Glaúcia Maria Machado-Santelli

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Abstract We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera , on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted Hs578T malignant phenotype to polarized spheroid‐like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time‐lapse microscopy showed that the peptide inhibited migration of these cells in a dose‐dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide. J. Cell. Physiol. 216: 583–594, 2008, © 2008 Wiley‐Liss, Inc.

The Spike protein (S protein) is a critical component in the infection of the new coronavirus (SARS-CoV-2). The objective of this work was to evaluate whether peptides from S protein could cause negative impact in the aquatic animals. The aquatic toxicity of SARS-CoV-2 Spike protein peptides derivatives has been evaluated in tadpoles (n = 50 tadpoles/5 replicates of 10 animals) from species Physalaemus cuvieri (Leptodactylidae). After synthesis, purification, and characterization of peptides (PSDP2001, PSDP2002, PSDP2003) an aquatic contamination has been simulated with these peptides during 24 h of exposure in two concentrations (100 and 500 ng/mL). The control group ("C") was composed of tadpoles kept in polyethylene containers containing de-chlorinated water. Oxidative stress, antioxidant biomarkers and AChE activity were assessed. In both concentrations, PSPD2002 and PSPD2003 increased catalase and superoxide dismutase antioxidants enzymes activities, as well as oxidative stress (nitrite levels, hydrogen peroxide and reactive oxygen species). All three peptides also increased acetylcholinesterase activity in the highest concentration. These peptides showed molecular interactions in silico with acetylcholinesterase and antioxidant enzymes. Aquatic particle contamination of SARS-CoV-2 has cholinesterasic effect in P. cuvieri tadpoles. These findings indicate that the COVID-19 can constitute environmental impact or biological damage potential.

To determine, through the micronucleus (MN) test, the cytogenetic effects of cigarette smoking on exfoliated cells from the uterine cervix in women with normal smears and women with inflammatory atypia, squamous intraepithelial lesion (SIL) (cervical intraepithelial neoplasia [CIN] 1-3) and cervical cancer.The study group consisted of 200 women divided into three subgroups: group 1 (n = 116), women periodically undergoing cervical cytology and residents of Salvador-Bahia; group II (n = 57), women residing in São Paulo and previously selected because of a possible cytopathologic test positive for such conditions as human papillomavirus infections or malignant or premalignant cervical lesions (CIN 1-3); group III (n = 27), inmates of the Tatuapé Penal Institution, São Paulo. All the women underwent cytologic and colposcopic examination, and biopsies were performed on 68 of them.Considering the samples as a whole and using the chi(2) test for rare events, the number of MNs in smokers was significantly greater than in nonsmokers. It was also greater in women with larger exposure to smoking. The occurrence of MN was significantly lower in women with normal smears (smokers and nonsmokers) than in those showing any kind of pathologic alteration. In nonsmokers the occurrence of MN was similar between those with inflammatory atypia (IA) or low grade (L) SIL (CIN 1) and significantly higher in women with more severe lesions or high grade (H) SIL (CIN 2 and 3). Smokers with LSIL (CIN 1) showed a higher number of MNs than nonsmokers with a comparable diagnosis and smokers with IA. No differences were observed when compared with smokers with HSIL (CIN 2 and 3). MN occurrence was not associated with other risk factors for SIL or cancer development, such as age at first coitus, number of sexual partners, multiparity and use of hormonal contraceptives.These results suggest that the mutagenic effect of cigarette smoking occurs in cervical cells and that the progression of SIL is associated with increased frequency of chromosomal damage. Moreover, the data suggest that cigarette smoking introduces an additional risk to the progression of low grade LSIL (CIN 1). MN testing would be helpful in monitoring smokers with this kind of lesion.Previous studies have shown that cigarette smoking increases the risk of developing squamous intraepithelial lesion (SIL) and cervical cancer. The present study used the micronucleus test to assess the cytogenic effects of smoking on exfoliated cells from 3 subgroups of Brazilian women: group 1 (n = 116), women periodically undergoing cervical cytology; group 2 (n = 57), women with a possibly positive cytologic test for human papillomavirus or malignant or premalignant cervical intraepithelial neoplasia (CIN 1-3); and group 3 (n = 27), inmates of the Tatuape Penal Institute. Overall, micronucleus frequency was significantly greater in smokers than in nonsmokers. The occurrence of micronuclei was significantly lower in women with normal smears (regardless of smoking status) than in women with any evidence of pathologic alterations. In nonsmokers, micronucleus frequency was similar in women with inflammatory atypia or low-grade CIN and significantly higher in women with more severe lesions and CIN 2-3. Smokers with CIN 1 had more micronuclei than nonsmokers with a comparable diagnosis and smokers with inflammatory atypia. No differences were observed in comparisons with smokers with CIN 2-3. Micronucleus occurrence was not associated with age at first coitus, number of sexual partners, multiparity, or use of hormonal contraception. These findings suggest that the mutagenic effect of smoking occurs in cervical cells and that SIL progression is associated with an increased frequency of chromosomal damage. The data further suggest that smoking adds to the risk of progression of low-grade SIL (CIN 1). Micronucleus testing, along with the cervical cytologic smear, is recommended to monitor smokers with this type of lesion.

