Biology subjects

Sousa, M.

Publications and source records attributed to Sousa, M..

15 recordsLinked to original sources

bioRxiv2026

A pro-inflammatory effector-memory T cell program links peripheral EBV priming to CNS autoimmunity

T and B cell-mediated immunosurveillance of the central nervous system (CNS) can become maladaptive in multiple sclerosis (MS). Epstein-Barr virus (EBV) is a likely driver of MS, yet how exactly EBV-specific T cells contribute remains unclear. We generated an antigen-resolved atlas of blood and cerebrospinal fluid (CSF) T cells from untreated MS patients and controls. T cell clones carrying a GZMK effector-memory program in blood were CSF-biased, particularly in MS, and associated with EBV reactivity. In CSF, EBV-reactive GZMK CD8 T cells engaged B lineage cells. Activated CD8 T cells promoted atypical B cell and plasmablast formation, whereas GZMK-enriched effector-memory cells induced IL-6 and IL-8 release from myeloid cells. Together, these findings identify an infection-imprinted, CNS-biased state and support a model in which EBV-specific CD8 T cells contribute to inflammation through a non-canonical mode of B cell and myeloid cell activation.

immunology↗

bioRxiv2025

Endogenous Osteocyte-Osteoclast Signaling Enables Growth Factor-Free Bone Remodeling, Drug Response, and Cancer Invasion in a Nanoscale Calcified Bone-on-a-Chip Model

Bone homeostasis depends on spatially orchestrated interactions among osteoclasts, osteoblasts, and osteocytes that are embedded within a unique extracellular matrix that is mineralized on the nanoscale. Reconstructing these interactions to enable autonomous cell differentiation and tissue remodeling has remained a significant challenge towards mimicking adequate bone physiology and disease in-vitro. Here, we present an engineered model that spatially defines the paracrine communication of heterogeneous cell populations within bone tissue that supports the rapid maturation of primary osteoblasts into osteocytes, the differentiation of immune cells into osteoclasts, and calcified tissue resorption within a mineralized cell-laden bone-like tissue. We demonstrate that nanoscale mineralization of cell-laden collagen hydrogels on-a-chip enhances osteoblast to osteocyte differentiation, whereas osteocytes in the matrix accelerate osteoclastogenesis and remodeling in a spatially defined manner without the need for exogenous growth factors. Osteocyte-dependent osteoclastogenesis on-a-chip outperformed conventional stimulation with RANKL and M-CSF, reproduced the clinical response of anti-resorptive drugs, and mimicked established tumor-bone interactions observed in invasive oral cancer. By replicating essential aspects of bone composition and function, this system provides a robust, self-regulated microphysiologic model to investigate bone remodeling, cancer-bone crosstalk, and therapeutic interventions.

bioengineering↗

bioRxiv2025

The Insertion of an ATTTC Repeat in an Alu Element Hyperactivates a Primate-Specific Neurodevelopmental Enhancer in Spinocerebellar Ataxia Type 37

Alu are evolutionarily very old primate-specific interspersed repeat elements that constitute [~]11% of the human genome. They are a source of short tandem repeats (STRs), which often expand in size and originate inherited neuromuscular and neurodegenerative disorders. How expanded STR insertion mutations within Alu STRs culminate in disease remains unknown. Here we report an Alu STR located in an intron of DAB1 that functions as a neurodevelopmental enhancer. We demonstrated that an ATTTC repeat insertion in this DAB1 Alu STR, known to cause spinocerebellar ataxia type 37 (SCA37), hyperactivates a neurodevelopmental DAB1 enhancer. Importantly, we showed that neurons derived from SCA37 subjects have higher levels of DAB1 expression and DAB1 overexpression causes abnormal axonal pathfinding in vivo. Overall, these results establish that neuronal dysregulation of a developmental DAB1 Alu STR enhancer contributes to SCA37 pathogenesis, an unexplored mechanism likely acting in many Alu STR diseases, potentially reshaping the therapeutic landscape.

