Biology subjects

Gomes, M.

Publications and source records attributed to Gomes, M..

13 recordsLinked to original sources

bioRxiv2026

Atf3 Integrates Lipid and Cytoskeletal Remodeling to Drive Macrophage Fusion

Macrophages are highly plastic innate immune cells that can form multinucleated giant cells (MGCs) under physiological and pathological conditions, such as osteoclasts and foreign body giant cells. The mechanisms governing macrophage multinucleation remain incompletely understood. Here, we identify activating transcription factor 3 (Atf3) as an essential regulator of MGC formation via cell-cell fusion in response to persistent Interleukin 4 (IL-4) and STAT6 signaling, characteristic of the foreign body reaction. Atf3-deficient macrophages activate STAT6-dependent transcriptional programs in response to IL-4, including fusion-associated genes, yet fail to undergo multinucleation. This defect is associated with impaired actin cytoskeleton remodeling, abnormal nuclear morphology, reduced lamin A/C expression, and genome instability. Mechanistically, loss of Atf3 derepresses the Cholesterol-25-hydroxylase (Ch25h), elevating 25-hydroxycholesterol (25-HC), suppressing the mevalonate pathway, and reducing cholesterol and isoprenoid biosynthesis essential for cytoskeletal dynamics. Deletion of Ch25h in Atf3-deficient macrophages restores cholesterol levels and actin turnover, but not lamin A/C or fusion. These findings establish Atf3 as a central transcriptional node integrating lipid metabolism with cytoskeletal and nucleoskeletal remodeling to control IL-4-driven macrophage multinucleation.

cell biology↗

bioRxiv2026

Nicotinamide phosphoribosyltransferase activates the mitochondrial unfolded protein response to promote pulmonary arterial endothelial cell proliferation

Vascular remodeling leading to occlusion of the pulmonary artery and increased pulmonary vascular resistance is the central feature of Pulmonary Arterial Hypertension (PAH) which leads to death due to right heart failure. The expression of Nicotinamide phosphoribosyltransferase (Nampt) is increased in the lungs and isolated PAECs of PAH patients. Inhibition of NAMPT is protective in preclinical models of pulmonary hypertension. NAMPT regulates multiple mitochondrial processes that control cell proliferation and survival. Using rodent models of PH, we demonstrated that the mitochondrial unfolded protein response (UPRmt) promotes vascular remodeling. Hence we, hypothesized that NAMPT activates the UPRmt to promote abnormal pulmonary arterial endothelial cell proliferation. Analysis of PAECs isolated from PAH patients show increased expression of the UPRmt pathway mediators, ATF-5, mtHSP70 and ClpP. Human PAEC cell lines were exposed to recombinant Nampt or transduced with lentiviral-Nampt. We observed increased phosphorylation and activation of eIF2 which permits preferential translation of the ATF-5 transcription factor. ATF-5 expression and nuclear localization was increased. Further, we observed increased expression of ATF-5 target genes, mtHSP70, HSP60, ClpP and LonP1 as well as increased PAEC proliferation. Blocking mtHSP70 function reversed the Nampt induced increase in proliferation. Neither UPRmt activation nor proliferation was increased in the presence of enzymatically inactive-Nampt. Our observations demonstrate that Nampt can promote PAEC proliferation by activating the UPRmt and uncovers novel potential targets to address vascular remodeling in PAH.

cell biology↗

bioRxiv2025

The Staphylococcus aureus LXG-domain toxins EsxX and SAR0287 do not promote virulence in a zebrafish larval infection model

The Staphylococcus aureus type VIIb secretion system (T7SSb) is a multiprotein secretion system that secretes toxins with antibacterial activity, but which is also required for full virulence in animal models of infection. S. aureus strains carry one of four T7SSb locus types, named essC1 to essC4, each of which encodes a characteristic LXG-family substrate at the T7SS locus. In essC2 strains this LXG-domain protein is EsxX, which has a glycine zipper sequence in its C-terminus and has potent antibacterial membrane-depolarising activity. In this work we recognise conserved features of the essC2 and essC3 systems, identifying the LXG protein SAR0287 as structurally and functionally similar to EsxX. Using a zebrafish larval hindbrain ventricle infection model we demonstrate that the T7SSb of essC2 and essC3 representative strains contribute to bacterial replication and zebrafish mortality. However, there is no significant loss of virulence in the model system if EsxX or SAR0287 are absent. These findings indicate there is no discernible role for either toxin in this virulence model.

