Biology subjects

Serrano, R.

Publications and source records attributed to Serrano, R..

8 recordsLinked to original sources

bioRxiv2025

Multiparametric Assessment of TNNI3 Variant Phenotypes in Human iPSC-Cardiomyocytes Correlates with Disease Severity in Patients

BackgroundThe routine genetic testing of cardiomyopathy patients has significantly accelerated the identification of causative cardiomyopathy variants. However, translating these genetic insights into effective patient management poses significant challenges, since the impact of gene variants on physiological function and clinical outcomes is not yet fully understood. Therefore, there is an urgent need for large-scale methods to assess the effects of genetic variants on cardiomyocyte physiology and to establish correlations between functional phenotypes and clinical severity. MethodsWe developed a high throughput imaging platform to measure force generation and calcium handling throughout the cardiac cycle of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). By expressing variants of a sarcomeric protein [cardiac Troponin-I (TNNI3)] in a healthy genetic background, we were able to assess sarcomeric calcium sensitivity as well as systolic and diastolic function. Analysis of these parameters distinguished subgroups of variants, and permitted the correlation of in vitro physiological effects with a measure of disease severity in a single-center cardiomyopathy cohort. ResultsCombining contractile force and calcium cycling measurements accurately distinguished known pathogenic from non-pathogenic TNNI3 variants and also revealed pathogenicity of two variants of unknown significance (VUS) that occurred in two families, suggesting the ability to prospectively discern pathogenicity. Clustering of TNNI3 variants based on quantitative physiological phenotypes identified subgroups that correlated with age of disease onset across a well-characterized cardiomyopathy patient cohort, showing clinical relevance of the in vitro phenotypes. Interestingly, normalized measures of in vitro diastolic function correlated with age of onset (R2 = 0.6), but calcium sensitivity, which accurately predicted pathogenicity, did not translate into disease severity. ConclusionsA high throughput in vitro platform that measures multidimensional cardiomyocyte function can link subgroups of human genetic variants in TNNI3 with differential patient outcomes. Comprehensive determination of variant effects on disease-relevant cardiomyocyte function will help classify variants into different pathogenic mechanisms leading to variable disease severity, and potentially lead to class-targeted ameliorative strategies.

genetics↗

bioRxiv2024

Uncertainty-Aware Traction Force Microscopy

Traction Force Microscopy (TFM) is a versatile tool to quantify cell-exerted forces by imaging and tracking fiduciary markers embedded in elastic substrates. The computations involved in TFM are ill-conditioned, and data smoothing or regularization is required to avoid overfitting the noise in the tracked substrate displacements. Most TFM calculations depend critically on the heuristic selection of regularization (hyper)parameters affecting the balance between overfitting and smoothing. However, TFM methods rarely estimate or account for measurement errors in substrate deformation to adjust the regularization level accordingly. Moreover, there is a lack of tools to quantify how these errors propagate to the recovered traction stresses. These limitations make it difficult to interpret TFM readouts and hinder comparing different experiments. This manuscript presents an uncertainty-aware TFM technique that estimates the variability in the magnitude and direction of the traction stress vector recovered at each point in space and time of each experiment. In this technique, substrate deformation and its uncertainty are quantified using a non-parametric bootstrap PIV method by resampling the microscopy image pixels (PIV-UQ). This information is passed to a hierarchical Bayesian framework that automatically selects its hyperparameters to perform spatially adaptive regularization conditioned on image quality and propagates the uncertainty to the traction stress readouts (TFM-UQ). We validate the performance of PIV-UQ and TFM-UQ using synthetic datasets with prescribed image quality variations and demonstrate the application of PIV-UQ and TFM-UQ to experimental datasets. These studies show that TFM-UQ locally adapts the level of smoothing, outperforming traditional regularization methods. They also illustrate how uncertainty-aware TFM tools can be used to objectively choose key image analysis parameters like PIV-UQ interrogation window size. We anticipate that these tools will allow for decoupling biological heterogeneity from measurement variability and facilitate automating the analysis of large datasets by parameter-free, input data-based regularization.

