Biology subjects

Romero, S.

Publications and source records attributed to Romero, S..

13 recordsLinked to original sources

bioRxiv2026

Physiological limits of localized hypothermia in the human cochlea: The role of vascular heat transport

PurposeMild therapeutic hypothermia (MTH) preserves cochlear function in animal models and is now entering early-phase human trials for hearing preservation. However, the extent to which the human cochlea can actually be cooled, and the mechanisms underlying MTH, remain unclear, in part because blood perfusion is expected to oppose localized cooling. In this study we evaluated the impact of blood flow on human cochlear temperature exposed to the MTH device using a combined experimental and computational approach. MethodsTemperature measurements were obtained from a human cadaver skull exposed to a commercial MTH device. These data were used to validate a three-dimensional bioheat transfer model incorporating realistic skull anatomy. The validated model was subsequently extended to include physiological blood perfusion in the internal carotid artery; a major heat source located near the cochlea. Finally, the in silico model was further expanded to incorporate the surrounding skin and brain tissues. ResultsIncorporating blood flow in internal carotid artery substantially altered predicted cochlear temperature distributions, highlighting the importance of localized vascular heat transport in the human cochlea during MTH. Although cochlear cooling was attenuated in the presence of perfusion, the therapeutic effects of MTH may not depend solely on the magnitude of local intracochlear temperature reduction. Additional mechanisms, such as reduced facial surface temperature, may also contribute to its efficacy. ConclusionThe validated in silico model provides a physiologically realistic framework for evaluating human cochlear thermal responses, investigating MTH mechanisms, and optimizing temperature-based strategies for hearing preservation.

bioengineering↗

bioRxiv2026

Vinculin organises apical Transcellular Actin Fibres to coordinate collective migration

Collective cell migration is coordinated by adherens junctions (AJs) which serve both as structural links between the actin cytoskeleton of adjacent cells, as well as mechano-transductory structures allowing cells to transmit mechanical signals. Vinculin contributes to AJ maturation in multiple ways, binding to actin and the cadherin-catenin complex, bundling actin filaments, antagonising branched actin polymerisation and recruiting late AJ proteins. Here we have analysed the effect of vinculin on junctional actin organisation and its role in collective migration during unjamming in the human epithelial cell line MCF10A. At the apical surface of MCF10A monolayers, we found transcellular actin fibres (TAFs) that are directionally coordinated across long ranges, up to 10 cells. These TAFs are contractile and "cross" cell-cell contacts at AJs. Analysis of a vinculin knockout cell line revealed that this protein is essential for the coordination of TAFs across multiple cells. Arp2/3 activity must be tightly regulated to establish a long-range network of TAFs, since its downregulation by CK666 treatment, as well as its upregulation by the expression of an activated Rac1 mutant or a mutation that prevents the vinculin-Arp2/3 interaction, all impair TAF formation. During hypotonic unjamming of monolayers, we found that MCF10A cells with long-range TAFs migrate more collectively than vinculin KO cells in which TAFs only connect adjacent cells. Similarly, space-induced unjamming of MCF10A and vinculin knockout monolayers showed that cells connected by the long-range TAFs can collectively coordinate the direction in which they extend their lamellipodia. Thus, we show that vinculin plays a novel role in organising long-range actin networks across multiple cells and coordinating collective migration within cell monolayers.

cell biology↗

bioRxiv2026

Proximity-Informed Graph Learning Defines Spatial Protein Communities for Tumor-Associated Proximity Antigen Discovery

The spatial organization of membrane proteins is an underexplored dimension of cell-surface biology. Spatial proximity shapes cellular function and therapeutic targetability, yet efforts to identify tumor-associated antigens (TAAs) have largely focused on expression alone. Here, we developed an industrialized surface-protein proximity mapping workflow to interrogate TAAs within their membrane microenvironments. In the process, we generated 248 proximity maps across 12 receptor tyrosine kinases (RTKs) and 28 tumor cell systems. This proximity atlas enabled two advances: first, MetaMap, a correlation-based analytical framework that defines spatial protein communities and infers non-targeted proximal proteins from reproducible proximity signatures; and second, tumor-associated proximity antigens (TAPAs), a conceptual class of co-targets defined by disease-specific spatial proximity to TAAs rather than expression alone. Applying these proximity-derived relationships within a multimodal prioritization framework, we identified and validated an EGFRxCDCP1 TAA-TAPA pair that enhanced tumor cell killing across therapeutic modalities. By integrating spatial organization with multimodal data, this work expands the design space for precision-guided therapeutic strategies.

cancer biology↗

bioRxiv2025

Mapping Gene Impact on Single-cell Transcriptomic Networks via Perturbation Response Scanning

