Biology subjects

Rubio, A.

Publications and source records attributed to Rubio, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Evolutionary expansion of the bacterial ompW gene family enables functional diversification during host adaptation

The outer membrane protein W (OmpW) family comprises minor porins essential for environmental adaptation and host interactions in Gram-negative bacteria. However, ompW is conventionally treated as a ubiquitous single-copy gene, masking its true evolutionary complexity. Through comprehensive phylogenetic analysis across thousands of reference genomes, we reveal that ompW is present in nearly half of Gram-negative lineages, exhibiting a patchy distribution governed by a birth-and-death model of evolution with recurrent duplications and horizontal transfer. Notably, copy number expansions concentrate in lineages associated with eukaryotic hosts, linking paralog accumulation to niche adaptation. Experimental validation in Acinetobacter baumannii reveals four distinct loci that maintain high structural conservation despite sequence divergence, yet undergo profound functional specialization spanning from baseline physiological maintenance to modulating host colonization and persistence in vitro and in vivo. These findings dismantle the single-copy paradigm, demonstrating that widespread gene family expansion and paralog diversification drive the phenotypic plasticity required for bacteria to colonize hostile host environments.

microbiology↗

bioRxiv2026

Scaling Functional Annotation Across Proteomes, Pangenomes and Metagenomes with Sma3s v3

High-throughput sequencing has generated protein datasets whose scale increasingly exceeds the practical limits of conventional functional annotation workflows. We present Sma3s v3, a scalable reimplementation of the Sma3s three-step annotation strategy, which combines transfer from highly similar homologs, orthology-based inference, and functional enrichment among homologous proteins. Sma3s v3 replaces BLAST-based searches with MMseqs2 and introduces parallel processing, reusable SQLite caches, taxonomic filtering, and traceable outputs that retain the evidence underlying each assignment. We evaluated the method on a Vibrio cholerae pangenome comprising 50,415 gene clusters from 11,295 quality-filtered genomes and on a metagenomic catalogue containing 843,935 proteins. After excluding non-informative assignments, Sma3s v3 annotated 30,662 pangenome clusters (60.8%), comparable to InterProScan (60.2%) and exceeding eggNOG-mapper (41.4%), while providing 5,747 annotations not recovered by either comparator. Gene Ontology comparisons showed broad semantic agreement between methods, with Sma3s v3 frequently contributing more non-redundant information in Molecular Function and Biological Process. Within the pangenome, annotation coverage reached 97.1% for core clusters and approximately 59% for accessory and unique clusters. Exact protein matches to non-Vibrio genera identified 1,838 candidate horizontally transferred clusters enriched in genetic mobility, antimicrobial resistance, and metal tolerance functions. In the metagenomic catalogue, Sma3s v3 annotated 728,014 proteins (86.3%), compared with 616,895 (73.1%) using InterProScan 2026, and recovered approximately 20,000 unique functional terms. These results establish Sma3s v3 as a scalable and interpretable tool for functional annotation and re-annotation of proteomes, pangenomes, and metagenomic protein catalogues.

bioinformatics↗

bioRxiv2026

SNMF: Ultrafast, Spatially-Aware Deconvolution for Spatial Transcriptomics

Sequencing-based spatial transcriptomics has revolutionized the study of tissue architecture, but its spots often contain multiple cells, creating a key computational challenge, termed deconvolution, to decipher each spots cell-type composition. Reference-free deconvolution methods avoid the need for a matched single-cell RNA-seq dataset, but typically neglect the spatial correlation between neighboring spots and do not leverage modern hardware for efficient computation. Here, we propose SNMF (Spatial Non-negative Matrix Factorization): a rapid, accurate, and reference-free deconvolution method. SNMF extends the standard NMF framework with a spatial mixing matrix that models neighborhood influences, guiding the factorization toward spatially coherent solutions. Our R package is, to our knowledge, the first spatial transcriptomics deconvolution tool to natively support GPU execution, completing benchmark analyses in under one minute--over two orders of magnitude faster than the slowest competing methods-- with moderate memory requirements. On synthetic and real benchmark datasets, SNMF significantly outperforms state-of-the-art methods in deconvolution accuracy, and on a human melanoma dataset it recovers biologically meaningful cell-type signatures--including a tumor-boundary transition zone-- without any reference input. The proposed mehtod is publicly available at https://github.com/ML4BM-Lab/SNMF.