genetics↗

bioRxiv2025

Unraveling the Gardnerella Pangenome: Insights into Genetic Diversity, Virulence, and Antibiotic Resistance

In recent years, the development of user-friendly and accessible bioinformatic tools has significantly advanced Gardnerella genome analysis. Despite the taxonomic reclassification of the genus in 2019, many studies continue to rely on subgroups/ clades for species identification. This leads to frequent misidentifications, hindering accurate assessments of species-specific features, such as antimicrobial resistance and pathogenic potential. This study aims to address these gaps by employing precise species-level identification to improve our understanding on Gardnerella and its clinical implications. We performed the most extensive pangenome analysis of Gardnerella to date, analyzing 153 genomes (120 from NCBI and 33 newly sequenced). G. vaginalis, G. leopoldii, G. swidsinskii, and G. pickettii species exhibited open pangenomes. The Gardnerella pangenome is highly diverse, with 90% of its genes belonging to the accessory genome. Carbohydrate transport and metabolism contributed the most for the primary genomic differences observed among Gardnerella species. Additionally, the search for antibiotic resistance genes (ARGs) revealed lsaC gene as the most prevalent ARG, potentially conferring resistance to clindamycin, a commonly prescribed antibiotic for treating bacterial vaginosis (BV). The clustering of Gardnerella genomes mimics the core genome phylogeny, pointing to species specific virulence-associated factors, highlighting important factors such as vaginolysin and sialidases. Our comprehensive pangenome analysis shows that Gardnerella species are well-adapted within the urogenital tract, with identified virulence factors and metabolic capabilities potentially enhancing the pathogenicity of some strains. Future studies should explore Gardnerellas ecological interactions and pathogenic mechanisms within the urogenital microbiome. ImportanceIn recent years, the availability of user-friendly and accessible bioinformatics tools has led to a surge in studies analyzing the Gardnerella genome. Despite the redefinition of the genus in 2019, many researchers continue to rely on subgroups or clades for Gardnerella identification. Our study builds upon the updated taxonomy of Gardnerella, offering a more comprehensive understanding of its genetic diversity. By conducting a pangenome analysis of 153 Gardnerella genomes, we uncover significant genetic variability, species-specific virulence factors, and the widespread presence of antibiotic resistance genes, highlighting the urgent need for precise species-level identification. The identification of novel genomic features and antibiotic resistance profiles not only enhances our understanding of Gardnerellas clinical implications but also sheds light on its metabolic and virulence potential. These insights help clarify the pathogenic mechanisms of Gardnerella and provide a more refined understanding of its role in the urogenital microbiome.

microbiology↗

bioRxiv2024

Perivascular cells function as mechano-structural sensors of vascular capillaries

