Biology subjects

Almeida, L.

Publications and source records attributed to Almeida, L..

11 recordsLinked to original sources

bioRxiv2026

Biomechanical stress in the retinal pigment epithelium surrounding laser scars in humans

Purpose : The wound response of an epithelium comprises tissue-scale activation of stress fibers whose contraction regionally reconfigures the epithelium and drives closure of the wound. Recent in vitro studies have shown that similar biomechanical processes can paradoxically lead to enlargement of epithelial holes. In patients having undergone laser photocoagulation of the retinal pigment epithelium (RPE), laser scars may expand over years. Here, to document the implication of epithelial biomechanics in the enlargement of laser scars we analyzed the dynamic changes affecting the RPE around scars in patients and performed confocal microscopy on a RPE flat-mount from a donor eye. Methods: We analyzed the image files of five patients (age 61 to 85 years) showing enlargement of laser scars in the RPE (follow-up, 13 to 60 months). In three of them, prospective imaging was performed using transscleral illumination adaptive optics ophthalmoscopy (AOO), an imaging technology enabling visualization of RPE cells. Time-lapse sequences were analyzed to track dynamic processes in the RPE surrounding scars. A flat-mounted phalloidin-labeled RPE from a human donor eye with a history of scatter photocoagulation was examined by confocal microscopy to document the distribution of stress fibers and cell metrics. Results: Time-lapse sequences showed that enlargement of laser scars was accompanied by the displacement of microscopic features in margins of atrophy. AOO imaging suggested locally coordinated displacement of mosaic of darks spots, with a generally radial or tangent direction relative to the edges of scars. Such movements were detected up to several hundred microns from the edges of scars. On the histology sample, elongated RPE cells with stress fibers forming actin cables were found along edges of scars, which formed fascicles between adjacent scars. Conclusion: Enlargement of RPE scars may be accompanied by long-lasting, largescale changes in the distribution of microscopic features in the margins of lesions. In particular, transscleral illumination AOO showed coordinated displacement of a mosaic similar to the one described for the RPE. The finding by histology of RPE cell with a tractional phenotype along edges further suggests that epithelial biomechanics contribute to such movements and may hence contribute to scar enlargement.

pathology↗

bioRxiv2025

Bright Cells, and Inefficient Enzymatic Activity in a Renin Hypomorph Mouse

Juxtaglomerular (JG) cells are crucial regulators of blood pressure and fluid-electrolyte homeostasis. Under normal conditions, renin secretion by JG cells is sufficient to maintain homeostasis. However, under physiological stress such as narrowing of one of the renal arteries, heart failure, dehydration, or chronic administration of renin-angiotensin system (RAS) inhibitors, additional cells along the renal arterioles are transformed to the renin phenotype to meet the demands for renin and regain homeostasis. In cases of prolonged and persistent stimulation of renin cells, concentric arteriolar hypertrophy develops. The study of renin cell identity, plasticity and function often requires the isolation of this rare cell type. Here, we report on the generation of a mouse model to label renin-expressing cells with a bright fluorescent reporter under control of the Ren1c locus for the tracking and isolation of renin cells. Kidneys from adult heterozygous (Het) Ren1ctdTomato/+ mice showed tdTomato signal confined to the JG area under basal conditions, and extending along the afferent arterioles and in the intraglomerular mesangium upon treatment with captopril + low-salt diet to induce the endocrine transformation of renin cells. Unexpectedly, homozygous (Homo) Ren1ctdTomato/tdTomato mice exhibited increased tdTomato signal that extended along the afferent arterioles and into the mesangium even under normal physiological conditions, with progressive thickening of the kidney arterioles with age. Despite reduced renin immunostaining in the renal cortex, Ren1ctdTomato/tdTomato Homo mice exhibited significantly higher kidney Ren1 mRNA and circulating renin levels when compared to Het controls. Moreover, Homo mice showed significantly lower blood pressure measured under anesthesia and angiotensin I (Ang I) plasma levels, indicating compromised renin activity. In addition, Homo mice developed interstitial fibrosis and compromised kidney function. The concentric arteriolar hypertrophy phenotype observed in these mice is identical to that described when RAS is genetically or pharmacologically inhibited, including the presence of mutations in the renin gene. Unlike mice with global deletion of renin, these animals did not require neonatal saline injections to survive and did not develop other kidney abnormalities, indicating that the bicistronic approach rendered a renin hypomorphic mouse. Ren1ctdTomato mice constitute an excellent model for the bright and strong labeling of renin-expressing cells and for the study of the mechanisms involved in the development of concentric vascular hypertrophy under RAS inhibition. In addition, this model may provide a better understanding of factors controlling renin protein folding, stability, packaging, and release.