biophysics↗

bioRxiv2024

Personalized transcriptome signatures in a cardiomyopathy stem cell biobank

BACKGROUNDThere is growing evidence that pathogenic mutations do not fully explain hypertrophic (HCM) or dilated (DCM) cardiomyopathy phenotypes. We hypothesized that if a patients genetic background was influencing cardiomyopathy this should be detectable as signatures in gene expression. We built a cardiomyopathy biobank resource for interrogating personalized genotype phenotype relationships in human cell lines. METHODSWe recruited 308 diseased and control patients for our cardiomyopathy stem cell biobank. We successfully reprogrammed PBMCs (peripheral blood mononuclear cells) into induced pluripotent stem cells (iPSCs) for 300 donors. These iPSCs underwent whole genome sequencing and were differentiated into cardiomyocytes for RNA-seq. In addition to annotating pathogenic variants, mutation burden in a panel of cardiomyopathy genes was assessed for correlation with echocardiogram measurements. Line-specific co-expression networks were inferred to evaluate transcriptomic subtypes. Drug treatment targeted the sarcomere, either by activation with omecamtiv mecarbil or inhibition with mavacamten, to alter contractility. RESULTSWe generated an iPSC biobank from 300 donors, which included 101 individuals with HCM and 88 with DCM. Whole genome sequencing of 299 iPSC lines identified 78 unique pathogenic or likely pathogenic mutations in the diseased lines. Notably, only DCM lines lacking a known pathogenic or likely pathogenic mutation replicated a finding in the literature for greater nonsynonymous SNV mutation burden in 102 cardiomyopathy genes to correlate with lower left ventricular ejection fraction in DCM. We analyzed RNA-sequencing data from iPSC-derived cardiomyocytes for 102 donors. Inferred personalized co-expression networks revealed two transcriptional subtypes of HCM. The first subtype exhibited concerted activation of the co-expression network, with the degree of activation reflective of the disease severity of the donor. In contrast, the second HCM subtype and the entire DCM cohort exhibited partial activation of the respective disease network, with the strength of specific gene by gene relationships dependent on the iPSC-derived cardiomyocyte line. ADCY5 was the largest hubnode in both the HCM and DCM networks and partially corrected in response to drug treatment. CONCLUSIONSWe have a established a stem cell biobank for studying cardiomyopathy. Our analysis supports the hypothesis the genetic background influences pathologic gene expression programs and support a role for ADCY5 in cardiomyopathy.

genomics↗

bioRxiv2022

Kaempferol over-accumulation in the flavonoid 3' hydroxylase tt7 mutant disrupts seed coat outer integument differentiation and compromises seed longevity

O_LISeeds slowly accumulate damage during storage, which ultimately results in germination failure. The seed coat is the barrier between the embryo and the external environment, and its composition is critical for seed longevity. Flavonols accumulates in the outer integument, but the effect of altering flavonol composition on outer integument development has not been explored. C_LIO_LIGenetic, biochemical, ultrastructural and transcriptomics assays on a battery of loss-of-function mutants in the flavonoid biosynthesis pathway were used to study the effect of altered flavonoid composition in seed development and seed longevity. C_LIO_LIControlled deterioration assays indicate that loss-of-function of the flavonoid 3 hydroxylase TT7 gene dramatically affects seed longevity and seed development. Seed outer integument differentiation is compromised from nine days after pollination in tt7 seeds, with a defective suberine layer and incomplete degradation of seed-coat starch. These distinctive phenotypes are not shared by other mutants showing also altered flavonoid composition. Double-mutant analysis indicate that over-accumulation of kaempferol is the primary cause of the observed phenotypes. Expression analysis suggest that the tt7 flavonoid pattern affects transcriptional and non-transcriptional levels of regulation. C_LIO_LIThe increase of kaempferols in the seed coat influences seed development. This positions TT7 as an essential player modulating seed coat development and seed longevity. C_LI

plant biology↗

bioRxiv2021

Comparative analysis of wild type accessions reveals novel determinants of Arabidopsis seed longevity

