Publications by authors named "Thiago P Silva"

18 Publications

  • Page 1 of 1

Whole slide imaging is a high-throughput method to assess Candida biofilm formation.

Microbiol Res 2021 Sep 10;250:126806. Epub 2021 Jun 10.

Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil. Electronic address:

New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyse fungal biofilm growth.
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http://dx.doi.org/10.1016/j.micres.2021.126806DOI Listing
September 2021

Changing our view of the Schistosoma granuloma to an ecological standpoint.

Biol Rev Camb Philos Soc 2021 Mar 22. Epub 2021 Mar 22.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, São Pedro, Juiz de Fora, MG, 36036-900, Brazil.

Schistosomiasis, a neglected parasitic tropical disease that has plagued humans for centuries, remains a major public health burden. A primary challenge to understanding schistosomiasis is deciphering the most remarkable pathological feature of this disease, the granuloma - a highly dynamic and self-organized structure formed by both host and parasite components. Granulomas are considered a remarkable example of how parasites evolved with their hosts to establish complex and intimate associations. However, much remains unclear regarding life within the granuloma, and strategies to restrain its development are still lacking. Here we explore current information on the hepatic Schistosoma mansoni granuloma in the light of Ecology and propose that this intricate structure acts as a real ecosystem. The schistosomal granuloma is formed by cells (biotic component), protein scaffolds, fibres, and chemical compounds (abiotic components) with inputs/outputs of energy and matter, as complex as in classical ecosystems. We review the distinct cell populations ('species') within the granuloma and examine how they integrate with each other and interact with their microenvironment to form a multifaceted cell community in different space-time frames. The colonization of the hepatic tissue to form granulomas is explained from the point of view of an ecological succession whereby a community is able to modify its physical environment, creating conditions and resources for ecosystem construction. Remarkably, the granuloma represents a dynamic evolutionary system that undergoes progressive changes in the 'species' that compose its community over time. In line with ecological concepts, we examine the granuloma not only as a place where a community of cells is settled (spatial niche or habitat) but also as a site in which the functional activities of these combined populations occur in an orchestrated way in response to microenvironmental gradients such as cytokines and egg antigens. Finally, we assert how the levels of organization of cellular components in a granuloma as conventionally defined by Cell Biology can fit perfectly into a hierarchical structure of biological systems as defined by Ecology. By rethinking the granuloma as an integrating and evolving ecosystem, we draw attention to the inner workings of this structure that are central to the understanding of schistosomiasis and could guide its future treatment.
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http://dx.doi.org/10.1111/brv.12708DOI Listing
March 2021

Antifungal Activity of the Natural Coumarin Scopoletin Against Planktonic Cells and Biofilms From a Multidrug-Resistant Strain.

Front Microbiol 2020 7;11:1525. Epub 2020 Jul 7.

Bioactive Natural Products Laboratory, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil.

is one the most relevant biofilm-forming fungal species increasingly associated with invasive mucosal candidiasis worldwide. The amplified antifungal resistance supports the necessity for more effective and less toxic treatment, including the use of plant-derived natural products. Scopoletin, a natural coumarin, has shown antifungal properties against plant yeast pathogens. However, the antifungal activity of this coumarin against clinically relevant fungal species such as remains to be established. Here, we investigated the potential antifungal properties and mechanisms of action of scopoletin against a multidrug-resistant strain (ATCC 28707). First, scopoletin was isolated by high-performance liquid chromatography from , a plant species (family ) distributed throughout South America. Next, scopoletin was tested on cultivated for 48h in both planktonic and biofilm forms. Fungal planktonic growth inhibition was analyzed by evaluating minimal inhibitory concentration (MIC), time-kill kinetics and cell density whereas the mechanisms of action were investigated with nucleotide leakage, efflux pumps and sorbitol and ergosterol bioassays. Finally, the scopoletin ability to affect biofilms was evaluated through spectrophotometric and whole slide imaging approaches. In all procedures, fluconazole was used as a positive control. MIC values for scopoletin and fluconazole were 50 and 250 μg/L respectively, thus demonstrating a fungistatic activity for scopoletin. Scopoletin induced a significant decrease of growth curves and cell density (91.7% reduction) compared to the growth control. Its action was related to the fungal cell wall, affecting plasma membrane sterols. When associated with fluconazole, scopoletin led to inhibition of efflux pumps at the plasma membrane. Moreover, scopoletin not only inhibited the growth rate of preformed biofilms (68.2% inhibition at MIC value) but also significantly decreased the extent of biofilms growing on the surface of coverslips, preventing the formation of elongated fungal forms. Our data demonstrate, for the first time, that scopoletin act as an effective antifungal phytocompound against a multidrug-resistant strain of with properties that affect both planktonic and biofilm forms of this pathogen. Thus, the present findings support additional studies for antifungal drug development based on plant isolated-scopoletin to treat candidiasis caused by
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http://dx.doi.org/10.3389/fmicb.2020.01525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359730PMC
July 2020