Abstract The Shwachman–Bodian–Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre‐60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co‐immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright © 2009 John Wiley & Sons, Ltd.

Breast cancer is a complex disease and encompassing different types of tumor. Although advances in understanding of the molecular bases of breast cancer biology, the therapeutic proposals available still are not effective. In this scenario, the present study aimed to evaluate the mechanisms associated to antitumor activity of 7-Epiclusianone (7-Epi), a tetraprenylated benzophenone, on luminal A (MCF-7) and claudin-low (Hs 578T) breast cancer cell lines. We found that 7-Epi efficiently inhibited cell proliferation and migration of these cells; however MCF-7 was slightly more responsive than Hs 578T. Cell cycle analysis showed accumulation of cells at G0/G1 phase with drastic reduction of S population in treated cultures. This effect was associated to downregulation of CDKN1A (p21) and cyclin E in both cell lines. In addition, 7-Epi reduced cyclin D1 and p-ERK expression levels in MCF-7 cell line. Cytotoxic effect of 7-Epi on breast cancer cell lines was associated to its ability to increase BAX/BCL-2 ratio. In conclusion, our findings showed that 7-Epi is a promising antitumor agent against breast cancer by modulating critical regulators of the cell cycle and apoptosis.

Summary Establishing new experimental animal models to assess the safety and immune response to the antigen used in the development of COVID-19 vaccine is an imperative issue. Based on the advantages of using zebrafish as a model in research, herein we suggest doing this to test the safety of the putative vaccine candidates and to study immune response against the virus. We produced a recombinant N-terminal fraction of the Spike SARS-CoV-2 protein and injected it into adult female zebrafish. The specimens generated humoral immunity and passed the antibodies to the eggs. However, they presented adverse reactions and inflammatory responses similar to severe cases of human COVID-19. The analysis of the structure and function of zebrafish and human Angiotensin-converting enzyme 2, the main human receptor for virus infection, presented remarkable sequence similarities. Moreover, bioinformatic analysis predicted protein-protein interaction of the Spike SARS-CoV-2 fragment and the Toll-like receptor pathway. It might help in the choice of future therapeutic pharmaceutical drugs to be studied. Based on the in vivo and in silico results presented here, we propose the zebrafish as a model for translational research into the safety of the vaccine and the immune response of the vertebrate organism to the SARS-CoV-2 virus.

Anticancer agents pose a great environmental risk due to their high toxicity. The aim of the current study is to assess the toxicity of trabectedin, a cytotoxic but atypical DNA binder, to liver cell line (ZFL) and embryo-larvae of the zebrafish Danio rerio employing an innovative approach. In ZFL cells, trabectedin cytotoxicity was measured using MTT and Trypan blue exclusion assay, and cell morphology was evaluated by fluorescence-activated cell sorting (FACS) and by immunofluorescence analysis. Trabectedin was 60-fold more toxic to ZFL cells than to zebrafish embryo-larvae in terms of mortality/cell viability, with mortality being observed in 42.7 µg.L−1 for embryo-larvae and non-viability in 0.04 µg.L−1 for cultured cells. Immunofluorescence staining showed morphology alterations of ZFL-cells exposed to trabectedin in a dose-dependent manner, from 0.04 to 0.15 µg.L−1. Furthermore, trabectedin induced morphological abnormalities to zebrafish embryo-larvae, such as tail malformations, pericardial edema and lack of equilibrium at concentrations lower than 50.3 µg.L−1. Regarding larvae behavior analysis, trabectedin increased velocity and total distance covered by zebrafish exposed to 42.7 µg.L−1 under dark conditions. These results reveal trabectedin to be toxic in both in vitro and in vivo zebrafish models, and thus the occurrence and persistence of this anticancer agent in the environment may represent a potential risk factor to the biota.