physiology↗

bioRxiv2024

A short noncoding RNA modulates gene expression and affects stress response and parasite differentiation in Leishmania braziliensis

The protozoan parasite Leishmania spp. is a causative agent of leishmaniasis, a disease that affects millions of people in more than 80 countries worldwide. Apart from its medical relevance, this organism has a genetic organization that is unique among eukaryotes. Studies of the mechanisms regulating gene expression in Leishmania led us to investigate noncoding RNAs (ncRNAs) as regulatory elements. We previously identified differentially expressed (DE) ncRNAs in Leishmania braziliensis with potential roles in the parasite biology and development. Herein, we present a functional analysis of one such DE ncRNA, the 147-nucleotide-long transcript ncRNA97, which is preferentially expressed in amastigotes, the replicative form within mammalian phagocytes. By RT-qPCR the ncRNA97 was detected in greater quantities in the nucleus under physiological conditions and in the cytoplasm under nutritional stress. Interestingly, the transcript is protected at the 5 end but is not processed by the canonical trypanosomatid trans-splicing mechanism, according to the RNA circularization assay. ncRNA97 knockout (KO) and addback (AB) transfectants were generated and subjected to phenotypic analysis, which revealed that ncRNA97 impairs the starvation response and differentiation to the infective form. Comparative transcriptomics of ncRNA97KO and parental cells revealed that transcripts encoding amastigote-specific proteins were affected. This pioneering work demonstrates that ncRNAs contribute to the developmental regulatory mechanisms of Leishmania.

molecular biology↗

bioRxiv2024

An efficient inducible model for the control of gene expression in renin cells

BackgroundFate mapping and genetic manipulation of renin cells have relied on either non-inducible Cre lines that can introduce developmental effects of gene deletion or BAC transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. MethodsWe generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells. ResultsWe evaluated the pattern of Cre expression in Akr1b7CreERT2/+;R26RmTmG/+ mice in which Akr1b7+/renin+ cells become GFP+ upon tamoxifen administration. At E18.5 and P5, GFP was found in Juxtaglomerular cells, along the arterioles, and in the glomerular mesangium. In adult kidneys, GFP was present mainly in Juxtaglomerular cells. In mice treated with captopril and a low sodium diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. In addition, we deleted renin in adult mice and found a marked reduction in kidney renin expression and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin+ cells. Most importantly, mice with renin deletion induced in the adult developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. ConclusionsRenin can be efficiently and specifically deleted in renin cells in adult life using our conditional model. Akr1b7CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells.

physiology↗

bioRxiv2024

Fitness adaptations of Japanese encephalitis virus in pigs following vector-free serial passaging