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

J Proteome Res 2020 11 4;19(11):4496-4515. Epub 2020 Aug 4.

GlycoProteomics Laboratory, Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.
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http://dx.doi.org/10.1021/acs.jproteome.0c00320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640952PMC
November 2020

Galectin-10, the protein that forms Charcot-Leyden crystals, is not stored in granules but resides in the peripheral cytoplasm of human eosinophils.

J Leukoc Biol 2020 07 28;108(1):139-149. Epub 2020 Feb 28.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

A predominant protein of human eosinophils is galectin-10 (Gal-10), also known as Charcot-Leyden crystal protein (CLC-P) because of its remarkable ability to form Charcot-Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC-P/Gal-10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC-P/Gal-10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre-embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC-P/Gal-10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC-P/Gal-10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250 nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High-resolution analysis of single cells revealed that CLC-P/Gal-10 interacts with the plasma membrane with immunoreactive microdomains of high CLC-P/Gal-10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF-α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC-P/Gal-10 as observed by both immunofluorescence and immuno-EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC-P/Gal-10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis).
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http://dx.doi.org/10.1002/JLB.3AB0220-311RDOI Listing
July 2020

Whole Slide Imaging and Its Applications to Histopathological Studies of Liver Disorders.

Front Med (Lausanne) 2019 8;6:310. Epub 2020 Jan 8.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil.

Histological analysis of hepatic tissue specimens is essential for evaluating the pathology of several liver disorders such as chronic liver diseases, hepatocellular carcinomas, liver steatosis, and infectious liver diseases. Manual examination of histological slides on the microscope is a classically used method to study these disorders. However, it is considered time-consuming, limited, and associated with intra- and inter-observer variability. Emerging technologies such as whole slide imaging (WSI), also termed virtual microscopy, have increasingly been used to improve the assessment of histological features with applications in both clinical and research laboratories. WSI enables the acquisition of the tissue morphology/pathology from glass slides and translates it into a digital form comparable to a conventional microscope, but with several advantages such as easy image accessibility and storage, portability, sharing, annotation, qualitative and quantitative image analysis, and use for educational purposes. WSI-generated images simultaneously provide high resolution and a wide field of observation that can cover the entire section, extending any single field of view. In this review, we summarize current knowledge on the application of WSI to histopathological analyses of liver disorders as well as to understand liver biology. We address how WSI may improve the assessment and quantification of multiple histological parameters in the liver, and help diagnose several hepatic conditions with important clinical implications. The WSI technical limitations are also discussed.
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http://dx.doi.org/10.3389/fmed.2019.00310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960181PMC
January 2020

Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies.

Front Mol Neurosci 2019 5;12:279. Epub 2019 Dec 5.

Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, United States.

Importance: Non-invasive techniques for retrieving ocular surface cells from babies infected by zika virus (ZIKV) during the gestational period remain to be determined.

