Biology subjects

Braga, L.

Publications and source records attributed to Braga, L..

12 recordsLinked to original sources

bioRxiv2026

Targeting hypoxia-responsive miR-155-5p and miR-210-3p restores alveolar regeneration and reverses pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an age-associated degenerative disease largely driven by failure of alveolar epithelial regeneration, yet current therapies slow fibrosis progression without restoring epithelial repair. Building on our previous unbiased microRNA screen in primary murine alveolar type II (ATII) cells, we identified miR-155-5p and miR-210-3p as previously unrecognized mediators of alveolar epithelial regenerative failure. Both microRNAs were markedly upregulated in ATII cells from patients with IPF and enriched within KRT17+/KRT5- aberrant transitional epithelial cells. Their expression also increased spontaneously with ageing in ATII cells from uninjured mice, linking these microRNAs to the age-dependent loss of ATII to ATI transdifferentiation capacity. Furthermore, we found that hypoxia-induced HIF signalling drives the expression of these microRNAs in ATII cells, locking them in a dysfunctional transitional state characterized by a profibrotic secretome that promotes paracrine myofibroblasts activation. Antisense oligonucleotide (ASO) inhibition of selected miRNAs restored ATII-to-ATI differentiation, eliminated aberrant transitional states, normalized epithelial-mesenchymal communication, promoted de novo alveolar regeneration and reversed fibrosis, including in both young and aged mice. Together, these findings identify the hypoxia-responsive miRNAs miR-155-5p and miR-210-3p as therapeutically actionable regulators of alveolar regenerative failure and establish their inhibition as a strategy to restore endogenous lung repair while disrupting pathological epithelial-mesenchymal crosstalk in pulmonary fibrosis.

cell biology↗

bioRxiv2026

AAV-miR-124 enhances endogenous alveolar epithelial regenerative plasticity and reverses bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by irreversible destruction of the alveolar epithelium and impaired regeneration. Although current therapies slow disease progression, they do not restore functional alveoli, highlighting the need for regenerative approaches that promote endogenous lung repair. Here, we performed the first unbiased functional screen of 2,042 human microRNA mimics in primary mouse alveolar type II (ATII) cells to identify regulators of ATII-to-alveolar type I (ATI) cell transdifferentiation. The screen identified miR-124-3p as the most effective promoter of ATI differentiation. In vitro, miR-124-3p promoted ATII-to-ATI transdifferentiation in healthy and bleomycin-injured ATII cells while also increasing the ATII cell pool, consistent with activity on epithelial progenitors. Using the engineered AAV6.2FF capsid, we generated a vector encoding miR-124-3p, which efficiently transduced ATII cells, MHC-II club distal progenitor cells, and injury-induced KRT8 epithelial intermediates. Therapeutic administration after fibrosis establishment reduced lung fibrosis, restored alveolar architecture, and showed greater efficacy than nintedanib in the bleomycin mouse model. Mechanistically, we propose a context-dependent model whereby miR-124-3p regulates epithelial cell states through the EZH2-C/EBP axis while attenuating epithelial transcriptional programs associated with IPF. Together, these findings support AAV-mediated delivery of miR-124 to promote alveolar repair in pulmonary fibrosis.

cell biology↗

bioRxiv2026

Molecular Profiling and High-Content Drug Screening of Metastatic Colorectal Cancer Organoids Reveal Evolutionary Mechanisms of Pan-KRAS Inhibitor Resistance

Metastatic colorectal cancer exhibits extensive intertumoral and intratumoral heterogeneity, which underlies variable treatment responses and the inevitable emergence of drug resistance. Preclinical models that accurately reflect this heterogeneity and enable the investigation of resistance mechanisms at the clonal level, while also providing sensitive and quantitative measurements of treatment response, remain limited. Here, we describe a living biobank of patient-derived organoids (PDOs), generated from metastatic colorectal cancer samples encompassing various genetic backgrounds, including different KRAS genotypes. These PDOs retain key phenotypic, genomic, and transcriptional features of their matched tumors, and, when coupled with a multiparametric high-content 3D drug-screening platform integrating volumetric growth, nuclear content, and proliferative readouts, enable sensitive detection of subtle responses that are not captured by conventional viability-based assays. Systematic phenotypic profiling identified KRAS mutational status as a major determinant of drug response, with KRAS wild-type organoids exhibiting predominant cytotoxic collapse, whereas KRAS-mutant PDOs displayed cytostatic growth arrest. Building on this phenotypic stratification, we investigated resistance to the pan-KRAS inhibitor BI-2865 by integrating longitudinal drug exposure with expressible DNA barcoding and single-cell RNA sequencing to resolve clonal and transcriptional dynamics. Resistance to BI-2865 arose primarily through transcriptional plasticity rather than widespread genomic alterations. Lineage tracing revealed genotype-dependent trajectories, with de novo emergent clones driving resistance in KRASWT PDO, whereas KRASG13D models were dominated by expansion of pre-existing clones. Single-cell analysis on KRASG13D demonstrated a shared transcriptional landscape comprising multiple resistant states rather than a single convergent identity. Together, our findings define resistance to pan-KRAS inhibition as a modular, state-resolved process driven by transcriptional plasticity and genotype-dependent clonal dynamics and establish PDO-based multiparametric phenotyping combined with lineage-resolved single-cell profiling as a robust framework for dissecting the evolutionary framework of therapeutic resistance in metastatic colorectal cancer.