Japanese encephalitis virus (JEV) is a zoonotic mosquito-transmitted Flavivirus circulating in birds and pigs. In humans, JEV can cause severe viral encephalitis with high mortality. Considering that vector-free direct virus transmission was observed in pigs, JEV introduction into an immunologically naive pig population could result in a series of direct transmissions disrupting the alternating host cycling between vertebrates and mosquitoes. To assess the potential consequences of such a realistic scenario, we passaged JEV ten times in pigs. This resulted in higher in vivo viral replication, increased shedding, and stronger innate immune responses in pigs. Nevertheless, the viral tissue tropism remained similar and frequency of direct transmission was not enhanced. Next generation sequencing showed single nucleotide deviations in 10% of the genome during passaging. In total, 25 point mutations were selected to reach a frequency of at least 35% in one of the passages. From these, six mutations resulted in amino acid changes located in the precursor of membrane, the envelope, the non-structural 3 and the non-structural 5 proteins. In a competition experiment with two lines of passaging, the mutation M374L in the envelope protein and N275D in the non-structural protein 5 showed a fitness advantage in pigs. Altogether, the interruption of the alternating host cycle of JEV caused a prominent selection of viral quasispecies as well as selection of de novo mutations associated with fitness gains in pigs, albeit without enhancing direct transmission frequency. Author summaryJapanese encephalitis virus (JEV) represents a major health threat in parts of Asia and Oceania. Primary vertebrate hosts are birds and pigs, but human infection also occurs and can cause severe encephalitis with high mortality. Like other Flaviviruses transmitted by insect bites, JEV requires replication in alternating cycles between mosquitoes on one side and birds or pigs on the other side. However, we previously reported that direct transmissions between pigs in absence of mosquitos can occur. Considering the increased risks for such events after the spread of JEV to a new region with immunologically naive pigs, the present study was performed to understand if and how a series of direct transmissions would promote JEV adaptations to pigs and change virus-host interactions. Pigs infected with JEV passaged ten times showed enhanced clinical symptoms and stronger antiviral immune response, but luckily no increase in direct transmission was observed. Nevertheless, genomic analysis demonstrated a complete change in dominant virus variants, as well as selection of six viral amino acid changes. This indicates that interruptions of the alternating lifestyle of JEV causes a strong evolutionary pressure, which through fitness adaptations can change the viral characteristics.

microbiology↗

bioRxiv2022

In Vitro Inhibition of SARS-CoV-2 Infection by Bromhexine hydrochloride

The world is enduring the SARS CoV-2 pandemic, and although extensive research has been conducted on the issue, only a few antivirals have been approved to treat patients with COVID-19. Bromhexine hydrochloride was previously identified as a potent inhibitor of TMPRSS2, an essential protease for ACE-2 virus receptor interactions. In the present study, we investigated whether bromhexine treatment could reduce SARS CoV-2 replication in vitro. To evaluate bromhexines effectiveness against SARS COV-2 infection, viral load was measured using Caco-2 cell lines expressing TMPRSS2. Our molecular docking results indicate that bromhexine displays an affinity with the active site of TMPRSS2. The drug significantly inhibited SARS CoV-2, both parental and P1 variant strains, infection in the Caco-2 cell line, reducing about 40% of SARS-CoV-2 entrance and about 90% of viral progeny in the supernatant 48h post-infection. Furthermore, bromhexine did not exhibit any direct virucidal activity on SARS CoV-2. In conclusion, bromhexine hydrochloride efficiently disrupts SARS CoV-2 infection in vitro and has the potential to become an effective antiviral agent in COVID-19 treatment.

pharmacology and toxicology↗

bioRxiv2022

Spectral flow cytometry reveals metabolic heterogeneity in tissue macrophages

Tissue-macrophage populations are constituted by a mosaic of phenotypes, yet new methods are needed to link metabolic status to the range of phenotypes in vivo. We therefore designed a high-dimensional panel for spectral flow cytometry to investigate the heterogeneity of tissue macrophage metabolism at steady-state, and their metabolic adaptation in response to infection. Distinct metabolic profiles were observed between tissue macrophages from different peripheral organs, as well as within populations from a specific site. As such, our data show multiple metabolic states in macrophages corresponding to relative stages of maturity in both the peritoneal cavity and small intestine. Immune perturbation with helminth infection further showed that peritoneal macrophages acquire an overall highly metabolically active profile, whereas responding intestinal macrophages displayed minimal changes in their metabolic phenotype. Thus, we demonstrate that high-dimensional, flow-based analysis is an exciting method to interrogate the metabolic heterogeneity and dynamics of tissue-macrophage populations.

immunology↗

bioRxiv2022

M2-polarized macrophages control LSC fate by enhancing stemness, homing, immune evasion and metabolic reprogramming

While it is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones, the tumor microenvironment (TME) in acute myeloid leukemias (AML) remains poorly understood. Here, we uncover the functional and prognostic relevance of an M2-polarized macrophage compartment. Intra bone marrow co-injection of M2d-macrophages together with leukemic blasts that fail to engraft on their own now induce fatal leukemia in mice. Even a short-term two-day in vitro exposure to M2d macrophages can "train" leukemic blasts after which cells are protected against phagocytosis, display increased mitochondrial metabolism and improved in vivo homing, resulting in full-blown leukemia. Single-cell RNAseq analysis of AML associated macrophages revealed metabolic-related pathways such as Fatty Acid Oxidation and NAD+ generation as therapeutical targetable vulnerabilities. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for effective targeting strategies.

