Biology subjects

Ruiz, L.

Publications and source records attributed to Ruiz, L..

12 recordsLinked to original sources

bioRxiv2026

EpCAM sustains crypt mechanics and prevents aberrant remodeling

Crypt morphogenesis is a fundamental process that drives intestinal growth and maintains its homeostasis. Although intestinal tissue mechanics is recognized as pivotal for early crypt initiation and budding, the molecular mechanisms that couple epithelial forces to mature crypt behavior remain largely unexplored. In particular, while Paneth cells have been proposed to be implicated in the mechanical control of crypt remodeling and fission, the underlying pathways have remained elusive. Here, we identified EpCAM (Epithelial Cell Adhesion Molecule) as a key regulator of epithelial contractility and crypt morphogenesis. EpCAM was preferentially enriched at lateral interfaces of Paneth cells in vivo and in organoid models, where it maintained cortical tension and contractile organization. Loss of EpCAM disrupted crypt cell cortical myosin II-A localization, leading to aberrant Paneth cell apical contractility, enlarged apical surfaces, distorted pyramidal morphology, and altered aspect ratios. Consequently, Paneth cells lose their tight clustering in crypt base, their spatial distribution along the crypt axis becomes irregular, and the collective architecture of the crypt epithelium was compromised. At the tissue level, EpCAM deficiency profoundly perturbed crypt fission, increasing fission frequency while favoring asymmetric outcome both in EPCAM-mutated Congenital Tufting Enteropathy (CTE) patients. Live imaging of EpCAM-knockout mouse organoids confirmed a failure in the spatial control of crypt bifurcation. Collectively, our findings revealed that EpCAM is not required to initiate fission, but is essential to coordinate the mechanical forces and ensure correct division of the crypt. We showed that EpCAM-dependent biomechanical integrity of Paneth cells generates a stabilizing force field at the crypt base, orchestrating coordinated tissue remodeling. Disruption of this program led to unbalanced force transmission, distorted crypt geometry, and aberrant fission outcomes, revealing a critical link between epithelial mechanics and intestinal morphogenesis.

developmental biology↗

bioRxiv2025

Borzoi-informed fine mapping improves causal variant prioritization in complex trait GWAS

1Genome-wide association studies (GWAS) have identified thousands of trait-associated loci. Prioritizing causal variants within these loci is critical for characterizing trait biology. Statistical fine mapping identifies causal variants at trait-associated loci, but linkage disequilibrium (LD) and limited GWAS sample sizes prevent the resolution of many associations. Functionally informed approaches augment fine mapping by estimating variant prior causal probabilities based on overlap with trait-relevant functional annotations. However, functional enrichment provides only an indirect proxy for variant functional impact. Sequence-to-function models directly estimate variant effects on molecular phenotypes from underlying sequence context. Borzoi is a long-context model that predicts sequence determinants of transcription, splicing, and polyadenylation across diverse tissues and cell types. Here we present Sniff, a Borzoi-informed fine-mapping approach that integrates broad genomic functional annotations with Borzoi-predicted variant effects via PolyFun to estimate variant prior causal probabilities. Applied to 15 UK Biobank traits, Sniff identifies 9.45% additional fine-mapped variants compared to PolyFun-Baseline at posterior inclusion probability (PIP) > 0.8. Sniff-prioritized variants exhibit allele-specific activity in reporter assays and are predicted to have tissue-specific activity in trait-relevant tissues. For most traits, genes nominated by Sniff receive higher scores from the orthogonal gene prioritization method PoPS compared to genes nominated using functional annotations alone. Because differentially prioritized variants are driven by Borzoi predictions, we leverage attribution techniques to characterize sequence features underlying fine mapping and generate mechanistic hypotheses for GWAS associations.

genetics↗

bioRxiv2025

In silico drug sensitivity predicts subgroup-specific therapeutics in medulloblastoma patients