Objectives: The aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from Zika infected babies with and without Congenital Zika Syndrome (CZS) in satisfactory amount and quality to enable easy adoption in the field and application in the context of genomic and molecular approaches.

Design Settings And Participants: Ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. After an authorized written informed consent from the parents, samples were collected from both eyes of 12 babies (4 babies with maternal ZIKV exposure during gestation and presence of clinical signs which included ocular abnormalities and microcephaly; 4 babies with maternal ZIKV exposure during gestation but no clinical signs; and 4 unaffected control babies with negative PCR for Zika virus and without clinical signs). Cells were used for microscopy analyses and evaluated for their suitability for downstream molecular applications in transcriptomic and proteomic experiments.

Results: Our optimized impression cytology protocol enabled the capture of a considerable number of viable cells. The microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed potential morphological alterations (cell keratinization, pyknosis, karyolysis, anucleation, and vacuolization). Molecular approaches were also performed in parallel. Genomic DNA and RNA were successfully isolated from all samples to enable the establishment of transcriptomic and proteomic studies.

Conclusions And Relevance: Our method proved to be a suitable, fast, and non-invasive tool to obtain ocular cell preparations from babies with and without Zika infection. The method yielded sufficient cells for detailed morphological and molecular analyses of samples. We discuss perspectives for the application of impression cytology in the context of ZIKV studies in basic and clinical research.
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http://dx.doi.org/10.3389/fnmol.2019.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907025PMC
December 2019

Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Infection.

Front Immunol 2018 20;9:3019. Epub 2018 Dec 20.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil.

Eosinophils have been long associated with helminthic infections, although their functions in these diseases remain unclear. During schistosomiasis caused by the trematode , eosinophils are specifically recruited and migrate to sites of granulomatous responses where they degranulate. However, little is known about the mechanisms of eosinophil secretion during this disease. Here, we investigated the degranulation patterns, including the cellular mechanisms of major basic protein-1 (MBP-1) release, from inflammatory eosinophils in a mouse model of infection (acute phase). Fragments of the liver, a major target organ of this disease, were processed for histologic analyses (whole slide imaging), conventional transmission electron microscopy (TEM), and immunonanogold EM using a pre-embedding approach for precise localization of major basic protein 1 (MBP-1), a typical cationic protein stored pre-synthesized in eosinophil secretory (specific) granules. A well-characterized granulomatous inflammatory response with a high number of infiltrating eosinophils surrounding eggs was observed in the livers of infected mice. Moreover, significant elevations in the levels of plasma Th2 cytokines (IL-4, IL-13, and IL-10) and serum enzymes (alanine aminotransferase and aspartate aminotransferase) reflecting altered liver function were detected in response to the infection. TEM quantitative analyses revealed that while 19.1% of eosinophils were intact, most of them showed distinct degranulation processes: cytolysis (13.0%), classical and/or compound exocytosis identified by granule fusions (1.5%), and mainly piecemeal degranulation (PMD) (66.4%), which is mediated by vesicular trafficking. Immunonanogold EM showed a consistent labeling for MBP-1 associated with secretory granules. Most MBP-1-positive granules had PMD features (79.0 ± 4.8%). MBP-1 was also present extracellularly and on vesicles distributed in the cytoplasm and attached to/surrounding the surface of emptying granules. Our data demonstrated that liver-infiltrating mouse eosinophils are able to degranulate through different secretory processes during acute experimental infections with PMD being the predominant mechanism of eosinophil secretion. This means that a selective secretion of MBP-1 is occurring. Moreover, our study demonstrates, for the first time, a vesicular trafficking of MBP-1 within mouse eosinophils elicited by a helminth infection. Vesicle-mediated secretion of MBP-1 may be relevant for the rapid release of small concentrations of MBP-1 under cell activation.
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http://dx.doi.org/10.3389/fimmu.2018.03019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306457PMC
November 2019

Antibacterial and Antibiofilm Activities of Psychorubrin, a Pyranonaphthoquinone Isolated From (Rubiaceae).

