Biology subjects

Freire, M.

Publications and source records attributed to Freire, M..

16 recordsLinked to original sources

bioRxiv2026

maniFasta and the AllOralsDB: simplifying construction of comprehensive reference databases for metaproteomics

Metaproteomics aims to capture a taxonomically comprehensive snapshot of proteins in a sample. Design of reference databases is a key aspect of metaproteomic workflows, as these define what is ultimately seen. Databases tailored to focal biomes offer optimal performance, yet their construction often requires drawing on heterogeneous data sources, posing a challenge to reproducibility and documentation. Here we present maniFasta, a tool enabling users to generate standardized, reproducible, and robustly documented protein reference sets from diverse input sources and datatypes. Users provide information on their desired input types and sources, and the output is an integrated database comprising a protein sequence file (FASTA), with harmonized identifiers and standardized headers, and an associated provenance metadata table (manifest). We highlight the value of maniFasta in the context of salivary metaproteomics, addressing the need for a taxonomically comprehensive reference database. The AllOralsDB resource includes human proteins, as well as proteins from bacteria and archaea, fungi and other microeukaryotes, viruses and viroid-like elements, dietary sources, and common contaminants. Together, this work provides a community resource for oral and salivary metaproteomics (https://www.homd.org/ftp/AllOralsDB/), and a versatile and accessible tool for constructing protein databases for metaproteomics generally (https://github.com/KauffmanLab/maniFasta).

microbiology↗

bioRxiv2026

Persistent SARS-CoV-2 Spike is Associated with Localized Immune Dysregulation in Long COVID Gut Biopsies

SARS-CoV-2 persistence is a proposed driver of Long COVID (LC), but the in-situ relationship between residual viral antigen and immune dysregulation remains poorly defined. To address this critical gap, we employed a high-resolution, multi-modal approach--combining RNAscope, GeoMx Digital Spatial Profiling (DSP), spatial transcriptomics, and multiplex immunofluorescence--on 25 terminal ileum and left colon biopsies from a clinical cohort of 8 LC participants and 5 healthy controls. We confirmed the persistence of SARS-CoV-2 Spike transcript and protein in the gut tissue of all LC cases and controls tested. Yet, comparison of Spike-positive (Spike+) regions in LC versus healthy control colon tissues revealed a differential, symptomatic state-associated signature, with 57 differentially expressed genes (DEGs) (26 upregulated, 31 downregulated), revealing genes that disrupt the immune response in LC subjects. LC colon Spike+ regions demonstrated increased expression of AQP8 and other absorptive-related genes (SLC26A3, SLC26A2, and CLCA4) which are involved with Chrons disease along with transcripts involved in tumorigenesis (GUCA2A, S100P, TSPAN1). Simultaneous downregulation of key homeostatic chemokines (CXCL13, CCL19, CCL21), and other transcripts reported to exhibit low expression in colorectal cancers (TMEM88B, NIBAN3, DMBT1), suggesting a paradox of epithelial tissue stress yet dysfunctional immune trafficking. Further analysis comparing Spike+ versus Spike-regions within LC colon tissue demonstrated an active, localized, antigen-driven immune microenvironment, identifying 122 DEGs (82 upregulated, 40 downregulated), including tumorigenesis genes. Cellular deconvolution of Spike+ regions revealed a statistically significant focal enrichment of myeloid-derived cells (macrophages, non-classical/intermediate monocytes), plasma cells, and regulatory T cells, coupled with significant enrichment in T-cell-related pathways, including "Antigen processing and presentation," and "Th1/Th2/Th17 cell differentiation." The ileum displayed a similar, though less pronounced, signature, demonstrating these statistically significant findings are specific to the colon of LC subjects. In contrast, corresponding Spike+ vs. Spike-analysis in healthy control colon tissues showed a more modest transcriptional response with 38 DEGs. Our data provide robust evidence that persistent SARS-CoV-2 Spike protein detection in the gut is not immunologically inert. Instead, it is actively associated with distinct, immune cell composition shifts and a dysfunctional pro-inflammatory transcriptional profile, supporting the hypothesis that retained viral antigen drives chronic immune dysregulation in tissue of Long COVID subjects.

genomics↗

bioRxiv2025

Bridging Gaps in Soil Ecology: Metagenomic Insights into Microbial Diversity and Functionality Across Brazil's Biomes.