A wide range of conditions, including chronic inflammatory diseases and cancer, are characterized by the fibrotic microarchitecture and increased stiffness of collagen type I extracellular matrix. These conditions are typically accompanied by altered vascular function, including vessel leakiness, abnormal capillary morphology and stability. The dynamic cell-matrix interactions that regulate vascular function in healthy tissues have been well documented. However, our understanding of how the gradual mechanical and structural alterations in collagen type I affect vascular homeostasis remains elusive, especially as a function of the interactions between endothelial and perivascular cell with the altered matrix. Here we hypothesized that perivascular cells might function as mechano-structural sensors of the microvasculature by mediating the interaction between endothelial cells and altered collagen type I. To test that, we utilized an organotypic model of perivascular cell-supported vascular capillaries in collagen scaffolds of controlled microarchitecture and mechanics. Our results demonstrate that capillaries cultured in soft reticular collagen exhibited consistent pericyte differentiation, endothelial cell-cell junctions, and barrier function. In contrast, capillaries embedded in stiff and bundled collagen fibrils to mimic a more fibrotic matrix induced abluminal migration of perivascular cells, increased leakage, and marked expression of vascular remodeling and inflammatory markers. These patterns, however, were only observed when endothelial capillaries were engineered with perivascular cells. Silencing of NOTCH3, a mediator of endothelial-perivascular cell communication, largely re-established normal vascular morphology and function. In summary, our findings point to a novel mechanism of perivascular regulation of vascular dysfunction in fibrotic tissues which may have important implications for anti-angiogenic and anti-fibrotic therapies in cancer, chronic inflammatory diseases and regenerative medicine. Significance StatementThe fibrotic alterations in extracellular matrix structure and mechanics that are common to many chronic and inflammatory conditions are often associated with a decrease in vascular homeostasis. The mechanisms regulating these abnormalities remain poorly understood. Here, we demonstrate that perivascular cells play a critical role in sensing progressive microarchitectural and mechanical changes occurring in the ECM, drastically altering vascular capillary morphology and barrier function, and exacerbating the production of inflammatory and remodeling markers. These results point to a previously unknown mechano-structural sensory mechanisms mediated by perivascular cells in vascular capillaries that may help elucidate the progression of many profibrotic conditions, and point to possible new targets for antiangiogenic and antifibrotic therapies in cancer, chronic inflammatory conditions and regenerative medicine.

bioengineering↗

bioRxiv2023

Dopamine neurons encode a multidimensional probabilistic map of future reward

Learning to predict rewards is a fundamental driver of adaptive behavior. Midbrain dopamine neurons (DANs) play a key role in such learning by signaling reward prediction errors (RPEs) that teach recipient circuits about expected rewards given current circumstances and actions. However, the algorithm that DANs are thought to provide a substrate for, temporal difference (TD) reinforcement learning (RL), learns the mean of temporally discounted expected future rewards, discarding useful information concerning experienced distributions of reward amounts and delays. Here we present time-magnitude RL (TMRL), a multidimensional variant of distributional reinforcement learning that learns the joint distribution of future rewards over time and magnitude using an efficient code that adapts to environmental statistics. In addition, we discovered signatures of TMRL-like computations in the activity of optogenetically identified DANs in mice during a classical conditioning task. Specifically, we found significant diversity in both temporal discounting and tuning for the magnitude of rewards across DANs, features that allow the computation of a two dimensional, probabilistic map of future rewards from just 450ms of neural activity recorded from a population of DANs in response to a reward-predictive cue. In addition, reward time predictions derived from this population code correlated with the timing of anticipatory behavior, suggesting the information is used to guide decisions regarding when to act. Finally, by simulating behavior in a foraging environment, we highlight benefits of access to a joint probability distribution of reward over time and magnitude in the face of dynamic reward landscapes and internal physiological need states. These findings demonstrate surprisingly rich probabilistic reward information that is learned and communicated to DANs, and suggest a simple, local-in-time extension of TD learning algorithms that explains how such information may be acquired and computed.

animal behavior and cognition↗

bioRxiv2022

Long-term mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells

DNA damage response (DDR) during interphase involves active signalling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy analysis we found that mitotic cells exposed to several cancer chemotherapy compounds acquire and signal DNA damage, regardless of how they interact with DNA. In-depth analysis upon long-term DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but DDR-independent, mitotic delay. This delay was due to the presence of misaligned chromosomes that ultimately satisfy the SAC and missegregate, leading to micronuclei formation. Mechanistically, we show that long-term mitotic DNA damage specifically stabilizes kinetochore-microtubule attachments in cancer cells, causing the missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Overall, these findings unveil that long-term therapeutic DNA damage regimens contribute to genomic instability through a surprising link between the stabilization of kinetochore-microtubule attachments and chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

cell biology↗

bioRxiv2022

Targeted Anatomical and Functional Identification of Antinociceptive and Pronociceptive Serotonergic Neurons that project to the Superficial Dorsal Horn