BackgroundMedulloblastoma is the most common malignant pediatric brain tumor. Survival rates vary widely between subgroups, with an average overall survival of 70%. Recurrent medulloblastoma is highly aggressive, treatment-resistant, and usually fatal. In addition, current treatments are highly toxic to the developing brain and surviving patients suffer from lifelong side effects. Therefore, novel therapeutic options are urgently needed. MethodsTo inform risk-based, personalized therapy, we developed a novel platform called DrugSeq, which allows predictions of drug sensitivities in patients across medulloblastoma subgroups. We used a perturbagen-response dataset to calculate transcriptional response signatures for each drug and compared this to patient medulloblastoma tumor gene expression. We then stratified patients by molecular subgroup and used an ANOVA analysis to identify drugs that selectively targeted each subgroup. ResultsWe found distinct differences in transcriptional profiles and predicted drug sensitivity for each medulloblastoma subgroup. We identified several kinase inhibitors, epigenetic inhibitors, and several drugs that have been investigated in drug repositioning studies for cancer. ConclusionsWe posit that DrugSeq may identify novel therapies and facilitate patient stratification in clinical trials, leading to more successful targeted medulloblastoma therapies that improve tumor response while minimizing late toxicities. This computational tool can also be used for other cancers to stratify patients based on any clinical or molecular feature. Key points DrugSeq calculates drug sensitivity for medulloblastoma tumors stratified by subgroup. DrugSeq platform may inform patient stratification strategies in clinical trials. Importance of the StudyMedulloblastoma is the most common malignant pediatric brain tumor. Current standard-of-care typically includes surgical resection, multi-agent chemotherapy, and radiation. However, survival rates vary widely between subgroups, ranging from 45 to 90%, depending on age and molecular features. In addition, surviving children frequently suffer from debilitating late side effects of therapy including neurocognitive impairment, epilepsy, stroke, subsequent cancer, endocrinopathies, and early mortality. Therefore, novel therapeutic options are urgently needed. However, a one-size-fits-all approach for therapy is unlikely to be effective given the well-characterized intertumor heterogeneity of medulloblastoma.

cancer biology↗

bioRxiv2025

RAC1 Regulates Shh-Medulloblastoma Growth via GLI-Mediated Transcription

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor. Current therapies are ineffective for targeting proliferation, leptomeningeal migration, and metastasis of MB cancer cells to visceral organs and therefore, novel treatments are needed. The small GTPase, RAC1, has emerged as an important regulator of actin cytoskeletal dynamics, proliferation, and migration in several cancers. However, it has not been characterized in MB and no clinical drug candidates have been described for RAC1 in MB. Here we demonstrate that RAC1 levels are higher in MB tissue relative to normal cerebellum. Further, RAC1 depletion significantly reduces proliferation and migration of Shh-MB cells in vitro. Mechanistically, RAC1 controls the mRNA and protein levels of the main transcription factors in the Shh pathway, GLI1 and GLI2. RAC1 binds to the GLI1 promoter highlighting a novel role in transcriptional regulation in Shh-dependent cancers. We demonstrate that the RAC1 inhibitor, GYS32661, is brain penetrant, and reduces MB growth and increases mouse survival in an orthotopic model of Shh-MB. Importantly, GYS32661 is a non-toxic clinical candidate, suggesting that it may be a novel potential drug for the treatment of either the pediatric or adult forms of MB. Collectively, our studies identify RAC1 as a druggable target in Shh-dependent MB. Graphical abstractRAC1 controls Shh-MB progression by binding to the GLI1 promoter. Graphical abstract demonstrating RAC1 localizes to the nucleus and binds to the GLI1 promoter and plays an important role in its transcription. RAC1 genetic or GYS32661 mediated inhibition leads to RAC1 dissociation from the GLI1 promoter and transcriptional repression of Shh-MB biomarkers GLI2, DNMT1 and UHRF1. This eventually causes inhibition of Shh-MB tumor cell proliferation and migration, which leads to a decrease in Shh-MB development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/655563v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@e4def9org.highwire.dtl.DTLVardef@1eab826org.highwire.dtl.DTLVardef@dfe68borg.highwire.dtl.DTLVardef@198efb9_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Divergent Effects of APOE3 and APOE4 Human Astrocytes on Key Alzheimer's Disease Hallmarks in Chimeric Mice