Front Microbiol 2018 13;9:724. Epub 2018 Apr 13.

Bioactive Natural Products Laboratory, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil.

Psychorubrin, a natural pyranonaphthoquinone found in different plants, has become an interesting compound in the search for new antimicrobial therapeutic agents. Here, we investigated the potential antagonistic activity of psychorubrin against planktonic and biofilm bacteria. First, psychorubrin was tested against several Gram-positive and Gram-negative bacteria strains by a broth microdilution susceptibility method. Second, bacterial killing assay, bacterial abundance, and membrane viability were evaluated. The nucleotide leakage assay was used to verify membrane destabilization while antibiofilm activities were analyzed by the effect on established biofilm, static biofilm formation, isolation of biofilm matrix assay and scanning electron microscopy. In parallel, the combinatorial effect of psychorubrin and chloramphenicol was evaluated by the checkerboard method. Psychorubrin was active against Gram-positive bacteria, showing rapid time-dependent kinetics of bacterial killing, amplified nucleotide leakage, and greater activity against the methicillin-resistant species (MRSA) 33591 and 33592 and 10096. Psychorubrin also interfered with the composition of the biofilm matrix by reducing the total content of carbohydrates and proteins. A synergic effect between psychorubrin and chloramphenicol was observed for 33592 and 10096 while an additive effect was detected for 33591. Our findings demonstrate, for the first time, an antagonistic activity of psychorubrin against bacteria not only in their planktonic forms but also in biofilms, and identify bacterial membranes as primary targets for this compound. Based on these observations, psychorubrin has a good potential for the design of novel antimicrobial agents.
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http://dx.doi.org/10.3389/fmicb.2018.00724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908958PMC
April 2018

The Cyanobacterium (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution.

Front Microbiol 2018 21;9:272. Epub 2018 Feb 21.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil.

Secretion of membrane-limited vesicles, collectively termed extracellular vesicles (EVs), is an important biological process of both eukaryotic and prokaryotic cells. This process has been observed in bacteria, but remains to be better characterized at high resolution in cyanobacteria. In the present work, we address the release of EVs by (CYRF-01), a filamentous bloom-forming cyanobacterium, exposed to environmental stressors. First, non-axenic cultures of (CYRF-01) were exposed to ultraviolet radiation (UVA + UVB) over a 6 h period, which is known to induce structural damage to this species. Second, was co-cultured in interaction with another cyanobacterium species, (MIRF-01), over a 24 h period. After the incubation times, cell density and viability were analyzed, and samples were processed for transmission electron microscopy (TEM). Our ultrastructural analyses revealed that constitutively releases EVs from the outer membrane during its normal growth and amplifies such ability in response to environmental stressors. Both situations induced significant formation of outer membrane vesicles (OMVs) by compared to control cells. Quantitative TEM revealed an increase of 48% (UV) and 60% (interaction) in the OMV numbers compared to control groups. Considering all groups, the OMVs ranged in size from 20 to 300 nm in diameter, with most OMVs showing diameters between 20 and 140 nm. Additionally, we detected that OMV formation is accompanied by phosphatidylserine exposure, a molecular event also observed in EV-secreting eukaryotic cells. Altogether, we identified for the first time that has the competence to secrete OMVs and that under different stress situations the genesis of these vesicles is increased. The amplified ability of cyanobacteria to release OMVs may be associated with adaptive responses to changes in environmental conditions and interspecies cell communication.
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http://dx.doi.org/10.3389/fmicb.2018.00272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826386PMC
February 2018

Histological assessment of granulomas in natural and experimental Schistosoma mansoni infections using whole slide imaging.

PLoS One 2017 13;12(9):e0184696. Epub 2017 Sep 13.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, Brazil.