bioinformatics↗

bioRxiv2026

YBX3 overexpression in mesothelioma drives aberrant cell proliferation

Malignant pleural mesothelioma (MpM) is a lethal tumour closely linked to asbestos exposure and is a cancer of unmet clinical need with no known oncogenic drivers. Recent advancements in technologies to identify RNA-binding proteins (RBPs) has uncovered an emerging role for RBP-RNA interactions in cancer progression and we therefore assessed changes in the RBPome of patient-derived MpM cell lines. We identify over 350 RBPs showing altered RNA binding, with functions consistent with key cancer hallmarks, and discovered YBX3 as a potential oncoprotein driving cell proliferation in MpM. Mechanistically we show the impact of YBX3 on cell growth is achieved through its control of the expression of the amino acid transporter SLC7A5/LAT1, with increased amino acid uptake increasing protein synthesis rates. Notably, we show the inhibition of cell growth by YBX3 deletion is recapitulated by the clinically-relevant SLC7A5/LAT1 inhibitor JPH203. Finally, we demonstrate that JPH203 sensitizes MpM cells to radiotherapy, which could provide a promising therapeutic strategy for MpM. TeaserHigher levels of YBX3 expression in mesothelioma increases cell proliferation and protein synthesis rates by upregulation of amino acid uptake.

molecular biology↗

bioRxiv2025

Discovery of a stress-response integrative and conjugative element from Sphingopyxis granuli TFA broadly conserved across Sphingomonadales and Rhizobiales

Horizontal gene transfer is a pivotal element in the evolution of microbes, enabling them to acquire novel genes and phenotypes. Integrative and conjugative elements (ICEs) are a type of mobile genetic element that can integrate into the host genome and propagate during chromosome replication and cell division. The induction of ICE gene expression results in the excision of the ICE gene, the production of conserved conjugation machinery, a Type IV Secretion System, and the potential for DNA transfer to appropriate receptors. It has been observed that ICEs frequently contain cargo genes that do not typically align with the ICE life cycle. These genes often result in the manifestation of phenotypes that are of particular interest. The bacterium Sphingopyxis granuli strain TFA is being studied for its ability to degrade the contaminant tetralin present in crude oils. Genomic analysis identified eight possible integrative mobile elements in S. granuli TFA. Most of these regions exhibited a distribution pattern that was restricted to the species, and they lacked some functional modules that are characteristic of a complete ICE. This finding suggests the presence of degenerate structures or limited mobilization capacity. However, only two of the detected elements exhibited the capacity to retain all the modules necessary for transfer, integration, and maintenance. These elements also contained a cargo module that included genes associated with lipid metabolic pathways and resistance mechanisms. Among them, ICE3 was distinguished as the sole complete functional ICE that was also present in other species. Transcriptomic analysis under multiple stress conditions revealed differential and consistent activation of ICE3 genes, demonstrating their direct contribution to bacterial resilience and suggesting a key adaptive role in response to adverse environmental changes.

bioinformatics↗

bioRxiv2024

The defensome of Acinetobacter baumannii reveals two genome groups according to their innate defense systems and phage profile

Phages are guilty of killing daily almost half of bacterial cells, while bacteria have developed defense mechanisms that number in the dozens. Individual defense systems are gained and lost by genomes of the same species, depending on their fitness advantage. Thus, some genomes have a certain combination of defense systems, while other genomes act as a reservoir for the rest of the systems, thus constituting the so-called pan-immune system of the species. Here we have analyzed thousands of genomes of the bacterium Acinetobacter baumannii, an opportunistic pathogen of humans of great clinical concern, and we have found 81 different defense systems heterogeneously distributed. By analyzing how these systems combine, we have found that more than half of the genomes lack the universal DNA-methylating restriction-modification systems (R-M) and harbor an alternative innate SspBCDE system that performs a DNA phosphorothioate modification. In addition, the adaptive CRISPR-Cas systems could act synergistically to the R-M systems, based on their frequency of co-appearance. The presence of one or the other innate system could modulate the evolution of the genomes of this species, causing them to present a different profile of phages integrated into the bacterial genome. We have also observed that the presence of many defense systems is associated with the presence of a higher number of prophages, which could be due to the fact that the prophage carries the system, or that the bacterium would not need these systems in environments where the phage is absent.

microbiology↗

bioRxiv2023

A biological warfare between two bacterial viruses in a particular genomic region using a CRISPR-Cas system as a weapon

CRISPR-Cas systems are acquired immunity systems of bacteria and archaea that prevent infection by phages and other mobile genetic elements. It is currently known that this defense system has also been co-opted by viruses. These viruses could use CRISPR-Cas systems to compete against other rival viruses. We have discovered a virus in the bacterium Acinetobacter baumannii that incorporates a CRISPR-Cas system into an integration hotspot of the host genome. Once integrated, this could prevent the infection of the most frequent viruses in this bacterial species, especially one that competes with the CRISPR-Cas system itself for the same integration site. This latter virus is prevalent in strains of the species belonging to the so-called Global Clone 2, which causes the most frequent outbreaks worldwide. Knowledge of this new viral warfare using CRISPR-Cas systems, known to limit the entry of antibiotic resistance genes into bacteria, could be useful in the fight against the infections they cause. But it would also shed light on the way in which these defense systems expand in bacteria.