Despite strong evidence supporting that both astrocytes and apolipoprotein E (APOE) play crucial roles in the pathogenesis and progression of Alzheimers disease (AD), the impact of astrocytes carrying different APOE variants on key AD pathological hallmarks remains largely unknown. To explore such effects in a human relevant context, we generated a chimeric model of AD. We transplanted isogenic APOE3 or APOE4 human induced pluripotent stem cell (hiPSC)-derived astrocyte progenitors into neonatal brains of AD model mice. We show that at five to six months after transplantation, transplanted cells have differentiated into mature astrocytes (h-astrocytes) that often integrate in upper layers of one cortical hemisphere. APOE3 and APOE4 h-astrocytes differentially express and secrete the APOE protein, which binds to A{beta} plaques with an isoform-dependent affinity. Remarkably, APOE3 h-astrocytes ameliorate A{beta} pathology, Tau pathology and neuritic dystrophy. In contrast, APOE4 h-astrocytes aggravate these AD processes. Moreover, APOE3 and APOE4 h-astrocytes modulate microglia responses to A{beta} pathology in opposite ways. APOE4 h-astrocytes enhance microglia clustering around A{beta} plaques and exacerbate DAM state whereas APOE3 h-astrocytes reduce microglia clustering and induce a more homeostatic state on plaque-associated microglia. These findings highlight a critical contribution of h-astrocytes not only to A{beta} pathology but also to other key AD hallmarks in chimeric mice. In addition, our findings reveal that h-astrocytes with different APOE variants and the different forms of APOE they secrete have a crucial role in AD progression.

neuroscience↗

bioRxiv2024

High-Resolution Spatial Transcriptomic Atlas of Mouse Soleus Muscle: Unveiling Single Cell and Subcellular Heterogeneity in Health and Denervation

Skeletal muscle exhibits pronounced cellular and subcellular heterogeneity, but comprehensive spatial mapping has been constrained by cell/nuclei dissociation-based methods that lose tissue architecture and by spatial platforms with insufficient resolution or limited transcriptome coverage. Here we present a high-resolution spatial transcriptomic atlas of mouse soleus muscle in longitudinal sections with unbiased whole-transcriptome coverage, enabling myofiber-resolved transcriptomes while preserving subcellular expression domains across the length of fibers. Combining histology-guided myofiber segmentation with unbiased grid-based mapping, we recover canonical fiber types and reveal widespread hybrid myofiber states in situ, including type IIb-associated signatures that are rare in soleus muscle and evident only when intramyofiber heterogeneity is assessed. At subcellular scale, we delineate the neuromuscular junction (NMJ) as a multi-compartment niche comprising postsynaptic myonuclei and spatially distinct peri-synaptic and myelinating Schwann cell-associated regions, each with characteristic gene programs. Applying this framework to denervation (3 and 7 days) identifies robust fiber-type-specific stress responses, coordinated remodeling of macrophage and fibroblast transcriptomes, and marked intramyofiber heterogeneity, including spatially nonuniform activation of damage-response genes along individual myofibers, with distinct transcriptional domains proximal and distal to the NMJ and associated degenerative histological features. Together, this atlas provides a high-resolution reference for muscle biology and clarifies how denervation reshapes myofiber, synaptic, and stromal-immune programs across cells and within cells in intact tissue.

physiology↗

bioRxiv2024

Hippocampal reactive neural stem cells are able to phagocytose and have an immunological molecular signature

Hippocampal neural stem cells (NSCs) are the drivers of neurogenesis in the dentate gyrus (DG) of most mammals including humans. During neuronal hyperactivity NSCs become reactive NSCs (react-NSCs), characterized by their activation, morphological changes, and symmetric division, abandoning their neurogenic programme and transforming into reactive astrocytes. Here, using different pathological models that induce react-NSCs in the DG, we looked for novel features of react-NSCs both histologically and by total RNA sequencing. We report that in two pathological models were react-NSCs emerge (mesial temporal lobe epilepsy (MTLE) and traumatic brain injury (TBI)) react-NSCs are capable of phagocytosis of dead cells, a typical immunological function carried out mainly by microglia in the brain. Importantly, MTLE-induced react-NSCs show phagocytic function in tissue and a predominantly immunological molecular signature, with a broad upregulation of phagocytosis-related gene expression. Our results describe a new function of react-NSCs as phagocytic and immunologically active cells in the hippocampal neurogenic niche.