Microorganisms participate in complex interactions involving different kingdoms, so rhizosphere biodiversity mapping is essential for understanding how microbes interact with each other in the soil and with roots. Although soil microbial communities are remarkably diverse and technological advances have provided a high capacity to acquire reliable sequence data, unique microbial taxa in soil, root and rhizosphere samples remain poorly described. For the first time, we organized a consortium to collect soil samples covering all Brazilian biomes, providing a comprehensive and unprecedented view of soil microbial diversity. This understanding is critical, especially within the context of climate change, which affects plant physiology, root exudation and, consequently, the composition and functionality of soil microbial communities. The interactions between soil, roots and rhizosphere are influenced by evolutionary and adaptive forces and shape the production of microbial natural products, which exhibit great therapeutic potential and Mapping and studying rhizosphere microbial biodiversity not only increases our knowledge of soil ecology but also offers valuable insights for developing sustainable practices. We employed both 16S/18S/ITS amplicon and metagenomic short-read shotgun sequencing methods to examine and catalogue the large-scale genomes of culture-independent rhizosphere microbes and their interactions with roots in six terrestrial Brazilian biomes, namely, the Amazon, Atlantic Forest, Cerrado, Caatinga, Pampa and Pantanal. Our results revealed the ubiquity of Proteobacteria, which reflects their adaptability to contrasting environments. Biomes with greater moisture availability, such as the Amazon and Pantanal, exhibited greater diversity and abundance of fast-growing bacteria, such as Proteobacteria, and nutrient cyclers, such as Thaumarchaeota. Arid and semiarid biomes, such as the Caatinga, were dominated by microorganisms tolerant to drought and nutrient-limited environments, such as Actinobacteria. Acidobacteria, which thrive in acidic, nutrient-poor soils, were very abundant in forest biomes. The Planctomycetes phylum also occurred more frequently in areas with a relatively high soil organic matter content, such as the Cerrado. Bacteroidetes was significantly more abundant in Pampa than in the other biomes. The results provide comprehensive insights into soil, root and rhizosphere biodiversity and not only enhance the knowledge of the fundamental biological processes sustaining plant life but also constitute a reliable sequencing databank to address present-day agricultural and environmental challenges.

microbiology↗

bioRxiv2024

The Soil Microbiome of the Caatinga Drylands in Brazil

Drylands represent a significant part of the Earths surface and include essential and vulnerable ecosystems for the global ecological balance. The Caatinga, with its unique biodiversity adapted to the extreme conditions of this semi-arid region, offers a valuable opportunity to expand our knowledge about these ecosystems. Here, this work reveals the high microbial diversity in the soil and rhizosphere of the Caatinga, with the roots presenting more specialized communities. Bacteria such as Bacilli, Alphaproteobacteria and Firmicutes excelled in critical functions such as nutrient cycling. Interplant differences suggested the influence of root exudates. The metagenomic study of interactions between microorganisms in the rhizosphere of selected plants revealed microbial biodiversity and contributed to our understanding of nutrient cycling, plant growth and resistance to water stress. In addition, they demonstrate biotechnological potential to address global challenges such as desertification and food security.