cancer biology↗

bioRxiv2021

Functional study of Leishmania braziliensis protein arginine methyltransferases (PRMTs) reveals that PRMT1 and PRMT5 are required for macrophage infection

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are major targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other macromolecular interactions of RBPs by transferring methyl groups to protein arginine residues. Herein, we present the results of a study that functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that arginine methylation profiles vary among Leishmania species and that target protein methylation changes across different L. braziliensis life cycle stages, with higher PRMT expression in the promastigote stages than in the axenic amastigote stage. Knockout of some of the L. braziliensis PRMTs led to significant changes in global arginine methylation patterns without affecting promastigote axenic growth. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine (MMA) levels, which is mainly catalyzed by PRMT7. Putative targets and/or PRMT-interacting proteins were identified by coimmunoprecipitation using HA-tagged PRMTs, revealing a network of target RBPs and suggesting functional interactions between them and a relevant participation in epigenetic control of gene expression. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and that in the absence of PRMT1 and PRMT5, axenic amastigote proliferation is impaired. The results indicate that arginine methylation is modulated across life cycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

molecular biology↗

bioRxiv2020

A genome-wide association study highlights a regulatory role for IFNG-AS1 contributing to cutaneous leishmaniasis in Brazil

BackgroundCutaneous leishmaniasis (CL) caused by Leishmania braziliensis remains an important public health problem in Brazil. The goal of this study was to identify genetic risk factors for CL. MethodsGenome-wide analysis was undertaken using DNAs from 956 CL cases and 868 controls (phase 1) and 1110 CL cases and 1178 controls (phase 2) genotyped using Illumina HumanCoreExome BeadChips. Imputation against 1000G data provided 4,498,586 quality-controlled single nucleotide variants (SNVs) common across phase 1 and phase 2 samples. Linear mixed models in FastLMM were used to take account of genetic diversity/ethnicity/admixture. Cellular cytokines were measured using flow cytometry. ResultsCombined analysis across cohorts found no associations that achieved genome-wide significance, commonly accepted as P<5x10-8. Support for variants at wound-healing genes previously studied as candidate genes for CL included SMAD2 (rs115582038/rs75753347; Pimputed_1000G=1.47x10-4). Top novel GWAS hits at P<5x10-5 in plausible candidate genes for CL included SERPINB10 (rs62097497;Pimputed_1000G=2.67x10-6), CRLF3 (rs75270613; Pimputed_1000G=5.12x10-6), STX7 (rs144488134;Pimputed_1000G=6.06x10-6), KRT80 (rs10783496 Pimputed_1000G=6.58x10-6), LAMP3 (rs74285558;Pimputed_1000G=6.54x10-6) and IFNG-AS1 (rs4913269;Pimputed_1000G=1.32x10-5). Of these, LAMP3 (Padjusted=9.25x10-12; +6-fold), STX7 (Padjusted=7.62x10-3; +1.3-fold) and CRLF3 (Padjusted=9.19x10-9; +1.97-fold) were all expressed more highly in CL biopsies compared to normal skin, whereas expression of KRT80 (Padjusted=3.07x10-8; -3-fold) was lower. Notably, the percent peripheral blood CD3+ T cells making interferon-{gamma} in response to Leishmania antigen differed significantly by IFNG-AS1 genotype. ConclusionsIn addition to supporting variants in wound-healing genes as genetic risk factors for CL, our GWAS results provide important novel leads to understanding pathogenesis of CL including through the regulation of interferon-{gamma} responses.

genetics↗

bioRxiv2019

Ribosome-targeting antibiotics impair T cell effector function and ameliorate autoimmunity by blocking mitochondrial protein synthesis

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive, for reasons that remain unclear. Here we show that linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of Th17 effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromises the integrity of the electron transport chain (ETC). Ultimately, this leads to loss of mitochondrial metabolism and cytokine production in differentiating Th cells. In accordance, mice lacking Gfm1 in T cells are protected from EAE, demonstrating that this pathway plays a key role in maintaining T cell function and pathogenicity.

immunology↗