bioinformatics↗

bioRxiv2023

A Systematic Identification of RBPs Driving Aberrant Splicing in Cancer

Alternative Splicing (AS) is a post-transcriptional process by which a single RNA can lead to different mRNA and, in some cases, several proteins. Various processes (probably many of them yet to be discovered) are involved in the regulation of alternative splicing. This work focuses on the regulation by RNA-binding proteins (RBPs). In addition to splicing regulation, these proteins are related to cancer prognosis and are emerging therapeutic targets for cancer treatment. CLIP-seq experiments target selected RBPs and result in uncovering the loci of the nascent transcriptome to where the RBP binds to. The presence of changes in the splicing status surrounding these loci is a good starting point to establishing a causal relationship. The selection of the specific RBP(s) to target in the CLIP-seq experiment is not straightforward; in many cases, this selection is driven by apriori hypotheses. In this work, we have developed an algorithm to detect RBPs that are likely related to the splicing changes between conditions. To do this we have integrated several databases of CLIP-seq experiments with an algorithm that detects differential splicing events to discover RBPs that are especially enriched in these events. This is a follow-up of a previous work that is refined by 1) improving the algorithm to predict the splicing events and 2) testing different enrichment statistics, and 3) performing additional validation experiments. As a result, the new method provides more accurate predictions, and it is also included in the Bioconductor package EventPointer. We tested the algorithm in four different experiments where seven different RBPs were knocked down. The algorithm accurately states the statistical significance of these RBPs using only the alterations in splicing. We also applied the algorithm to study sixteen cancer types from The Cancer Genome Atlas (TCGA). We found relationships between RBPs and several cancer types like CREBBP and MBNL2 alterations in adenocarcinomas of the lung, liver, prostate, rectum, stomach, and colon cancer. Some of these relationships have been validated in the literature but other ones are novel. AvailabilityThis method is integrated EventPointer, an available Bioconductor R package.

bioinformatics↗

bioRxiv2023

A machine learning toolbox for the analysis of sharp-wave ripples reveal common features across species

The study of sharp-wave ripples (SWRs) has advanced our understanding of memory function, and their alteration in neurological conditions such as epilepsy and Alzheimers disease is considered a biomarker of dysfunction. SWRs exhibit diverse waveforms and properties that cannot be fully characterized by spectral methods alone. Here, we describe a toolbox of machine learning (ML) models for automatic detection and analysis of SWRs. The ML architectures, which resulted from a crowdsourced hackathon, are able to capture a wealth of SWR features recorded in the dorsal hippocampus of mice. When applied to data from the macaque hippocampus, these models were able to generalize detection and revealed shared SWR properties across species. We hereby provide a user-friendly open-source toolbox for model use and extension, which can help to accelerate and standardize SWR research, lowering the threshold for its adoption in biomedical applications.

neuroscience↗

bioRxiv2022

In vitro activity and in vivo efficacy against non-tuberculous mycobacteria of SPR719, the active moiety of the novel oral benzimidazole prodrug SPR720

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is increasing globally, with Mycobacterium avium complex (MAC) accounting for 80% of cases and Mycobacterium abscessus among the next most prevalent pathogens. Current treatment necessitates long-term administration of poorly tolerated and modestly effective antibiotic combinations, highlighting the need for new oral agents. SPR719, the active moiety of the benzimidazole phosphate prodrug SPR720, inhibits the ATPase subunits of DNA gyrase B, a target with no human homolog and not exploited by current antibiotics. We demonstrated SPR719 activity against MAC (MIC90, 2 {micro}g/mL) and M. abscessus clinical isolates (MIC90, 4 {micro}g/mL), including those resistant to current standard-of-care (SOC) agents. In vivo efficacy of SPR720 was demonstrated against M. avium ATCC 700898 in a chronic C3HeB/FeJ murine model of pulmonary infection, both as a monotherapy and in combination with clarithromycin, ethambutol and rifabutin. SPR720 monotherapy exhibited a dose-dependent reduction in bacterial burden, with the largest reduction observed when combined with clarithromycin and ethambutol. Efficacy of SPR720 was also demonstrated against M. abscessus subspecies abscessus 1513, a virulent multidrug-resistant strain in a prolonged acute model of pulmonary infection in mice. SPR720 monotherapy exhibited a dose-dependent reduction in bacterial burden with further reductions when combined with SOC agents, clarithromycin and amikacin {+/-} clofazimine. Taken together, the in vitro activity of SPR720 against common NTM pathogens in concert with the proof-of-concept efficacy in murine infection models warrants the continued clinical evaluation of SPR720 as a new oral option for the treatment of NTM-PD.