neuroscience↗

bioRxiv2022

Accurate Genome-Wide Phasing from IBD Data

As genotype databases increase in size, so too do the number of detectable segments of identity by descent (IBD): segments of the genome where two individuals share an identical copy of one of their two parental haplotypes, due to shared ancestry. We show that given a large enough genotype database, these segments of IBD collectively overlap entire chromosomes and can be used to phase them accurately. Furthermore, primarily using instances of IBD that span multiple chromosomes, we can accurately phase an entire genome. We are able to separate the DNA inherited from each parent completely, across the entire genome, with 98% median accuracy in a test set of 30,000 individuals. We estimate the IBD data requirements for accurate phasing, and we propose a method for estimating confidence in the resulting phase. We show that our methods do not require the genotypes of close family, and that they are robust to genotype errors and missing data. In fact, our method can impute missing data accurately and correct genotype errors. Author summaryWe present a method for phasing, separating the DNA inherited from each parent, of an entire genome using short segments of DNA that are shared between the genome of the person we wish to phase and those of distant cousins. Essentially, if we have enough of these distant cousins genotypes available, we can piece together overlapping segments until we have phased the entire genome. We have developed a method that can phase large numbers of individuals, and that makes special considerations for potential close family and potential genotype errors in data. We report results on experiments phasing 30,000 individuals. We analyze how many such segments are required to phase accurately, and propose a model-based method to estimate accuracy. We also show that our method can accurately impute missing data and correct genotype errors.

genetics↗

bioRxiv2021

Conformational landscape of full-length Smad proteins

Smad transcription factors, the main effectors of the TGF{beta} (transforming growth factor {beta}) network, have been shaped along the evolution of multicellular animals to regulate essential processes. Smad proteins have a mixed architecture of globular domains and flexible linkers and adopt distinct quaternary structures depending on their activation state and cellular context. Here we studied the structures of full-length Smad4 and Smad2 proteins through an integrative approach combining small-angle X-ray scattering and detailed atomic information obtained from Nuclear Magnetic Resonance spectroscopy, X-ray and molecular dynamic simulations. Both Smad4 and Smad2 populate ensembles of expanded/compact conformations, with the MH1 and MH2 domains tethered by intrinsically disordered linkers that provide conformational freedom to the proteins. In solution, Smad4 is monomeric, whereas Smad2 coexists as monomer-dimer-trimer association states, even without activation. Smad2 dimers, which were previously overlooked, are proposed as key building blocks that define the functional quaternary structures of Smad proteins. HighlightsO_LISolution ensembles of full-length Smad2 and Smad4 proteins C_LIO_LISmad4 populates dynamic extended and compact monomeric conformations C_LIO_LISmad2 dimers may be the intermediate state to form heterotrimers with Smad4 C_LIO_LINew integrative methodological strategy for examining multi-domain and flexible proteins C_LI

molecular biology↗

bioRxiv2021

Follow-up of a hospital cohort during the first 3,530 suspected cases of COVID-19 in Sao Jose do Rio Preto, Sao Paulo, Brazil

IntroductionIn a global context, COVID-19 is the most significant health threat in the present days, evidenced by the fact that, in just over four months, SARS-CoV-2 has spread to 171 countries, reaching a Pandemic status. Most patients with COVID-19 have a mild course of the disease. However, approximately 20% develop severe illness with a high mortality rate which is associated with age, comorbidities, and immunosuppression. Epidemiological studies are used to reveal the extent of viral spread in homes, communities, and hospitals. Thus, preventive and control measures can be established by the authorities. ObjectiveIn this study, patients with suspect COVID-19 symptoms who search for hospital care at the city of Sao Jose do Rio Preto (Sao Paulo, Brazil) were monitored, in order to identify the first case of this new disease in the region. In the first two months (March and April), more than 3000 individuals looked for the public and private health system with suspected respiratory symptoms, but only 164 (8.4%) were COVID-19 confirmed. ResultsFrom those, males (56.1%) and patients of the age distribution of 16-59 (91.2%), with diarrhea (22.2%), runny nose (25%), altered taste (15.9%), and anosmia (11.6%) presented statistical significance, although none comorbidities were related with COVID-19 occurrence. The odds ratio analysis supports this finding. Days of onset of symptoms are positively associated with whit viral load, and the same happens with the occurrence of symptoms (dyspnea and low saturation).

molecular biology↗

bioRxiv2020

Structures of the germline-specific Deadhead and Thioredoxin T proteins from Drosophila melanogaster reveal unique features among Thioredoxins