microbiology↗

bioRxiv2025

Engineering NIR probes to enhance affinity and clinical workflow compatibility for prostate cancer imaging

Positive surgical margins following radical prostatectomy increase the risk of biochemical recurrence and subsequent disease progression. Fluorescence guided surgery (FGS) using targeted contrast agents has shown clinical benefits for several cancer types. However, current prostate cancer targeted imaging probes exhibit long pharmacokinetic (PK) profiles, necessitating extended waiting periods or repeated hospital visits, limiting their integration into standard clinical workflow. To overcome this critical clinical compatibility challenge, we developed an innovative tri-compartment, chemistry-driven probe design strategy. Specifically, we developed a congeneric library of near infrared (NIR) water soluble fluorescent probes incorporating: (1) a glutamic acid-urea-lysine (EuK) ligand targeting prostate specific membrane antigen (PSMA); (2) a NIR heptamethine cyanine fluorophore optimized for enhanced PSMA binding via secondary binding sites interactions; and (3) distinct PK modulators residing outside the PSMA binding pocket to promote rapid off-target tissue clearance. While molecular docking scores, photophysical properties and live-cell staining results showed similar overall performance, probes bearing PK modulators produced stronger tumor-specific fluorescence in vivo than the control lacking a PK modulator. This effort enabled identification of a lead probe with robust tumor targeting and accelerated off-target clearance, providing optimal tumor-specific signal and contrast in a timeframe, fully compatible with robotic-assisted radical prostatectomy (RARP) timelines.

pharmacology and toxicology↗

bioRxiv2025

Partial Wwox Loss of Function Increases Severity of Murine Sepsis and Neuroinflammation

RationaleWW domain-containing oxidoreductase (WWOX) is a gene associated implicated in both neurologic and inflammatory diseases and is susceptible to environmental stressors. We hypothesize partial loss of Wwox function will result in increased sepsis severity and neuroinflammation. MethodsWwox WT/P47T mice, generated by CRISPR/Cas9, and Wwox WT/WT mice were treated with intraperitoneal PBS vs LPS (10mg/kg) and euthanized 12 hours post-injection. Open Field Testing (OFT) and Murine Sepsis Severity Scores (MSS) were utilized to measure sickness behavior and sepsis severity, respectively. Brain tissue was analyzed using immunohistochemistry and PCR to measure neuroinflammation and apoptosis. ResultsWwox WT/P47T LPS mice demonstrated a more significant response to sepsis with an increase in sickness behavior, sepsis severity, gliosis, and apoptosis compared to Wwow WT/WT LPS littermates. ConclusionsPartial loss of Wwox function increases risk for severe sepsis and neuroinflammation. Given the susceptibility of WWOX to environmental stressors, this may be a target for future therapeutic interventions.

neuroscience↗

bioRxiv2024

A computationally guided approach to improve expression of VHH binders

The variable heavy chain fragments derived from camelid antibodies, called VHHs or nanobodies, have recently shown promise as high-affinity reagents. They offer higher stability compared to conventional antibodies and fragments thereof. Furthermore, their smaller size ([~]15-20 kDa) allows better targeting of molecules localized inside the cell and in crowded environments, like tissues and protein aggregates. Despite these advantages, nanobody clones screened using phage display can suffer from poor soluble expression, which we hypothesized, is due to the presence of hydrophobic hotspots on their surface. In this work, we propose a novel computationally guided workflow for screening and production of nanobody binders for optimized expression. After an initial round of phage display screens against our target (K-Ras), we modeled the lead candidates to generate Spatial Aggregation Propensity (SAP) maps to highlight the hydrophobic hotspots with single amino acid resolution, which were subsequently used to guide mutagenesis of the binders for soluble expression. We followed two approaches to perform point hydrophilic mutations: i) performing point hydrophilic mutations in the hydrophobic hotspots; ii) combining point mutation resulting from a round of random mutagenesis that show favorable SAP scores. Both approaches led a remarkable increase in soluble expression which allowed production and characterization of their binding to their target (K-Ras) on soluble ELISA, and biolayer interferometry. We observed that the latter approach resulted in clones with stronger binding affinity compared to the former approach. Our results emphasize the need to perform a round of random mutagenesis to identify point mutations, which can then be used in an in-silico guided pipeline to identify the right combination of mutations for high soluble expression.