microbiology↗

bioRxiv2022

An outstanding bacterial membranome requires CRISPR-Cas systems to avoid the intervention of phages

Antimicrobial resistance is widely recognized as a serious global public health problem. To combat this threat, a thorough understanding of bacterial genomes is necessary. The current wide availability of bacterial genomes provides us with an in-depth understanding of the great variability of dispensable genes and their relationship with antimicrobials. Some of these accessory genes are those involved in CRISPR-Cas systems, which are acquired immunity systems that are present in part of bacterial genomes. They prevent viral infections through small DNA fragments called spacers. But the vast majority of these spacers have not yet been associated with the virus they recognize, and this has been named CRISPR dark matter. By analyzing the spacers of tens of thousands of genomes from six bacterial species highly resistant to antibiotics, we have been able to reduce the CRISPR dark matter from 80-90% to as low as 15% in some of the species. In addition, we have observed that, when a genome presents CRISPR-Cas systems, this is accompanied by particular collections of membrane proteins. Our results suggest that when a bacterium presents membrane proteins that make it compete better in its environment, and these proteins are in turn receptors for specific phages, it would be forced to acquire CRISPR-Cas immunity systems to avoid infection by these phages.

bioinformatics↗

bioRxiv2021

Identifying Lethal Dependencies with HUGE Predictive Power from Large-Scale Functional Genomic Screens

Recent functional genomic screens -such as CRISPR-Cas9 or RNAi screening-have fostered a new wave of targeted treatments based on the concept of synthetic lethality. These approaches identified LEthal Dependencies (LEDs) by estimating the effect of genetic events on cell viability. The multiple-hypothesis problem related to the large number of gene knockouts limits the statistical power of these studies. Here, we show that predictions of LEDs from functional screens can be dramatically improved by incorporating the "HUb effect in Genetic Essentiality" (HUGE) of gene alterations. We analyze three recent genome-wide loss-of-function screens - Project Score, CERES score and DEMETER score-identifying LEDs with 75 times larger statistical power than using state-of-the-art methods. HUGE shows an increased enrichment in a recent harmonized knowledgebase of clinical interpretations of somatic genomic variants in cancer (with an AUROC up to 0.87). Our approach is effective even in tumors with large genetic heterogeneity such as acute myeloid leukemia, where we identified LEDs not recalled by previous pipelines, including FLT3-mutant genotypes sensitive to FLT3 inhibitors. Interestingly, in-vitro validations confirm lethal dependencies of either NRAS or PTPN11 depending on the NRAS mutational status. HUGE will hopefully help discover novel genetic dependencies amenable for precision-targeted therapies in cancer.

genomics↗

bioRxiv2021

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.

genetics↗

bioRxiv2021

L-arginine ameliorates defective autophagy in GM2 gangliosidoses by mTOR modulation

Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause fatal and progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by {beta}-hexosaminidase A (HexA) leads to the accumulation of its specific substrate, GM2 ganglioside, in neuronal lysosomes. Despite the development of a florid storage phenotype, the role of autophagy and its regulation by the mammalian target of rapamycin (mTOR) has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. Pathological autophagosomes, with enhanced expression of the p62/SQSTM1 protein, suggested impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed inappropriately diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities - and immediately suggests an avenue for therapeutic exploration in this cruel disease. We also contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of systemic treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy

cell biology↗

bioRxiv2020

BOSO: a novel feature selection algorithm for linear regression with high-dimensional data

MotivationWith the frenetic growth of high-dimensional datasets in different biomedical domains, there is an urgent need to develop predictive methods able to deal with this complexity. Feature selection is a relevant strategy in machine learning to address this challenge. ResultsWe introduce a novel feature selection algorithm for linear regression called BOSO (Bilevel Optimization Selector Operator). We conducted a benchmark of BOSO with key algorithms in the literature, finding a superior performance in highdimensional datasets. Proof-of-concept of BOSO for predicting drug sensitivity in cancer is presented. A detailed analysis is carried out for methotrexate, a well-studied drug targeting cancer metabolism. AvailabilityA Matlab implementation of BOSO is available as a Supplementary Material. Contactfplanes@tecnun.es Supplementary InformationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2020