Understanding the genetic factors involved in seed longevity is of paramount importance in agricultural and ecological contexts. The polygenic nature of this trait suggests that many of them remain undiscovered. Here, we exploited the contrasting seed longevity found amongst wild type Arabidopsis thaliana accessions to further understand this phenomenon. Concentrations of the antioxidant glutathione were consistently higher in longer-lived than shorter-lived accessions, supporting that redox poise plays a prominent role in seed longevity. However, high seed permeability, normally associated with shorter longevity, is also present in accessions with longer seed longevity. Transcriptome analysis indicated that the detrimental effect on longevity caused by seed coat permeability may be counterbalanced by higher levels of specific mRNAs stored in dry seed, particularly those of heat-shock proteins. Indeed, reverse genetics demonstrated that heat-shock factors HSF1A and 1B contributed to longevity. Furthermore, loss-of-function mutants of RNA-binding proteins, such as the stress-granule zinc-finger protein TZF9, or the spliceosome subunits MOS4 or MAC3A/MAC3B, extended seed longevity, positioning RNA as a novel player in the regulation of seed viability. mRNAs of proteins with putative relevance to longevity were also abundant in shorter-lived accessions, reinforcing the idea that resistance to ageing is determined by multiple factors.

plant biology↗

bioRxiv2019

A novel microRNA-based strategy to expand the differentiation potency of stem cells

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

cell biology↗

bioRxiv2019

Drug related and psychopathological symptoms in HIV-positive men who have sex with men who inject drugs during sex (Slamsex): Data from the U-SEX GESIDA 9416 Study.

ObjectivesIntravenous sexualized drug use also known as slamsex seems to be increasing among HIV-positive men who have sex with men (MSM). This practice may entail severe consequences for physical and mental health in this population. Research on the subject is scarce. The aim of our study was to describe the psychopathological background of a sample of HIV positive MSM who practiced slamsex during the previous year and compare the physical and psychological symptoms between these participants and those who practiced sexualized drug use (SDU) or chemsex without slamsex.\n\nDesign and MethodsParticipants (HIV-positive MSM) were recruited from the U-Sex study in 22 HIV clinics in Madrid during 2016-17. All participants completed an anonymous cross-sectional survey on sexual behaviour and recreational drug use. The present analysis is based on HIV-positive MSM who had practiced SDU.\n\nResultsThe survey sample comprised 742 participants. Of all the participants who completed the survey, 216 (29.1%) practiced chemsex, and of these, 34 (15.7%) had practiced slamsex. Participants who practiced slamsex were more likely to have current psychopathology (depression, anxiety and drug related disorders) than chemsex users. In addition, participants who practiced slamsex had more high-risk sexual behaviours, polydrug use and were more often diagnosed with sexually transmitted infections (STIs) and hepatitis C than those who did not inject drugs. Compared with patients who did not inject drugs, patients who engaged in slamsex showed more severe drug related symptoms (withdrawal and dependence), symptoms of severe intoxication (loss of consciousness), and severe psychopathological symptoms related to SDU, such as paranoid thoughts and suicidal behaviour.\n\nConclusionSlamsex (intravenous SDU) is closely associated with current psychiatric disorders and severe drug-related and psychiatric symptoms.

scientific communication and education↗

bioRxiv2019

Three-dimensional Monolayer Stress Microscopy

Many biological processes involve the collective generation and transmission of mechanical stresses across cell monolayers. In these processes, the monolayer undergoes lateral deformation and bending due to the tangential and normal components of the cell-generated stresses. Monolayer Stress Microscopy (MSM) methods have been developed to measure the intracellular stress distribution in cell monolayers. However, current methods assume plane monolayer geometry and neglect the contribution of bending to the intracellular stresses.\n\nThis work introduces a three-dimensional (3D) MSM method that calculates monolayer stress from measurements of the 3D traction stresses exerted by the cells on a flexible substrate. The calculation is carried out by imposing equilibrium of forces and moments in the monolayer, subject to external loads given by the 3D traction stresses. The equilibrium equations are solved numerically, and the algorithm is validated for synthetic loads with known analytical solutions.\n\nWe present 3D-MSM measurements of monolayer stress in micropatterned islands of endothelial cells of different sizes and shapes. These data indicate that intracellular stresses caused by lateral deformation emerge collectively over long distances; they increase with the distance from the island edge until they reach a constant value that is independent of island size. On the other hand, bending-induced intracellular stresses are more concentrated spatially and remain confined to within 1-2 cell lengths of bending sites. The magnitude of these bending stresses is highest at the edges of the cell islands, where they can exceed the intracellular stresses caused by lateral deformations. Our data from non-patterned monolayers suggests that biomechanical perturbations far away from monolayer edges also cause significant localized alterations in bending tension. The localized effect of bending-induced stresses may be important in processes like cellular extravasation, which are accompanied by significant normal deflections of a cell monolayer (i.e. the endothelium), and which require localized changes in monolayer permeability.

biophysics↗