The pathology of schistosomiasis mansoni, a neglected tropical disease of great clinical and socioeconomic importance, results from the parasite eggs that become trapped in host tissues, particularly in the liver and intestines. Continuous antigenic stimulation from these eggs leads to recruitment of inflammatory cells to the sites of infection with formation of periovular granulomas. These complex structures have variable size and composition and are the most striking histopathological feature of schistosomiasis mansoni. However, evaluation of granulomas by conventional microscopy methods is time-consuming and limited, especially in large-scale studies. Here, we used high resolution Whole Slide Imaging (WSI), which allows fast scanning of entire histological slides, and multiple morphometric evaluations, to assess the granulomatous response elicited in target organs (liver, small and large intestines) of two models of schistosomiasis mansoni. One of the advantages of WSI, also termed virtual microscopy, is that it generates images that simultaneously offer high resolution and a wide field of observation. By using a model of natural (Nectomys squamipes, a wild reservoir captured from endemic areas in Brazil) and experimental (Swiss mouse) infection with Schistosoma mansoni, we provided the first detailed WSI characterization of granulomas and other pathological aspects. WSI and quantitative analyses enabled a fast and reliable assessment of the number, evolutional types, frequency and areas of granulomas and inflammatory infiltrates and revealed that target organs are differentially impacted by inflammatory responses in the natural and experimental infections. Remarkably, high-resolution analysis of individual eosinophils, key cells elicited by this helminthic infection, showed a great difference in eosinophil numbers between the two infections. Moreover, features such as the intestinal egg path and confluent granulomas were uncovered. Thus, WSI may be a suitable tool for detailed and precise histological analysis of granulomas and other pathological aspects for clinical and research studies of schistosomiasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184696PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597217PMC
October 2017

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts.

An Acad Bras Cienc 2017 14;89(3 Suppl):2053-2073. Epub 2017 Aug 14.

Laboratório de Produtos Naturais Bioativos, Departamento de Bioquímica, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/n, São Pedro, 36036-900 Juiz de Fora, MG, Brazil.

This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.
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http://dx.doi.org/10.1590/0001-3765201720150702DOI Listing
April 2018

Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

Microbiol Res 2017 Jan 3;194:38-46. Epub 2016 Nov 3.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG 36036-900, Brazil. Electronic address:

Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation.
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http://dx.doi.org/10.1016/j.micres.2016.08.002DOI Listing
January 2017

Natural Schistosoma mansoni Infection in the Wild Reservoir Nectomys squamipes Leads to Excessive Lipid Droplet Accumulation in Hepatocytes in the Absence of Liver Functional Impairment.

PLoS One 2016 23;11(11):e0166979. Epub 2016 Nov 23.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, Brazil, 36036-900.

Schistosomiasis is a neglected tropical disease of a significant public health impact. The water rat Nectomys squamipes is one of the most important non-human hosts in the schistosomiasis mansoni transmission in Brazil, being considered a wild reservoir. Cellular mechanisms that contribute to the physiological adaptation of this rodent to the Schistosoma mansoni parasite are poorly understood. Here we identified, for the first time, that a hepatic steatosis, a condition characterized by excessive lipid accumulation with formation of lipid droplets (LDs) within hepatocytes, occurs in response to the natural S. mansoni infection of N. squamipes, captured in an endemic region. Significant increases of LD area in the hepatic tissue and LD numbers/hepatocyte, detected by quantitative histopathological and ultrastructural analyses, were paralleled by increased serum profile (total cholesterol and triglycerides) in infected compared to uninfected animals. Raman spectroscopy showed high content of polyunsaturated fatty acids (PUFAs) in the liver of both groups. MALDI-TOFF mass spectroscopy revealed an amplified pool of omega-6 PUFA arachidonic acid in the liver of infected animals. Assessment of liver functional activity by the levels of hepatic transaminases (ALT and AST) did not detect any alteration during the natural infection. In summary, this work demonstrates that the natural infection of the wild reservoir N. squamipes with S. mansoni elicits hepatic steatosis in the absence of liver functional harm and that accumulation of lipids, markedly PUFAs, coexists with low occurrence of inflammatory granulomatous processes, suggesting that lipid stores may be acting as a protective mechanism for dealing with the infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166979PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120838PMC
June 2017

Extracellular Microvesicle Production by Human Eosinophils Activated by "Inflammatory" Stimuli.