bioengineering↗

bioRxiv2024

Genetically encoded reporters of actin filament organization in living cells and tissues

The cytoskeletal protein actin is crucial for cell shape and integrity throughout eukaryotes. Actin filaments perform essential biological functions, including muscle contraction, cell division and tissue morphogenesis. These diverse activities are achieved through the ability of actin filaments to be arranged into precise architectures. Much progress has been made in defining the proteome of the actin cytoskeleton, but a detailed appreciation of the dynamic organizational state of the actin filaments themselves has been hindered by available tools. Fluorescence polarization microscopy is uniquely placed for measuring actin filament organization by exploiting the sensitivity of polarized light excitation to the orientation of fluorophores attached to actin filaments. By engineering fusions of five widely used actin localization reporters to fluorescent proteins with constrained mobility, we have succeeded in developing genetically-encoded, green- and red-fluorescent-protein-based reporters for non-invasive, quantitative measurements of actin filament organization in living cells and tissues by fluorescence polarization microscopy.

cell biology↗

bioRxiv2022

Cellular, Molecular, and Enzymatic Signatures ofThrombi are Vascular Bed-Dependent

BackgroundThe contribution of arterial and venous thrombi to vascular remodeling is unclear. While catheter-extraction of thrombus in cerebrovascular accident (CVA) is time-sensitive, similar urgency is rare in managing venous thromboembolism (VTE). ObjectivesOur goal was to determine molecular cellular signatures of thrombus extracted by catheter from various vascular beds to gain insight into vascular remodeling. MethodsTwenty-five patients underwent catheter-directed thrombectomy (CDT), 13 for acute CVA, 8 for pulmonary embolism (PE), and 4 for deep vein thrombosis (DVT). Protein and RNA extracted from thrombus was evaluated by immunoblotting and sequencing, respectively. Thrombus-derived enzymes for which substrate is present in the blood vessel wall were examined for enzymatic activity. ResultsTime from symptom onset to thrombus extraction was 7.7 {+/-} 1.9 hours for CVA and 109 {+/-} 55 hours for VTE. Protein concentration, white blood cell and red blood cell content were all greater in venous compared with arterial thrombus while platelet content was similar. Both venous and arterial thrombus contained multiple Matrix Metalloproteinase (MMP) isoforms. MMP9 specific activity was greater in venous than in arterial thrombus (57 {+/-} 6 ng/mL.g protein-1 vs. 24 {+/-} 8 ng/mL.g protein-1, P=0.0051). ConclusionsArterial and venous thrombus have dissimilar phenotypes, each with biologically-active enzymes known to remodel blood vessels, and enzymatic activity proportional to the white blood cell content which increases with thrombus age. These data suggest a mechanistically-important role for early CDT to avoid the consequences of irreversible vascular remodeling. Condensed AbstractEmergent extraction of acute thrombus from arterial vascular beds restores limb and end-organ perfusion and is widely-accepted to be the standard of care. Extraction of thrombus from venous vascular beds, however, is rarely considered urgent, even though many patients subsequently develop debilitating symptoms. By capitalizing on privileged thrombus extracted from multiple vascular beds, we gained mechanistic insight regarding the cellular composition and cell-derived enzymes secreted from thrombus that may remodel the vessel wall. This study shows thrombi are biologically-active entities, continuously recruiting circulating cells that secrete enzymes both proportional to thrombus age and the time of patient presentation.

biochemistry↗

bioRxiv2022

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.

cell biology↗

bioRxiv2021

OrtSuite - from genomes to prediction of microbial interactions within targeted ecosystem processes

The high complexity found in microbial communities makes the identification of microbial interactions challenging. To address this challenge, we present OrtSuite, a flexible workflow to predict putative microbial interactions based on genomic content of microbial communities and targeted to specific ecosystem processes. The pipeline is composed of three user-friendly bash commands. OrtSuite combines ortholog clustering with genome annotation strategies limited to user-defined sets of functions allowing for hypothesis-driven data analysis such as assessing microbial interactions in specific ecosystems. OrtSuite matched, on average, 96 % of experimentally verified KEGG orthologs involved in benzoate degradation in a known group of benzoate degraders. Identification of putative synergistic species interactions was evaluated using the sequenced genomes of an independent study which had previously proposed potential species interactions in benzoate degradation. OrtSuite is an easy to use workflow that allows for rapid functional annotation based on a user curated database and can easily be extended to ecosystem processes where connections between genes and reactions are known. OrtSuite is an open-source software available at https://github.com/mdsufz/OrtSuite.