Spinally-projecting serotonergic neurons play a key role in controlling pain sensitivity and can either increase or decrease nociception depending on physiological context. It is currently unknown how serotonergic neurons mediate these opposing effects. Utilizing virus-based strategies, we identified two anatomically separated populations of serotonergic hindbrain neurons located in the lateral paragigantocellularis (LPGi) and the medial hindbrain, which respectively innervate the superficial and deep spinal dorsal horn and have contrasting effects on sensory perception. Our tracing experiments revealed that serotonergic neurons of the LPGi were much more susceptible to transduction with spinally injected AAV2retro vectors than medial hindbrain serotonergic neurons. Taking advantage of this difference, we employed intersectional chemogenetic approaches to demonstrate that activation of the LPGi serotonergic projections decreases thermal sensitivity, whereas activation of medial serotonergic neurons increases sensitivity to mechanical von Frey stimulation. Together these results suggest that there are functionally distinct classes of serotonergic hindbrain neurons that differ in their anatomical location in the hindbrain, their postsynaptic targets in the spinal cord, and their impact on nociceptive sensitivity. The LPGi neurons that give rise to rather global and bilateral projections throughout the rostrocaudal extent of the spinal cord appear to be ideally posed to contribute to widespread systemic pain control.

neuroscience↗

bioRxiv2022

High resolution micro-CT imaging in mice stroke models: from 3D detailed infarct characterization to automatic area segmentation

Characterization of brain infarct lesion in rodent models of stroke is crucial to assess the outcome of stroke therapies and to study the disease pathophysiology. However, so far it has been mostly performed by: 1) histological methods, a time-consuming process that lead to significant flaws and tissue distortion; or 2) via MRI imaging, which is faster, yielding 3D information but at high costs. High-resolution micro-CT imaging became, in the last decade, a simple, fast, and cheaper solution, allowing 3D analysis of samples. Here, we describe that high-resolution micro-CT, either using iodine/OsO4 as contrast agents, can be successfully applied for fine quantification and localization of lesion size and edema volume in preclinical stroke models. We successfully correlated this new approach with the standard histological method TTC. In transient MCAO mouse stroke model, we were able to identify/quantify large lesioned areas (segmented in core and penumbra), up to degenerated finer striatal myelinated fibers in a transient ischemic attack (TIA) mouse model, at different timepoints post-ischemia, through manual and automatic segmentation approaches (deep learning). Furthermore, 3D reconstructions of the whole brain allow for brain atlas co-registration of the specific affected brain areas. Hence, the presented methodology, through iodine/OsO4 micro-CT imaging, constitutes a valuable advance in tools for precise and detailed assessment of stroke outcomes in preclinical animal studies.

neuroscience↗

bioRxiv2022

Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

The recognition of microbiome inhabiting the healthy female bladder engendered the need for comprehensive characterization of the female urinary microbiome (FUM) in health and disease. Although previous studies reported FUM composition at different taxonomic levels, progress towards reliable identification at species level is highly required. The aim of this study was to comprehensively characterize bacterial species of FUM of healthy reproductive-age European women by two complementary methodologies i.e., extended culturomics and long-read third generation sequencing of near full-length 16S rRNA gene. A wide diversity of bacterial species was captured (297 species) with a median of 53 species/sample, including 16 putative uropathogens. Clustering FUM into community structure types revealed high inter-individual differences. Notably, there was not a single species common to all samples, although the Lactobacillus genus was detected in all samples. Lactobacillus crispatus, Lactobacillus iners and Lactobacillus mulieris were observed in high relative abundance in several samples as well as other species (e.g., Streptococcus agalactiae, Atopobium vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while more prevalent species were often low abundant members (e.g., Finegoldia magna). We captured remarkable richness within Corynebacterium spp. (25 species) and Lactobacillaceae (4 genera, 14 species). While amplicon sequencing allowed detection of more anaerobic species (e.g., 11 Peptoniphilus spp.), culturomics enabled the identification of recently recognized Gardnerella species and putative novel Corynebacterium species. This study provided fine-grained FUM profiling at species level and revealed detailed FUM structure, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders. IMPORTANCEDespite evidence of the resident microbial community in the female lower urinary tract, bacterial species diversity and abundance in healthy women is still unclear. This study demonstrated that complementarity between optimized culture-dependent and -independent approaches is highly beneficial for comprehensive FUM species profiling by detecting higher FUM species diversity than previously reported, including identification of unreported Lactobacillaceae species and putative novel Corynebacterium species. Although some particular species were present in high relative abundance, low-abundant members were more prevalent. FUM classification into community structure types demonstrated high inter-individual differences in urinary microbiome composition among healthy women. We also report moderate correlation between culture-dependent and -independent derived data highlighting drawbacks resulting from each methodological approach. Our findings suggest that FUM bacterial diversity reported from previous studies may be underestimated. Finally, our results contribute to the fundamental knowledge of healthy FUM required for further exploration of the urinary microbiome role in urinary tract diseases.