Abstract The diptera Rhynchosciara americana (sciaridae) is an important model organism in polyteny and gene amplification research, but up to now a limited amount of data regarding DNA sequences and molecular aspects of this species is available. Considering the importance of going further on the DNA puffs biological meaning, we proposed to generate EST sequences from a DNA library constructed from salivary glands. After their categorization in gene ontology terms, they were used to construct an ‘electronic Northern’ that represents a general view of the salivary gland metabolic status in an important phase of larval development: the spinning of communal cocoon. In this phase occurs the last polytene DNA replication cycle concomitantly with the specific loci amplification related to protein secretion.

The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic processes including the promotion of proliferation and bypass of senescence. Importantly, based on compelling biological evidences, TBX2 has been considered as a potential target for new anticancer therapies. There has therefore been a substantial interest to identify molecules with TBX2-modulatory activity, but no such substance has been found to date. Here, we adopt a targeted approach based on a reverse-affinity procedure to identify the ability of chromomycins A5 (CA5) and A6 (CA6) to interact with TBX2. Briefly, a TBX2-DNA-binding domain recombinant protein was N-terminally linked to a resin, which in turn, was incubated with either CA5 or CA6. After elution, bound material was analyzed by UPLC-MS and CA5 was recovered from TBX2-loaded resins. To confirm and quantify the affinity (KD) between the compounds and TBX2, microscale thermophoresis analysis was performed. CA5 and CA6 modified the thermophoretic behavior of TBX2, with a KD in micromolar range. To begin to understand whether these compounds exerted their anti-cancer activity through binding TBX2, we next analyzed their cytotoxicity in TBX2 expressing breast carcinoma, melanoma and rhabdomyosarcoma cells. The results show that CA5 was consistently more potent than CA6 in all tested cell lines with IC50 values in the nM range. Of the cancer cell types tested, the melanoma cells were most sensitive. The knockdown of TBX2 in 501mel melanoma cells increased their sensitivity to CA5 by up to 5 times. Furthermore, inducible expression of TBX2 in 501mel cells genetically engineered to express TBX2 in the presence of doxycycline, were less sensitive to CA5 than the control cells. Together, the data presented in this study suggest that, in addition to its already recognized DNA-binding properties, CA5 may be binding the transcription factor TBX2, and it can contribute to its cytotoxic activity.

Advanced chronic kidney disease is associated with high rates of cardiovascular disease. Considering the crucial role of capillaries in renal function, our study aimed to evaluate microparticles related to vascular physiology examining the link between stages of chronic kidney disease with circulating endothelial (EMP), platelet (PMP) and monocytic (MMP) microparticles. Cross-sectional study with blinded endpoints included subjects of both sexes, aged 40–75 years (n = 247), with established cardiovascular disease (coronary heart disease, ischemic stroke, or peripheral artery disease). They were stratified 1:1 by the presence or absence of decreased glomerular filtration rate (GFR < 60 mL/min/1.73 m2) estimated by the CKD-EPI criteria, and according to the stages of CKD. Microparticles were quantified by flow-cytometry using specific antibodies to identify endothelial, platelet, and monocytic derived microparticles. Higher percentages of circulating MMP (p = 0.036), but not for EMP or PMP, were observed in subjects with reduced GFR. Circulating MMP were also related to the stages of chronic kidney disease (trend analysis across renal stages, p = 0.038). Higher percentages of circulating MMP were found in subjects with reduced GFR, and their percentages were progressively higher according to the stage of chronic renal function.

Many molecular mechanisms in complex biological processes of diseases cannot be fully understood without direct visualization. In the last decades, advances in imaging principles and technologies have expanded our ability to capture and analyze the morphology of tissues and cells' components on conventional widefield optical and fluorescence microscopies and their derivatives confocal and multiphoton fluorescence laser-scanning microscopes, as well as transmission electron microscopes. Innovative imaging technologies are now emerging for constructing fine structural features, precise localization and the dynamic interplay of single and macromolecular assemblies that drive cell growth, differentiation and cell death as well as stromal and chromatin remodeling within many cellular context. The newer super-resolution microscopies capture images with unprecedented sensitivity and clarity allowing the exploration of interactions between individual molecules with a distance resolution as low as 20 nm. But these techniques are not robust enough to quantitate molecules on a genome-wide scale. Mass spectrometry imaging is a high-throughput chemical imaging technique for the identification, quantitation and distribution of proteins, lipids and chemical metabolites at picomol level within a single-cell and complex multicellular tissue. Here we provide an overview on imaging instrumentations and computational platforms to store, data mining, analyze and retrieving genomic, proteomic and immunohistochemistry digital image information which are available for multilevel academic-private collaborations. The expansion of these data sets will lead to a merge picture from it we will retrieve knowledge for more rational-design systems to basic and clinical research in near future.