bioinformatics↗

bioRxiv2021

Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers

Septins, a family of GTP-binding proteins assembling into higher order structures, interface with the membrane, actin filaments and microtubules, which positions them as important regulators of cytoarchitecture. Septin 9 (Sept9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short MAP-like motif unique to Sept9 isoform 1 (Sept9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring Sept9_i1. Although outside of the MAP-like motif, HNA mutations abrogates this association, identifying a putative regulatory domain. Removal of this domain from Sept9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin associated pathologies.

cell biology↗

bioRxiv2021

An anaphase surveillance mechanism prevents micronuclei formation from mitotic errors

Micronuclei are a hallmark of cancer and other human disorders and have recently been implicated in chromothripsis, a series of massive genomic rearrangements that may drive tumor evolution and progression. Here we show that Aurora B kinase mediates a surveillance mechanism that integrates error correction during anaphase with spatial control of nuclear envelope reformation to protect against micronuclei formation during human cell division. Using high-resolution live-cell imaging of human cancer and non-cancer cells we found that anaphase lagging chromosomes are often transient and rarely formed micronuclei. This strong bias against micronuclei formation relied on a midzone-based Aurora B phosphorylation gradient that assisted the mechanical transduction of spindle forces at the kinetochore-microtubule interface required for anaphase error correction, while delaying nuclear envelope reformation on lagging chromosomes, independently of microtubules. Our results uncover a new layer of protection against genomic instability and provide a strategy for the rational design of micronuclei-targeting therapies.

cell biology↗

bioRxiv2018

Photodynamic therapy effect on the ultrastructure of Trichomonas vaginalis trophozoites and their effectiveness in experimentally infected animals.

BackgroundTrichomonas vaginalis is an amitochondrial parasitic that causes human trichomoniasis, the most common non-viral sexually transmitted infection in the world. The therapy of choice is metronidazole (MTZ). Despite MTZ effectiveness, resistant cases are becoming more frequent. Another point to emphasize are the side effects that may result in treatment discontinuation, leading to further spread of infection and emergence of resistant strains. This scenario reveals the need to develop new therapeutic options. Photodynamic therapy (PDT) is an experimental treatment that involves the activation of photosensitive substances and the generation of cytotoxic oxygen species and free radicals to promote the selective destruction of target tissues. A previous study, from our group, identified an excellent in vitro PDT activity using methylene blue and light emitting diode against MTZ sensitive and resistant strains of T. vaginalis. The aim of this study was to evaluate the efficacy of PDT in vivo and clarify its high trichomonicidal potential by evaluating its action upon T. vaginalis trophozoites through transmission electron microscopy (TEM).\n\nMethodologySeven-week-old female Balb/c mice were infected intravaginally with T. vaginalis trophozoites. On the third day of infection, methylene blue was introduced into the vaginal canal of the animals, which then received 68.1 J / cm2 of radiation for 35.6 sec. Control groups without infection and infected, treated with metronidazole were also included for comparison. Twenty-four hours after treatment the vaginal canal of the animals was scraped and the samples processed by the immunocytochemistry technique. After in vitro photodynamic treatment, T. vaginalis trophozoites were processed for TEM. Ultrathin sections were collected in 400-mesh copper grids, contrasted with 5% uranyl acetate and 3% lead citrate, in aqueous solutions for 20 and 5 min., respectively and observed in a Jeol JEM 230 transmission electron microscope.\n\nResultsTEM showed morphological changes such as centripetal displacement of organelles, cannibalism, hydrogenosomal damage, intense cytoplasmic vacuolization, dilated endoplasmic reticulum cisternae and membrane discontinuity, in both resistant and sensitive strains, suggesting that trichomonicidal activity is mainly due to necrosis.\n\nPDT significantly reduced infection in animals treated with a single therapy session, compared to control groups, being statistically as efficient as MTZ.\n\nConclusionsOur results demonstrated high trichomonicidal activity of PDT with morphological alterations compatible with necrosis. Therefore these results indicate that PDT represents not only an alternative therapy for refractory trichomoniasis, but also routinely for this important neglected parasitic disease.

pharmacology and toxicology↗