microbiology↗

bioRxiv2021

In vitro neuronal networks show bidirectional axonal conduction with antidromic action potentials effectively depolarizing the soma

Recent technological advances are revealing the complex physiology of the axon and challenging long-standing assumptions. Namely, while most action potential (AP) initiation occurs at the axon initial segment in central nervous system neurons, initiation in distal parts of the axon has been shown to occur in both physiological and pathological conditions. However, such ectopic action potential (EAP) activity has not been reported yet in studies using neuronal cultures and its functional role, if exists, is still not clear. Here, we show the spontaneous occurrence of EAPs and effective antidromic conduction in hippocampal neuronal cultures. We also observe a significant fraction of bidirectional axonal conduction in dorsal root ganglia neuronal cultures. We investigate and characterize this antidromic propagation via a combination of microfluidics, microelectrode arrays, advanced data analysis and in silico studies. We show that EAPs and antidromic conduction can occur spontaneously, and after distal axotomy or physiological changes in the axon biochemical environment. Conduction velocity is asymmetrical, with antidromic conduction being slower than orthodromic. Importantly, EAPs may carry information and can have a functional impact on the neuron, as they consistently depolarize the soma. Thus, plasticity or gene transduction mechanisms triggered by soma depolarization can also be affected by these antidromic APs. Altogether these findings have important implications for the study of neuronal function in vitro, reshaping our understanding on how information flows in neuronal cultures.

neuroscience↗

bioRxiv2020

The mutability of demographic noise in microbial range expansions

Demographic noise, the change in the composition of a population due to random birth and death events, is an important driving force in evolution because it reduces the efficacy of natural selection. Demographic noise is typically thought to be set by the population size and the environment, but recent experiments with microbial range expansions have revealed substantial strain-level differences in demographic noise under the same growth conditions. Many genetic and phenotypic differences exist between strains; to what extent do single mutations change the strength of demographic noise? To investigate this question, we developed a high-throughput method for measuring demographic noise in colonies without the need for genetic manipulation. By applying this method to 191 randomly-selected single gene deletion strains from the E. coli Keio collection, we find that a typical single gene deletion mutation decreases demographic noise by 8% (maximal decrease: 81%). We find that the strength of demographic noise is an emergent trait at the population level that can be predicted by colony-level traits but not cell-level traits. The observed differences in demographic noise from single gene deletions can increase the establishment probability of beneficial mutations by almost an order of magnitude higher than the wild type. Our results show that single mutations can substantially alter adaptation through their effects on demographic noise and suggest that demographic noise can be an evolvable phenotype of a population.

evolutionary biology↗

bioRxiv2020

A 21-year survey of from bloodstream infections (BSIs) in a tertiary hospital reveals how community-hospital dynamics, influence local BSI rates, the trends of the B2 phylogroup and the STc131 pandemic clone