microbiology↗

bioRxiv2024

Aggressive conflict impacts path integration in homing desert ants

Across many species of the animal kingdom, the ability to navigate long distances is important for securing food and other resources. For example, ants, bees, and rats have long been known to use path integration, a mechanism to integrate distance and direction travelled, to find their way back to their nest. However, path integration is imperfect, and errors accumulate over time as animals forage. There are several ways to counteract this uncertainty, for example by utilizing local olfactory or visual landmarks along a familiar route to infer an individuals position relative to its nest, i.e. its homing vector. Local landmarks are thought to be effective if they are constant, granting a strong association with the discrete location it is located at. Here we show that ants can utilize something much sparser and more distributed than an individual constant landmark to inform its homing vector. Our data suggests that Tunisian desert ants integrate intraspecific contact, or social cues, to augment navigation. We demonstrate that contact with foreign conspecifics at a position where the nest is expected diminishes path integration certainty, thereby expanding the nest search pattern. Furthermore, we show that ants form associations between intraspecific conflict and discrete locations to create so called local vectors, within which the homing path to the nest can be stored. Highlights- Contact with foreign ants reduces path integrator certainty and alters homing strategy - Pattern of systematic search could be altered by persistent adaptions of walking speed - Foraging ants can form local vector memories using intraspecific conflict as a landmark

animal behavior and cognition↗

bioRxiv2024

Sputum production and salivary microbiome in COVID-19 patients reveals oral-lung axis

ObjectiveSARS-CoV-2 is a severe respiratory disease that primarily targets the lungs and was the leading cause of death worldwide during the pandemic. Investigating the intricate interplay between the oral microbiome and inflammatory cytokines during the acute phase of infection is crucial for understanding host immune responses. This study aimed to explore the relationship between the oral microbiome and cytokines in COVID-19 patients, specifically examining those with and without sputum production. MethodsSaliva and blood samples from 50 COVID-19 patients were subjected to 16S ribosomal RNA gene sequencing to analyze the oral microbiome. Additionally, 65 saliva and serum cytokines were assessed using Luminex multiplex analysis. The Mann-Whitney test compared cytokine levels between individuals with and without sputum production. ResultsOur study revealed significant differences in the membership (Jaccard dissimilarity: p=0.016) and abundance (PhILR dissimilarity: p=0.048; metagenomeSeq) of salivary microbial communities between COVID-19 patients with and without sputum production. Seven bacterial genera, including Prevotella, Streptococcus, Actinomyces, Atopobium, Filifactor, Leptotrichia, and Selenomonas, were present in statistically higher proportions of patients with sputum production (p<0.05, Fishers exact test). Eight bacterial genera, including Prevotella, Megasphaera, Stomatobaculum, Leptotrichia, Veillonella, Actinomyces, Atopobium, and Corynebacteria were significantly more abundant in the sputum-producing group, while Lachnoacaerobaculum was notably more prevalent in the non-sputum-producing group (p<0.05, ANCOM-BC).We observed a significant positive correlation between salivary IFN-gamma (Interferon-gamma) and Eotaxin2/CCL24 (chemokine ligand 24) with sputum production. Conversely, negative correlations were noted in serum MCP3/CCL7 (monocyte-chemotactic protein 3/Chemokine ligand 7), MIG/CXCL9 (Monokine induced by gamma/Chemokine ligand 9), IL1 beta (interleukin 1 beta), and SCF (stem cell factor) with sputum production (p<0.05, Mann-Whitney test). ConclusionSubstantial distinctions in salivary microbial communities were evident between COVID-19 patients with and without sputum production, emphasizing the notable impact of sputum production on the oral microbiome and cytokine levels during the acute phase of infection.

microbiology↗

bioRxiv2024

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

Single-molecule super-resolution imaging is instrumental for investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise, 3D nanoscale studies of a wide range of cellular structures. Here we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi-and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D singlemolecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin - a protein located in the focal adhesion complex - and actin in human osteosarcoma cells.

