects on brain dynamics of particular interest. Specifically, DBS reversibility is a major point of debate. Preclinical studies in obesity showed that the stimulation of the lateral hypothalamus (LH) and nucleus accumbens (NAcc), brain centers involved in satiety and reward circuits, are able to modulate the activity of brain structures impaired in this pathology. Nevertheless, the long-term persistence of this modulation after DBS withdrawal was unexplored. Here we examine the in vivo presence of such changes 1 month after LH- and NAcc-DBS, along with differences in synaptic plasticity, following an exploratory approach. Thus, both stimulated and non-stimulated animals with electrodes in the NAcc showed a common pattern of brain metabolism modulation, presumably derived from the electrodes' presence. In contrast, animals stimulated in the LH showed a relative metabolic invariance, and a reduction of neuroplasticity molecules, evidencing long-lasting neural changes. Our findings suggest that the reversibility or persistence of DBS modulation in the long-term depends on the selected DBS target. Therefore, the DBS footprint would be influenced by the stability achieved in the neural network involved during the stimulation.
2021-03-10T00:00:00ZEffects of Colchicine on Atherosclerotic Plaque Stabilization: a Multimodality Imaging Study in an Animal Model.
http://hdl.handle.net/20.500.12105/13380
Colchicine demonstrated clinical benefits in the treatment of stable coronary artery disease. Our aim was to evaluate the effects of colchicine on atherosclerotic plaque stabilization. Atherosclerosis was induced in the abdominal aorta of 20 rabbits with high-cholesterol diet and balloon endothelial denudation. Rabbits were randomized to receive either colchicine or placebo. All animals underwent MRI, 18F-FDG PET/CT, optical coherence tomography (OCT), and histology. Similar progression of atherosclerotic burden was observed in the two groups as relative increase of normalized wall index (NWI). Maximum 18F-FDG standardized uptake value (meanSUVmax) decreased after colchicine treatment, while it increased in the placebo group with a trend toward significance. Animals with higher levels of cholesterol showed significant differences in favor to colchicine group, both as NWI at the end of the protocol and as relative increase in meanSUVmax. Colchicine may stabilize atherosclerotic plaque by reducing inflammatory activity and plaque burden, without altering macrophage infiltration or plaque typology.
2021-02-01T00:00:00ZImmune synapse instructs epigenomic and transcriptomic functional reprogramming in dendritic cells.
http://hdl.handle.net/20.500.12105/13345
Understanding the fate of dendritic cells (DCs) after productive immune synapses (postsynaptic DCs) with T cells during antigen presentation has been largely neglected in favor of deciphering the nuances of T cell activation and memory generation. Here, we describe that postsynaptic DCs switch their transcriptomic signature, correlating with epigenomic changes including DNA accessibility and histone methylation. We focus on the chemokine receptor Ccr7 as a proof-of-concept gene that is increased in postsynaptic DCs. Consistent with our epigenomic observations, postsynaptic DCs migrate more efficiently toward CCL19 in vitro and display enhanced homing to draining lymph nodes in vivo. This work describes a previously unknown DC population whose transcriptomics, epigenomics, and migratory capacity change in response to their cognate contact with T cells.
2021-02-01T00:00:00ZHuman influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death.
http://hdl.handle.net/20.500.12105/13344
Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations.
We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase.
Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.
2021-02-01T00:00:00ZActivation of amino acid metabolic program in cardiac HIF1-alpha-deficient mice.
http://hdl.handle.net/20.500.12105/13343
HIF1-alpha expression defines metabolic compartments in the developing heart, promoting glycolytic program in the compact myocardium and mitochondrial enrichment in the trabeculae. Nonetheless, its role in cardiogenesis is debated. To assess the importance of HIF1-alpha during heart development and the influence of glycolysis in ventricular chamber formation, herein we generated conditional knockout models of Hif1a in Nkx2.5 cardiac progenitors and cardiomyocytes. Deletion of Hif1a impairs embryonic glycolysis without influencing cardiomyocyte proliferation and results in increased mitochondrial number and transient activation of amino acid catabolism together with HIF2α and ATF4 upregulation by E12.5. Hif1a mutants display normal fatty acid oxidation program and do not show cardiac dysfunction in the adulthood. Our results demonstrate that cardiac HIF1 signaling and glycolysis are dispensable for mouse heart development and reveal the metabolic flexibility of the embryonic myocardium to consume amino acids, raising the potential use of alternative metabolic substrates as therapeutic interventions during ischemic events.