Ribosome profiling reveals ribosome stalling on tryptophan codons upon oxidative stress in fission yeast

Modulation of translation is an essential response to stress conditions. We have investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe subject to five environmental stresses: oxidative stress, heavy metal, heat shock, osmotic shock and DNA damage. We also explored the contribution of two major defence pathways to these programmes: The Integrated Stress Response, which directly regulates translation initiation, and the stress-response MAPK pathway. To obtain a genome-wide and high-resolution view of this phenomenon, we performed ribosome profiling of control cells and of cells subject to each of the five stresses mentioned above, both in wild type background and in cells in which the Integrated Stress Response or the MAPK pathway were inactivated. Translational changes were partially dependent on the integrity of both signalling pathways. Interestingly, we found that the transcription factor Fil1, a functional homologue of the Gcn4 and Atf4 proteins (from budding yeast and mammals, respectively), was highly upregulated in most stresses. Consistent with this result, Fil1 was required for the normal response to most stresses. A large group of mRNAs were translationally downregulated, including many required for ribosome biogenesis. Overall, our data suggest that severe stresses lead to the implementation of a universal translational response, which includes energy-saving measures (reduction of ribosome production) and induction of a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons specifically upon oxidative stress, a phenomenon that is likely caused by a decrease in charged tRNA-Tryptophan. Tryptophan stalling led to a mild translation elongation reduction and contributed to the inhibition of initiation by the Integrated Stress Response. Taken together, our results show that different stresses elicit common and specific translational responses, revealing a special and so far unknown role in Tryptophan-tRNA availability.

molecular biology↗

bioRxiv2020

Reconstitution of intestinal stem cell niche in vitro with pharmacological inhibitors or L-WRN conditioned medium differentially regulates epithelial proliferation, differentiation and barrier function in rabbit caecum organoids

Intestinal organoids are self-organized 3-dimensional (3D) structures formed by a single layer of polarized epithelial cells. This innovative in vitro model is highly relevant to study physiology of the intestinal epithelium and its role in nutrition and barrier function. However, this model has never been developed in rabbits, while it would have potential applications for biomedical and veterinary research. Here, we cultured rabbit caecum organoids with either pharmacological inhibitors (2Ki medium) or L-WRN cells conditioned medium (L-WRN CM) to reconstitute the intestinal stem cell niche in vitro. Large spherical organoids were obtained with the 2Ki medium and this morphology was associated with a high level of proliferation and stem cells markers gene expression. In contrast, organoids cultured with L-WRN CM had a smaller diameter; a greater cell height and part of them were not spherical. When the L-WRN CM was used at low concentration (5%) for two days, the gene expression of stem cells and proliferation markers were very low, while absorptive and secretory cells markers and antimicrobial peptides were elevated. Epithelial cells within organoids were polarized in 3D cultures with 2Ki medium or L-WRN CM (apical side towards the lumen). We cultured dissociated organoid cells in 2D monolayers, which allowed accessibility to the apical compartment. Under these conditions, actin stress fibers were observed with the 2Ki medium, while perijonctionnal localization of actin was observed with the L-WRN CM suggesting, in 2D cultures as well, a higher differentiation level in the presence of L-WRN CM. In conclusion, rabbit caecum organoids cultured with the 2Ki medium were more proliferative and less differentiated than organoids cultured with L-WRN CM. We propose that organoids cultured with the 2Ki medium could be used to rapidly generate in vitro a large number of rabbit intestinal epithelial stem cells while organoids cultured with the L-WRN CM represent a suitable model to study differentiated rabbit epithelium.

cell biology↗

bioRxiv2020

Morphological profiling of human T and NK lymphocytes identifies actin-mediated control of the immunological synapse

The detection and neutralization of infected cells and tumors by cytotoxic lymphocytes is a vital immune defense mechanism. The immunological synapse orchestrates the target recognition process and the subsequent cytotoxic activity. Here, we present an integrated experimental and computational strategy to systematically characterize the morphological properties of the immunological synapse of human cytotoxic lymphocytes. Our approach combines high-content imaging with an unbiased, data-driven identification of high-resolution morphological profiles. Such profiling discriminates with high accuracy immunological synapse perturbations induced by an array of actin drugs in both model cell lines and primary lymphocytes. It reveals inter-individual heterogeneity in lymphocyte morphological traits. Furthermore, it uncovers immunological synapse alterations in functionally defective CD8+ T cells from immunodeficient patients carrying ARPC1B mutations. Our study thus provides a foundation for the application of morphological profiling as a powerful and scalable approach to monitor lymphocyte activation status in experimental and disease settings.

immunology↗