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and TrxT. Both proteins belong to the L(3)mbt malignant brain tumor signature and to the MMS survival network of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome. Here we present the first structures of Dhd and TrxT that unveil new features of these Thioredoxins. Dhd is highly positively charged, unusual in canonical Trxs. This positively charged surface can facilitate its approximation to DNA and to protamine oligomers, to promote chromatin remodeling. On the other hand, TrxT contains a C-terminal extension, mostly unstructured and highly flexible, which wraps the conserved core through a closed conformation. This extension partially covers the catalytic site and modulates the redox activity of the protein. The information provided by these structures can guide future work aimed at understanding how redox inputs modulate the initial steps of embryo development in Drosophila and may help in the design of molecular inhibitors through a structure-based approach. HighlightsO_LIWe have determined the first structures of the germline-specific Trxs Dhd and TrxT. C_LIO_LIDhd has a highly positively charged surface that facilitates its approximation to DNA and protamine oligomers, to promote chromatin remodeling. C_LIO_LITrxT contains a C-terminal extension, highly unusual in canonical Trxs, mostly unstructured and highly flexible. C_LIO_LIThe TrxT C-terminal extension partially covers the catalytic site and modulates the redox activity of the protein. C_LIO_LIThe differences observed in Thioredoxins can help in fine-tuning specific molecules to be active against selected insect species. C_LI

molecular biology↗

bioRxiv2019

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes

R-Smads are effectors of the transforming growth factor {beta} (TGF{beta}) superfamily and along with Smad4 form trimers to interact with other transcription factors and with DNA. The 5GC-DNA complexes determined here by X-ray crystallography for Smad5 and Smad8 proteins corroborate that all MH1 domains bind SBE and 5GC sites similarly, although Smad2/3/4 MH1 domains bind DNA as monomers whereas Smad1/5/8 form helix-swapped dimers. To examine the relevance of the dimerization phenomenon and to exclude a possible crystallography-induced dimeric state, we studied these MH1 domains in solution. The results show that Smad5/8 domains populate dimers and monomers in equilibrium, whereas their Smad2/3/4 counterparts adopt monomeric conformations. We also found that swapping the loop1 sequence between Smad5 and Smad3 results in the Smad5 chimera-DNA complex crystallizing as a monomer, revealing that the loop1 sequence determines the monomer/dimer propensity of Smad MH1-domains. We propose that the distinct MH1-dimerization status of TGF{beta} and BMP activated Smads influences the interaction with specific loci genome-wide by distinct R-Smad and Smad4 complexes. SignificanceTGF{beta}- and BMP-activated R-Smads were believed to have different preferences with respect to the recognition of DNA motifs and to respond to specific activation inputs. However, recent results indicate that several types of R-Smads can be activated by similar receptors and that all Smads might recognize various DNA motifs. These results pose new questions as to why different types of R-Smads have been conserved for more than 500 million years if they could have a redundant function. They also raise questions as to how different Smad complexes recognize specific clusters of DNA motifs genome-wide. Here, using structural biology approaches, we elucidate some of the rules that help define the dimers of Smad-DNA complexes and propose how dimers and monomers could influence the composition of Smad complexes, as well as the recognition of specific cis-regulatory elements genome-wide. HighlightsR-Smads and Smad4 interact with 5GC and GTCT sites using a conserved binding mode. Functional differences of TGF{beta}- and BMP-activated R-Smads are not exclusively related to DNA specificity. Dimer/monomer propensities are detected in solution and in the absence of DNA. Loop1 sequence determines the propensity of R-Smads to form monomers or dimers in complexes with DNA. Author ContributionsL.R., Z.K., and T.G. designed and performed most experiments and coordinated collaborations with other authors. L.R. and E.A. cloned, expressed and purified all proteins, L.R., R.F., C.T., N.M., and J.C performed EMSA experiments. L.R., E.A., T.G., T.C., P.M.M., and M.J.M. performed the SAXS and NMR measurements and analyzed the data. P.M.M. analyzed the clustering of DNA motifs in ChIP-Seq data. Z.K. B.B. and R.P. screened crystallization conditions, collected X-ray data and determined the structures. Z.K., R.F. R.P., T.G., J.A.M., and M.J.M. analyzed the structures. All authors contributed ideas to the project. M.J.M. and R.P. supervised the project. M.J.M. wrote the manuscript with contributions from all other authors. The authors declare no conflict of interest. Data DepositionNMR assignments and chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank, BMRB entry 27548, and the Small-angle scattering data and models have been deposited in SASBDB, entries SASDE32 (Smad5) and SASDE42 (Smad8). Densities and coordinates have been deposited in the Protein Data Bank, entries 6FZS (Smad5), 6FZT (Smad8), 6TBZ (Smad5_3 chimera), 6TCE (Smad5_gly mutant). This article contains supporting information.

molecular biology↗