biophysics↗

bioRxiv2023

Host-Microbiome Associations in Saliva Predict COVID-19 Severity

Established evidence indicates that oral microbiota plays a crucial role in modulating host immune responses to viral infection. Following Severe Acute Respiratory Syndrome Coronavirus 2 - SARS-CoV-2 - there are coordinated microbiome and inflammatory responses within the mucosal and systemic compartments that are unknown. The specific roles that the oral microbiota and inflammatory cytokines play in the pathogenesis of COVID-19 are yet to be explored. We evaluated the relationships between the salivary microbiome and host parameters in different groups of COVID-19 severity based on their Oxygen requirement. Saliva and blood samples (n = 80) were collected from COVID-19 and from non-infected individuals. We characterized the oral microbiomes using 16S ribosomal RNA gene sequencing and evaluated saliva and serum cytokines using Luminex multiplex analysis. Alpha diversity of the salivary microbial community was negatively associated with COVID-19 severity. Integrated cytokine evaluations of saliva and serum showed that the oral host response was distinct from the systemic response. The hierarchical classification of COVID-19 status and respiratory severity using multiple modalities separately (i.e., microbiome, salivary cytokines, and systemic cytokines) and simultaneously (i.e., multi-modal perturbation analyses) revealed that the microbiome perturbation analysis was the most informative for predicting COVID-19 status and severity, followed by the multi-modal. Our findings suggest that oral microbiome and salivary cytokines may be predictive of COVID-19 status and severity, whereas atypical local mucosal immune suppression and systemic hyperinflammation provide new cues to understand the pathogenesis in immunologically naive populations. Significance StatementThe oral mucosa is one of the first sites encountered by bacterial and viral infections, including SARS-CoV-2. It consists of a primary barrier occupied by a commensal oral microbiome. The primary function of this barrier is to modulate immunity and provide protection against invading infection. The occupying commensal microbiome is an essential component that influences the immune systems function and homeostasis. The present study showed that the host oral immune response performs unique functions in response to SARS-CoV-2 when compared to systemic responses during the acute phase. We also demonstrated that there is a link between oral microbiome diversity and COVID-19 severity. Additionally, the salivary microbiome was predictive of not only disease status but also severity.

immunology↗

bioRxiv2022

Minimal cell JCVI-syn3B as a chassis to reveal the mechanisms behind Mycoplasma host-pathogen interactions

Mycoplasmas are atypical bacteria that are obligate parasites of animals and plants. They are known for their capacity to contaminate and parasitize mammalian cell cultures, thereby offering fortuitous models to examine the host-microbe interaction. We used a near wildtype strain of the goat pathogen Mycoplasma mycoides (Mmc) and several minimized Mmc strains lacking genes not essential for growth in laboratory media to investigate host-mycoplasma interactions. Stains with near minimal genomes were incapable of surviving in co-culture with mammalian HEK-293T and HeLa cells. We identified a cluster of eight non-essential genes that when added back to the minimized strains enabled mycoplasma attachment to mammalian cells. Those genes did not restore the capacity of the minimal cell to grow when co-cultured with mammalian cells indicating processes of attaching to mammalian cells and parasitism involve different genes. Furthermore, minimized Mmc strains were not phagocytized by human myeloid cells unlike the near wildtype Mmc.

microbiology↗

bioRxiv2022

Does sexual conflict contribute to the evolution of novel warning patterns?

Why warning patterns are so diverse is an enduring evolutionary puzzle. Because predators associate particular patterns with unpleasant experiences, an individuals predation risk should decrease as the local density of its warning pattern increases, promoting pattern monomorphism. Distasteful Heliconius butterflies are known for their diversity of warning patterns. Here, we explore whether interlocus sexual conflict can contribute to their diversification. Male Heliconius use warning patterns as mating cues, but mated females may suffer costs if this leads to harassment, favoring novel patterns. Using simulations, we show that drift alone is unlikely to cause pattern diversification, but that sexual conflict can assist such process. We also find that genetic architecture influences the evolution of male preferences, which track changes in warning pattern due to sexual selection. When male attraction imposes costs on females, this affects the speed at which novel pattern alleles increase. In two experiments, females laid fewer eggs with males present. However, although males in one experiment showed less interest in females with manipulated patterns, we found no evidence that female coloration mitigates sex-specific costs. Overall, male attraction to conspecific warning patterns may impose an unrecognized cost on Heliconius females, but further work is required to determine this experimentally.

evolutionary biology↗

bioRxiv2022

Immune and Clotting Dysfunction Detected in Saliva and Blood Plasma after COVID-19.