Front Cell Dev Biol 2016 27;4:117. Epub 2016 Oct 27.

Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical SchoolBoston, MA, USA; Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de ForaJuiz de Fora, Brazil.

A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking, and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs), very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1) and tumor necrosis factor alpha (TNF-α). EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM) and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVs) outwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells. TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20 to 1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune responses. The contribution of the eosinophil-derived MVs to the regulation of immune responses awaits further investigation.
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http://dx.doi.org/10.3389/fcell.2016.00117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081571PMC
October 2016

Potential effects of UV radiation on photosynthetic structures of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii CYRF-01.

Front Microbiol 2015 30;6:1202. Epub 2015 Oct 30.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora Juiz de Fora, Brazil.

Cyanobacteria are aquatic photosynthetic microorganisms. While of enormous ecological importance, they have also been linked to human and animal illnesses around the world as a consequence of toxin production by some species. Cylindrospermopsis raciborskii, a filamentous nitrogen-fixing cyanobacterium, has attracted considerable attention due to its potential toxicity and ecophysiological adaptability. We investigated whether C. raciborskii could be affected by ultraviolet (UV) radiation. Non-axenic cultures of C. raciborskii were exposed to three UV treatments (UVA, UVB, or UVA + UVB) over a 6 h period, during which cell concentration, viability and ultrastructure were analyzed. UVA and UVA + UVB treatments showed significant negative effects on cell concentration (decreases of 56.4 and 64.3%, respectively). This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe. Over 90% of UVA + UVB- and UVA-treated cells died. UVB did not alter cell concentration, but reduced cell viability in almost 50% of organisms. Transmission electron microscopy (TEM) revealed a drastic loss of thylakoids, membranes in which cyanobacteria photosystems are localized, after all treatments. Moreover, other photosynthetic- and metabolic-related structures, such as accessory pigments and polyphosphate granules, were damaged. Quantitative TEM analyses revealed a 95.8% reduction in cell area occupied by thylakoids after UVA treatment, and reduction of 77.6 and 81.3% after UVB and UVA + UVB treatments, respectively. Results demonstrated clear alterations in viability and photosynthetic structures of C. raciborskii induced by various UV radiation fractions. This study facilitates our understanding of the subcellular organization of this cyanobacterium species, identifies specific intracellular targets of UVA and UVB radiation and reinforces the importance of UV radiation as an environmental stressor.
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http://dx.doi.org/10.3389/fmicb.2015.01202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627488PMC
November 2015

Visualizing aquatic bacteria by light and transmission electron microscopy.

Antonie Van Leeuwenhoek 2014 Jan 17;105(1):1-14. Epub 2013 Oct 17.

Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, 36036-900, Brazil.

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.
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http://dx.doi.org/10.1007/s10482-013-0047-6DOI Listing
January 2014

New substituted 4-arylaminoquinazolines as potent inhibitors of breast tumor cell lines: in vitro and docking experiments.

Eur J Med Chem 2010 Sep 13;45(9):4339-42. Epub 2010 May 13.

Department of Clinical Pathology, School of Medical Sciences, State University of Campinas, POB 6111, 13083-887 Campinas, SP, Brazil.

The arylquinazolines represent significant advances in the clinical management of breast cancer. Nevertheless some confirmatory studies must be considered to foster the use of anti-EGFR therapies including safety and clinical use. Two 4-arylaminoquinazoline derivatives, recently synthesized, were tested as kinase inhibitors and their citotoxicities showed potent growth inhibitory activity in breast tumor cell lines (MCF-7). The predicted complex structure of quinazoline inhibitors with EGFR protein from molecular docking provided a stereoview of the binding site correlated with structure activity, affording important information about structure-based drug design.
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http://dx.doi.org/10.1016/j.ejmech.2010.04.034DOI Listing
September 2010
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