2021-02-19T00:00:00ZTodos los caminos llevan a Roma: resultados de las terapias respiratorias no invasivas aplicadas en un centro terciario sin Unidad de Cuidados Intermedios durante la pandemia de COVID-19
http://hdl.handle.net/20.500.12105/13315
Introducción.
Las terapias respiratorias no invasivas (TRNI) fueron ampliamente utilizadas en la primera ola de la pandemia de COVID-19, en escenarios distintos según los medios disponibles. El objetivo fue presentar la supervivencia a 90 días y los factores asociados a esta de los pacientes tratados con TRNI en un centro de tercer nivel sin Unidad de Cuidados Respiratorios Intermedios. Como objetivo secundario comparar los resultados obtenidos de las distintas terapias.
Métodos.
Estudio observacional de pacientes tratados con TRNI fuera de un ambiente de Cuidados Intensivos o Unidad de Cuidados Respiratorios Intermedios, diagnosticados de COVID-19 y con síndrome de distrés respiratorio agudo por criterios radiológicos y de ratio SpO2/FiO2. Se desarrolló un modelo multivariante de regresión logística para determinar las variables independientemente asociadas, y se compararon los resultados de la terapia de alto flujo con cánula nasal y la presión positiva continua en la vía aérea.
Resultados.
Se trataron 107 pacientes y sobrevivieron 85 (79,4%) a los 90 días. Antes de iniciar la TRNI el ratio medio de SpO2/FiO2 fue de 119,8±59,4. Un mayor score de SOFA se asoció significativamente a la mortalidad (OR 2,09; IC95% 1,34 – 3,27), mientras que la autopronación fue un factor protector (OR 0,23; IC95% 0,06 – 0,91). La terapia de alto flujo con cánula nasal fue utilizada en 63 sujetos (58,9%), y la presión positiva continua en la vía aérea en 41 (38,3%). No se encontraron diferencias entre ellas.
Conclusión.
Aproximadamente cuatro de cada cinco pacientes tratados con TRNI sobrevivieron a los 90 días, y no se encontraron diferencias significativas entre la terapia de alto flujo con cánula nasal y la presión positiva continua en la vía aérea.
2021-01-01T00:00:00ZCovalently Labeled Fluorescent Exosomes for In Vitro and In Vivo Applications.
http://hdl.handle.net/20.500.12105/13313
The vertiginous increase in the use of extracellular vesicles and especially exosomes for therapeutic applications highlights the necessity of advanced techniques for gaining a deeper knowledge of their pharmacological properties. Herein, we report a novel chemical approach for the robust attachment of commercial fluorescent dyes to the exosome surface with covalent binding. The applicability of the methodology was tested on milk and cancer cell-derived exosomes (from U87 and B16F10 cancer cells). We demonstrated that fluorescent labeling did not modify the original physicochemical properties of exosomes. We tested this nanoprobe in cell cultures and healthy mice to validate its use for in vitro and in vivo applications. We confirmed that these fluorescently labeled exosomes could be successfully visualized with optical imaging.
2021-01-16T00:00:00ZThe Paternal Transition Entails Neuroanatomic Adaptations that are Associated with the Father's Brain Response to his Infant Cues.
http://hdl.handle.net/20.500.12105/13305
The transition into fatherhood is a life-changing event that requires substantial psychological adaptations. In families that include a father figure, sensitive paternal behavior has been shown to positively impact the infant's development. Yet, studies exploring the neuroanatomic adaptations of men in their transition into fatherhood are scarce. The present study used surface-based methods to reanalyze a previously published prospective magnetic resonance imaging dataset comprised of 20 first-time fathers (preconception-to-postpartum) and 17 childless men. We tested if the transition into fatherhood entailed changes in cortical volume, thickness, and area and whether these changes were related to 2 indicators of paternal experience. Specifically, we tested if such changes were associated with (1) the baby's age and/or (2) the fathers' brain activity in response to pictures of their babies compared with an unknown baby. Results indicated that first-time fathers exhibited a significant reduction in cortical volume and thickness of the precuneus. Moreover, higher volume reduction and cortical thinning were associated with stronger brain responses to pictures of their own baby in parental brain regions. This is the first study showing preconception-to-postpartum neuroanatomical adaptations in first-time fathers associated with the father's brain response to cues of his infant.
2020-11-01T00:00:00ZInterplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma.
http://hdl.handle.net/20.500.12105/13248
Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.
2020-12-01T00:00:00Z