A growing number of studies indicate that coronavirus disease 2019 (COVID-19) is associated with inflammatory sequelae, but molecular signatures governing the normal vs. pathologic convalescence process have not been well-delineated. We characterized global immune and proteome responses in matched plasma and saliva samples obtained from COVID-19 patients collected between 4-6 weeks after initial clinical symptoms resolved. Convalescent subjects showed robust IgA and IgG responses and positive antibody correlations between matched saliva and plasma samples. However, global shotgun proteomics revealed persistent inflammatory patterns in convalescent samples including dysfunction of salivary innate immune cells and clotting factors in plasma (e.g., fibrinogen and antithrombin), with positive correlations to acute COVID-19 disease severity. Saliva samples were characterized by higher concentrations of IgA, and proteomics showed altered pathways that correlated positively with IgA levels. Our study positions saliva as a viable fluid to monitor immunity beyond plasma to document COVID-19 immune, inflammatory, and coagulation-related sequelae.

immunology↗

bioRxiv2020

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral main protease is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. Although several mechanisms of self-cleavage had been proposed for SARS-CoV, the lack of structural information for each step is a setback to the understanding of this process. Herein, we used X-ray crystallography to characterize an immature form of the main protease, which revealed major conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis-cleavage of N-terminal residues within the dimer, leading to the mature active site. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the main protease bound to its endogenous N and C-terminal residues during the formation of the tetramer. This quaternary form is also present in solution, suggesting a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during maturation, which can guide the development of new inhibitors targeting its intermediary states.

biochemistry↗

bioRxiv2020

Light environment influences mating behaviours during the early stages of divergence in tropical butterflies

Species divergence is facilitated when traits under divergent selection also act as mating cues. Fluctuations in sensory conditions can alter signal perception independently of adaptation to contrasting sensory environments, but how this fine scale variation affects behavioural isolation has received less attention, especially in terrestrial organisms. The warning patterns of Heliconius butterflies are under selection for aposematism and act as mating cues. Using computer vision, we extracted behavioural data from 1481 hours of video footage for 387 individuals. We show that the putative hybrid species H. heurippa and its close relative H. timareta linaresi differ in their response to divergent warning patterns, and that these differences are strengthened with increased local illuminance. Trials with live individuals reveal low-level assortative mating that are sufficiently explained by differences in visual attraction. Finally, results from hybrid butterflies are consistent with linkage between a major warning pattern gene and the corresponding behaviour, though the differences in behaviour we observe are unlikely to cause rapid reproductive isolation as predicted under a model of hybrid trait speciation. Overall, our results highlight that the role of ecological mating cues for behavioural isolation may depend on the immediate sensory conditions during which they are displayed to conspecifics.

evolutionary biology↗

bioRxiv2020

Netome: The Molecular Characterization of Neutrophil Extracellular Traps (NETs)

Neutrophils are the most abundant type of white blood cells in humans with biological roles relevant to inflammation and fighting infections. The release of neutrophil extracellular DNA aims to control invasion by bacteria, viruses, fungi, and tissue damage. Neutrophil Extracellular Traps (NETs) act as antimicrobial agents triggering immune signaling through the release of the nuclear content into the extracellular space. Although intense investigations have elucidated the pathways preceding NET formation, the exact molecular composition of released NETs has not been mapped. We aimed to decode the sequences of DNA and proteins from NETs. With emerging needs to understand neutrophil functions precisely, we open the field of NETOMIC studies through isolation of NETs in combination with omics approaches including shotgun genomics and proteomics. Our in vitro NET isolation methodology allowed for unprecedented replicability with induction in a sterile inflammation model system. Enrichment of mitochondrial DNA and telomere sequences are significantly expressed in NET genomes. This study revealed that the genomic sequence released in the extracellular milieu is not stochastically serving as a scaffold for a repertoire of proteins involved in neutrophil protective functions. Collectively, we established the gene and protein signatures exclusive to the extracellular NETs in comparison to undifferentiated and differentiated neutrophil states, further guiding future detection of specific regions needed for diagnostics and targeted therapies of NET related conditions.