cancer biology↗

bioRxiv2024

Drug-repurposing screen identifies thiostrepton as a novel regulator of the tumor suppressor DAB2IP

The tumor suppressor DAB2IP, a RasGAP and cytoplasmic adaptor protein, modulates signal transduction in response to several extracellular stimuli, negatively regulating multiple oncogenic pathways. Accordingly, the loss of DAB2IP in tumor cells fosters metastasis and enhances chemo- and radio-resistance. DAB2IP is rarely mutated in cancer but is frequently downregulated or inactivated by multiple mechanisms. Solid experimental evidence show that DAB2IP reactivation can reduce cancer aggressiveness in tumors driven by multiple different oncogenic mutations, making this protein an interesting target for anti-cancer therapy. Based on these premises, we screened a library of FDA-approved drugs to search for molecules that can increase DAB2IP protein levels. We exploited CRISPR/Cas9 gene editing to generate two prostate cancer cell models in which endogenous DAB2IP is fused to HiBiT, a peptide tag that enables luminescence-based detection of protein levels in a sensitive and quantitative manner. Using this approach, we identified drugs able to increase DAB2IP levels. We focus our attention on thiostrepton, a natural cyclic oligopeptide antibiotic that has been reported to inhibit survival of various cancer cell lines. Functional experiments revealed that the cancer inhibitory effect of thiostrepton is reduced in the absence of DAB2IP, suggesting that the observed upregulation contributes to its action. These findings encourage the further development of thiostrepton for the treatment of solid cancers, and unveil a novel molecular mechanism underlying its anti-tumoral action.

cancer biology↗

bioRxiv2024

Functional screening identifies novel miRNAs inhibiting Vascular Smooth Muscle Cell proliferation

Proliferation of vascular smooth muscle cells (vSMCs) following injury is a crucial contributor to pathological vascular remodelling. MicroRNAs (miRNAs) are powerful gene regulators and attractive therapeutic agents. Here, we aim to systematically identify and characterise miRNAs with therapeutic potential in targeting aberrant vSMC proliferation. We performed a high-throughput in vitro screen using a library of 2042 human miRNA-mimics for their impact on vSMC proliferation and identified seven novel antiproliferative miRNAs i.e miR-323a-3p, miR449b-5p, miR-491-3p, miR-892b, miR-1827, miR-4774-3p, miR-5681b. Overexpression of these seven miRNAs affects proliferation of vSMCs from different vascular beds. Focusing on vein graft failure, a condition in which miRNA-based therapeutics can be applied to the graft ex-vivo, we showed that these miRNAs reduced human saphenous vein SMC (HSVSMC) proliferation without inducing apoptosis or senescence, and five of them also significantly decreased migration. HSVSMC transcriptomic analysis showed that each miRNA overexpression affects a core cell cycle gene network. However, this effect is mediated by distinct miRNA targets. In contrast to HSVSMC, miRNA overexpression in saphenous vein endothelial cells (ECs) led to no decrease or a less pronounced reduction in proliferation for the seven miRNAs. Transcriptomics analysis confirmed a distinct and limited response of ECs to the miRNA overexpression.

molecular biology↗

bioRxiv2023

Pauperization of Emerin from nuclear envelope drives prostate cancer cell migration and invasiveness

Micronuclei (MN) can arise from many causes, including the breakage of aberrant cytokinetic chromatin bridge. The frequent observation of MN in tumors raises the specter that they might not merely be passive elements but could instead play active roles in tumor progression. Here, we test the hypothesis that the presence of micronuclei could induce specific phenotypic and functional changes to the cell and lead to increased cancer invasive potential. With a variety of imaging and molecular methods in vitro and in clinical samples from prostate cancer (PCa) patients, we show that chromosome bridge resolution can lead to EMD accumulation and formation of EMD-rich MN. Such structure is negative for Lamin A/C and positive for LBR and Sec6{beta}. It can cause EMD pauperization from NE affecting migratory and invasive properties of a cell and can be translated to PCa patients poor prognosis.

cancer biology↗

bioRxiv2023

Intra and interspecific diversity in a tropical plant clade alter herbivory and ecosystem resilience