This is a longitudinal study comprising 649 Escherichia coli (EC) isolates representing all 7165 EC-BSI episodes recorded in a hospital (1996-2016). Strains analysis included clonal identification (phylogenetic groups/subgroups, STc131 subclades, PFGE, and WGS), antibiotic susceptibility (13 antibiotics), and virulence-associated genes (VAGs, 29 genes). The incidence of BSI-EC increased from 1996 to 2016 (5.5 to 10.8 BSI episodes/1000 hospitalizations, average 7-8/1000). B2 isolates predominate (53%), subgroups B2-I (STc131), B2-II, B2-IX, and B2-VI representing 25%, 25%, 14%, and 9%, respectively. Intertwined waves of community-acquired (CA) + healthcare-associated and community-onset healthcare-associated (HCA), and hospital-acquired (HA) episodes of both B2 and non-B2 phylogroups occurred. A remarkable increase was only observed for B2-I-STc131 (C1/C2 subclades), with oscillations for other B2 subgroups and phylogroups throughout the years. Epidemic and persistent clones (comprising isolates with highly similar/identical-PFGE types and genomes differing in 18-97 SNPs) of B2-I (STc131), B2-II (STc73), B2-III (STc127), B2-IX (STc95), and B2-VI (STc12) were recovered from different patients, most at hospital admission, for long periods (2-17 years), ESBL producers or resistance to ciprofloxacin in B2 isolates were almost restricted to B2-I (STc131) subclade C. STc131 contributed to increasing the B2 rates but only transiently altered the EC-population structure. The increase of EC-BSI was determined by waves of CA+HCA-BSI episodes that predate the waves of HA-BSI. Besides the risk of hospital transmission that led to temporal increases in BSIs, this study suggests that EC-populations/clones from community-based healthy individuals may occasionally have an epidemic structure and provide a source of transmissible strains influencing the HA-BSIs incidence. IMPORTANCESepsis is the third cause of mortality in Western countries and one of the Global Health threads recognized by the WHO since 2017. Despite Escherichia coli constitutes the most common cause of bloodstream infections (BSI), its epidemiology is not fully understood, in part due to the scarcity of local and longitudinal studies. Our work analyzes the long-term dynamics of E. coli causing bacteremia in a single institution and reveals waves of different clonal lineages that emerge periodically and successfully spread afterward in both the community and hospitals. Because the origin of BSI-E. coli infections is the gut, the microbiota of healthy individuals might occasionally have an epidemic structure, providing a source of E. coli strains to influence the incidence of hospital BSIs. The study complements previous fractionated observations focusing on specific E. coli lineages or antibiotic-resistant isolates in the last decades and helps to understand the epidemiology of E. coli BSIs and the dynamics of pandemic clones.

microbiology↗

bioRxiv2019

Worldwide recombination in emergent white-nose syndrome pathogen Pseudogymnoascus destructans

Pseudogymnoascus destructans (Pd), the emergent fungus causing bat "White-Nose Syndrome", responsible for [~]6 million mortalities in the United States (US), is thought to expand clonally in North America and Europe. Presence of distinct mating-types in Europe led to numerous research attempts searching for population sexuality worldwide. This study not only presents the first evidence of genetic recombination in Pd but also detects recombination in Pd genotype data generated by previous studies in Europe and North America, through clone-corrected linkage disequilibrium analysis. Portuguese and other European populations are apparently reproducing through sex between two mating-types. Seeming parasexual recombination in the invasive single mating-type US population rings alarms for the North American bat populations and deserves urgent attention. This study emphasizes on clone-correction in linkage disequilibrium analysis. One Sentence SummaryClone-correction yielded signs of elusive recombination in the global "clonal" populations of white-nose syndrome pathogen.

ecology↗

bioRxiv2018

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally breaks the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

biophysics↗