immunology↗

bioRxiv2019

Host-Microbial Interactions in Systemic Lupus Erythematosus and Periodontitis

Systemic lupus erythematosus (SLE) is a potentially fatal complex autoimmune disease, that is characterized by widespread inflammation manifesting tissue damage and comorbidities across the human body including heart, blood vessels, joints, skin, liver, kidneys, and periodontal tissues. The etiology of SLE is partially attributed to a deregulated inflammatory response to microbial dysbiosis and environmental changes. In the mouth, periodontal environment provides an optimal niche to assay local dynamic microbial ecological changes in health and disease important to systemic inflammation in SLE subjects. Our aim was to evaluate the reciprocal impact of periodontal subgingival microbiota on SLE systemic inflammation. Ninety-one female subjects were recruited, including healthy (n=31), SLE-inactive (n=29), and SLE-active (n=31). Patients were screened for probing depth (PD), bleeding on probing (BOP), clinical attachment level (CAL), and classified with or without periodontal dysbiosis, periodontitis. Serum inflammatory cytokines were measured by human cytokine panel and subgingival biofilm was examined by DNA-DNA checkerboard. The results showed significant upregulation of proinflammatory cytokines in individuals with SLE when compared to controls. Stratification of subjects into SLE-inactive (I) and SLE-active (A) phenotypes or periodontitis and non-periodontitis groups provided new insights into SLE pathophysiology. While low-grade inflammation was found in SLE-I subjects, a potent anti-inflammatory cytokine, IL-10 was found to control clinical phenotypes. Out of twenty-four significant differential oral microbial abundances found in SLE, fourteen unique subgingival bacteria profiles were found to be elevated in SLE. Pathogens from periodontal disease sites (Treponema denticola and Tannerella forsythia) showed increase abundance in SLE-A subjects when compared to controls. Cytokine-microbial correlations showed that periodontal pathogens dominating the environment increased proinflammatory cytokines systemically. Deeper clinical attachment loss and periodontal pathogens were found in SLE subjects, especially on SLE-I, likely due to long-term chronic and low-grade inflammation. Altogether, local periodontal pathogen enrichment was positively associated with high systemic inflammatory profiles, relevant to the overall health and SLE disease pathogenesis.

immunology↗

bioRxiv2019

The GRASP domain in Golgi Reassembly and Stacking Proteins: differences and similarities between lower and higher Eukaryotes

The Golgi complex is part of the endomembrane system and is responsible for receiving transport cargos from the endoplasmic reticulum and for sorting and targeting them to their final destination. To perform its function in higher eukaryotic cells, the Golgi needs to be correctly assembled as a flatted membrane sandwich kept together by a protein matrix. The correct mechanism controlling the Golgi cisternae assembly is not yet known, but it is already accepted that the Golgi Reassembly and Stacking Protein (GRASP) is a main component of the Golgi protein matrix. Unlike mammalian cells, which have two GRASP genes, lower eukaryotes present only one gene and distinct Golgi cisternae assembly. In this study, we performed a set of biophysical studies to get insights on both human GRASP55 and GRASP65 and compare them with GRASPs from lower eukaryotes (S. cerevisiae and C. neoformans). Our data suggest that both human GRASPs are essentially different from each other and GRASP65 is more similar to the subgroup of GRASPs from lower eukaryotes. GRASP55 is present mainly in the Golgi medial and trans faces, which are absent in both funguses, while GRASP65 is located in the cis-Golgi. We suggest that the GRASP65 gene is more ancient and the paralogue GRASP55 might have appeared latter in evolution, together with the medial and trans Golgi faces in mammalians.

biophysics↗