Gene knockout experiments are essential for dissecting gene function, and CRISPR has made targeted gene disruption more accessible than ever. Single-cell CRISPR screening enables the construction of rich genetic perturbation landscapes, facilitating the identification of genes whose perturbation strongly reshapes cellular states. However, due to the nonlinear dependencies within gene networks, identifying the most impactful tangible genes remains challenging. Existing virtual knockout methods estimate downstream effects of single-gene deletions but do not evaluate whether such perturbations disrupt global information flow or compromise cellular robustness. To address this limitation, we adapt a perturbation-response framework originally developed for protein structural dynamics to identify gene modules most susceptible to perturbation. We introduce the single-cell Perturbation Impact Index (scPII), a data-driven metric derived from gene regulatory networks that quantifies system-level responses to gene perturbations, without using any CRISPR screening information. Our results demonstrate that scPII effectively identifies genes whose perturbation has the greatest system-wide impact. Analysis of single-cell RNA-seq data from the Cancer Cell Line Encyclopedia revealed a strong correlation between scPII-derived gene impact scores and gene effect scores from genome-wide CRISPR screens. These findings indicate that scPII provides a robust metric for quantifying gene knockout effects. More broadly, integrating perturbation response scanning with gene regulatory networks offers a powerful framework for advancing single-cell data analytics in biomedical research.

bioinformatics↗

bioRxiv2024

Beyond Differential Expression: Embracing Cell-to-Cell Variability in Single-Cell Gene Expression Data Analysis

Single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of cellular variability by capturing gene expression profiles of individual cells. The importance of cell-to-cell variability in determining and shaping cell function has been widely appreciated. Nevertheless, differential expression (DE) analysis remains a cornerstone method in analytic practice, by focusing exclusively on mean expressional differences, current computational analyses overlook the rich information encoded by variability within the single-cell gene expression data. Thus, there is a need for more relevant approaches to assess data variability rather than the mean in order to offer a deeper understanding of cellular systems. Here we present spline-DV, a novel statistical framework for differential variability (DV) analysis using scRNA-seq data. The spline-DV method identifies genes exhibiting significantly increased or decreased expression variability among cells derived from two experimental conditions. Case studies show that DV genes identified using spline-DV are representative, and functionally relevant in the tested cellular conditions including obesity, fibrosis, and cancer.

bioinformatics↗

bioRxiv2023

Vinculin Antagonizes Branched Actin in Controlling Membrane Protrusion, Cell Migration and Cell Cycle Progression

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

cell biology↗

bioRxiv2023

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

molecular biology↗

bioRxiv2022

The adherens junction proteins α-catenin, vinculin and VASP cooperate to promote actin assembly

The cohesion of tissues requires that cells establish cell-cell junctions. Cells contact each other by forming Arp2/3-dependent lamellipodia before they initiate the formation of cadherin-based adherens junctions (AJs). Maturing AJs then assemble actin under force though the formation of a mechanosensitive complex comprising the actin-binding proteins -catenin, vinculin and VASP, which individually act on the nucleation, elongation and organisation of actin filaments in different ways. However, the activity of the ternary complex that these actin-regulatory proteins form has not been investigated due to the difficulty of assembling this complex in vitro in the absence of force. Here, we first designed mutants of these proteins that interact independently of force. We then studied their activity by combining actin polymerization kinetics in fluorescence spectroscopy with observation of single actin filaments in TIRF microscopy. Our results reveal how -catenin, vinculin and VASP combine their activities in a complex to inhibit Arp2/3-mediated branching, stimulate the nucleation and elongation of linear actin filaments from profilin-actin and crosslink these filaments into bundles. These findings shed light on the molecular mechanisms by which actin regulators synergistically control the transition of actin architecture and dynamics that accompanies the formation and maturation of AJs.

biochemistry↗

bioRxiv2022

Trypanosoma cruzi isolates naturally adapted to congenital transmission display a unique strategy of transplacental passage

Chagas disease is mainly transmitted by vertical transmission (VT) in non-endemic areas and in endemic areas where vector control programs have been successful. For the present study, we isolated natural strains vertically transmitted through three generations and proceeded to study their molecular mechanism of VT. No parasitemia was detected in immunocompetent mice, but they were able to induce an immune response and colonize different organs. VT experiments revealed that infection with the different strains did not affect mating, pregnancy or resorptions, but despiting low parasitemia, VT strains reached the placenta and resulted in higher vertical transmission rates than strains of either moderate or high virulence. While the virulent strain modulated more than 2500 placental genes, VT strains modulated 150, and none of the modulated genes is shared between them. VT strains downregulate genes associated with cell division and replication and upregulate immunomodulatory genes leading to anti-inflammatory responses and tolerance. The virulent strain stimulates a strong pro-inflammatory immune response, and this molecular footprint correlated with histopathological analyses. We herein describe a unique placental response regarding the passage of T cruzi VT isolates across the maternal-fetal interphase, challenging the current knowledge derived mainly from studies of laboratory-adapted or highly virulent strains.