Declines in biodiversity generated by anthropogenic stressors at both species and population levels can alter emergent processes instrumental to ecosystem function and resilience. As such, understanding the role of biodiversity in ecosystem function and its response to climate perturbation is increasingly important, especially in tropical systems where responses to changes in biodiversity are less predictable and more challenging to assess experimentally. Using large scale transplant experiments conducted at five neotropical sites, we documented the impacts of changes in intraspecific and interspecific plant richness in the genus Piper on insect herbivory, insect richness, and ecosystem resilience to perturbations in water availability. We found that reductions of both intraspecific and interspecific Piper diversity had measurable and site specific effects on herbivory, herbivorous insect richness, and plant mortality. The responses of these ecosystem-relevant processes to reduced intraspecific Piper richness were often similar in magnitude to the effects of reduced interspecific richness. Increased water availability reduced herbivory by 4.2% overall, and the response of herbivorous insect richness and herbivory to water availability were altered by both intra and interspecific richness in a site dependent manner. Our results underscore the role of intraspecific and interspecific richness as foundations of ecosystem function and the importance of community and location specific contingencies in controlling function in complex tropical systems.

ecology↗

bioRxiv2021

A microRNA program controls the transition of cardiomyocyte hyperplasia to hypertrophy and stimulates mammalian cardiac regeneration

Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. Here we report that the miR-106b[~]25 cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. In line, gene delivery of miR-106b[~]25 to the mouse heart provokes cardiomyocyte proliferation by targeting a network of negative cell cycle regulators including E2f5, Cdkn1c, Ccne1 and Wee1. Conversely, gene-targeted miR-106b[~]25 null mice display spontaneous hypertrophic remodeling and exaggerated remodeling to overload by derepression of the prohypertrophic transcription factors Hand2 and Mef2d. Taking advantage of the regulatory function of miR-106b[~]25 on cardiomyocyte hyperplasia and hypertrophy, viral gene delivery of miR-106b[~]25 provokes nearly complete regeneration of the adult myocardium after ischemic injury. Our data demonstrate that exploitation of conserved molecular programs can enhance the regenerative capacity of the injured heart.

molecular biology↗

bioRxiv2020

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells.

SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2 cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.

microbiology↗

bioRxiv2020

CRISPR-Csy4-mediated editing of rotavirus double-stranded RNA genome

CRISPR-nucleases have been widely applied for editing cellular and viral genomes, but nuclease-mediated genome editing of double-stranded RNA (dsRNA) viruses has not yet been reported. Here, by engineering CRISPR-Csy4 nuclease to localise to rotavirus viral factories, we achieved the first nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multi-segmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 were repaired through the formation of defined deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we labelled for the first time the products of rotavirus secondary transcription made by newly assembled viral particles during rotavirus replication, demonstrating that this step largely contributes to the overall production of viral proteins. We anticipate that the nuclease-mediated cleavage of dsRNA virus genomes will promote a new level of understanding of viral replication and host-pathogen interactions, offering the opportunity to develop new therapeutics.

microbiology↗

bioRxiv2020

Heparan sulfate structure is influenced by the ER-Golgi dynamics of its modifying enzymes

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control or influence crucial biological processes. Attempts to describe its structure-function relationships with HS binding proteins in a classical lock and key type manner, however, have been unsuccessful. HS chains are synthesized in a non-template driven process in the ER and Golgi apparatus, involving a large number of enzymes capable of fine-tuning structures. Changes in the localization of HS-modifying enzymes throughout the Golgi, rather than protein expression levels, were found to correlate with changes in the structure of HS. Following brefeldin A treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Further, shortly after treatment with heparin, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS trisulfated disaccharide content. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings highlight that the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking processes are critical prerequisite for the complete delineation of HS biosynthesis.

cell biology↗

bioRxiv2020

Glucocorticoid Receptor ablation promotes cardiac regeneration by hampering cardiomyocyte terminal differentiation

In mammals, glucocorticoid levels rise dramatically shortly before birth and prepare the foetus for post-natal life by promoting the maturation of the lungs and other organs. However, their impact on cardiac postnatal growth and regenerative plasticity is unknown. Here, we demonstrate that exposure to endogenous glucocorticoids facilitates cell cycle exit and reduces the proliferation of neonatal cardiomyocytes. This cytostatic activity is shared by several synthetic glucocorticoid receptor (GR) agonists routinely used in clinical settings. We also observed that GR levels increase in cardiomyocytes during early post-natal development. Importantly, in vivo cardiomyocyte-specific GR ablation delayed the transition from hyperplastic (increase in cell number) to hypertrophic (increase in cell size) growth. Further, GR ablation partially impaired cardiomyocyte maturation, reducing myofibrils-mitochondria organization along with the expression of genes involved in fatty acid metabolism, mitochondrial respiration and energy transfer from mitochondria to the cytosol. Finally, we show increased cardiomyocyte proliferation in GR ablated juvenile and adult cardiomyocytes in response to myocardial infarction in vivo, thus promoting cardiac tissue regeneration. We suggest that GR antagonization could serve as a strategy for heart regeneration based on endogenous cardiomyocyte renewal.

cell biology↗