microbiology↗

bioRxiv2022

PPP2R1A Regulates Migration Persistence through the WAVE Shell Complex

The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Using proteomics, we identified PPP2R1A among proteins differentially associated with the WAVE complex subunit ABI1 when RAC1 was activated and downstream generation of branched actin was blocked. PPP2R1A was found to associate at the lamellipodial edge with a novel form of WAVE complex, the WAVE Shell Complex (WSC), that contains NHSL1 instead of the Arp2/3 activating subunit WAVE as in the canonical WAVE Regulatory Complex (WRC). PPP2R1A was required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement was abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impaired WSC binding and migration regulation, suggesting that this novel function of PPP2R1A is critical for its tumor suppressor activity.

cell biology↗

bioRxiv2021

Parallel genetic changes underlie integrated craniofacial traits in an adaptive radiation of trophic specialist pupfishes

Many factors such as divergence time, shared standing genetic variation, frequency of introgression, and mutation rates can influence the likelihood of whether populations adapt to similar environments via parallel or non-parallel genetic changes. However, the frequency of parallel vs non-parallel genetic changes resulting in parallel phenotypic evolution is still unknown. In this study, we used a QTL mapping approach to investigate the genetic basis of highly divergent craniofacial traits between scale- and snail-eating trophic specialist species across similar hypersaline lake environments in an adaptive radiation of pupfishes endemic to San Salvador Island, Bahamas. We raised F2 intercrosses of scale- and snail-eaters from two different lake populations of sympatric specialists, estimated linkage maps, scanned for significant QTL for 30 skeletal and craniofacial traits, and compared the location of QTL between lakes to quantify parallel and non-parallel genetic changes. We found strong support for parallel genetic changes in both lakes for five traits in which we detected a significant QTL in at least one lake. However, many of these shared QTL affected different, but highly correlated craniofacial traits in each lake, suggesting that pleiotropy and trait integration should not be neglected when estimating rates of parallel evolution. We further observed a 23-52% increase in adaptive introgression within shared QTL, suggesting that introgression may be important for parallel evolution. Overall, our results suggest that the same genomic regions contribute to parallel integrated craniofacial phenotypes across lakes. We also highlight the need for more expansive searches for shared QTL when testing for parallel evolution.

evolutionary biology↗

bioRxiv2020

Cortical Organoids Model Early Brain Development Disrupted by 16p11.2 Copy Number Variants in Autism

Reciprocal deletion and duplication of 16p11.2 region is the most common copy number variation (CNV) associated with Autism Spectrum Disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV has mirror-opposite effect on neuronal maturation, proliferation, and synapse number, in concordance with its effect on brain growth in humans. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with excess of neurons and depletion of neural progenitors observed in deletions, and mirror phenotypes in duplications. Transcriptomic and proteomic profiling revealed multiple dysregulated pathways, including defects in neuron migration. Inhibition of activity of the small GTPase RhoA rescued migration deficits. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

neuroscience↗

bioRxiv2019

Cellular and widefield imaging of sound frequency organization in primary and higher-order fields of the mouse auditory cortex

The mouse auditory cortex (ACtx) contains two core fields - A1 and AAF - arranged in a mirror reversal tonotopic gradient. The best frequency (BF) organization and naming scheme for additional higher-order fields remain a matter of debate, as does the correspondence between smoothly varying global tonotopy and heterogeneity in local cellular tuning. Here, we performed chronic widefield and 2-photon calcium imaging from the ACtx of awake Thy1-GCaMP6s reporter mice. Data-driven parcellation of widefield maps identified five fields, including a previously unidentified area at the ventral posterior extreme of the ACtx (VPAF) and a tonotopically organized suprarhinal auditory field (SRAF) that extended laterally as far as ectorhinal cortex. Widefield maps were stable over time, where single pixel BFs fluctuated by less than 0.5 octaves during a one-month imaging period. After accounting for neuropil signal and frequency tuning strength, BF organization in neighboring layer 2/3 neurons was intermediate to the heterogeneous salt and pepper organization and the highly precise local organization that have each been described in prior studies. Multiscale imaging data suggest there is no ultrasonic field or secondary auditory cortex in the mouse. Instead, VPAF and a dorsoposterior field (DP) emerged as the strongest candidates for higher-order auditory areas.

neuroscience↗