ABSTRACT The Spike protein (S protein) is a critical component in the infection of the new coronavirus (SARS-CoV-2). The objective of this work was to evaluate whether peptides from S protein could cause negative impact in the aquatic animals. The aquatic toxicity of SARS-CoV-2 spike protein peptides derivatives has been evaluated in tadpoles (n = 50 tadpoles / 5 replicates of 10 animals) from species Physalaemus cuvieri (Leptodactylidae). After synthesis, purification, and characterization of peptides (PSDP2001, PSDP2002, PSDP2003) an aquatic contamination has been simulatedwith these peptides during 24 hours of exposure in two concentrations (100 and 500 ng/mL). The control group (“C”) was composed of tadpoles kept in polyethylene containers containing de-chlorinated water. Oxidative stress, antioxidant biomarkers and neurotoxicity activity were assessed. In both concentrations, PSPD2002 and PSPD2003 increased catalase and superoxide dismutase antioxidants enzymes activities, as well as oxidative stress (nitrite levels, hydrogen peroxide and reactive oxygen species). All three peptides also increased acetylcholinesterase activity in the highest concentration. These peptides showed molecular interactions in silico with acetylcholinesterase and antioxidant enzymes. Aquatic particle contamination of SARS-CoV-2 has neurotoxics effects in P. cuvieri tadpoles. These findings indicate that the COVID-19 can constitute environmental impact or biological damage potential. HIGHLIGHTS SARS-CoV-2 spike protein peptides (PSDP) were synthesized, purified, and characterized by solid phase peptide synthesis. PSDP peptides promoted REDOX imbalance and acute neurotoxicity in tadpoles (Physalaemus cuvieri) In silico studies have shown interactionsbetween peptides and acetylcholinesterase and antioxidant enzymes Aquatic particle contamination of SARS-CoV-2 can constitute additional environmental damage GRAPHICAL ABSTRACT

Abstract Metastasis is the leading cause of melanoma mortality. The metastatic cascade represents a multi-step process which includes several cellular and molecular processes including cell migration and invasion. Tumor cells can migrate either collectively, in a mesenchymal or in an amoeboid type of movement. The role of microfilaments is essential in mesenchymal migration, where it acts in the formation of membrane protrusions, and amoeboid migration, where they play a key role in cell contraction. In this context, the present work aimed to analyze the effects of the marine sponge-derived drug jaspamide, which has well-known properties on cytoskeleton (anti-polymerizing of the actin microfilaments). After the determination of the IC50 concentrations for the HT144 (derived from human melanoma) and NGM (from human benign nevus) cell lines, the effects of the treatment with jaspamide were studied through wound assays, transwell assays, Boyden's chamber assays, fluorescent cytochemistry, immunofluorescence and Western blotting. The treatments demonstrated the drug acts disorganizing the microfilament cytoskeleton in a dose-dependent way, leading the accumulation of F-actin in the perinuclear region. The wound assays indicated that drug treatment impaired the migration ability of both cell lines. However assays in Boyden's chambers showed increasing in cell migration in consequence of jaspamide treatment, result that could be explained by switch of the migration strategy from mesenchymal to ameboid. This fact was corroborated by the decreased expression of the IRSp53 protein after jaspamide treatment meanwhile MT1-MMP expression is not affected by the treatment. By treating thecell lines with the compounds Y-27632 or NSC23766 in addition to jaspamide, it is possible to conclude that NGM cells use mainly the RAc signaling pathway to control cellular migration process. In contrast, the melanoma cell line HT144 however could use either Rac or Rho pathway to migrate. Key words: Melanoma. Jaspamide. Migration. Invasion. Metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 481. doi:1538-7445.AM2012-481

Tunicates have been reported to be a rich source of biologically active compounds. In this study, we demonstrate the presence of cytotoxic substances in Phallusia nigra, a common tunicate from Brazilian coastal waters. An extract of tunicate tissue was obtained by homogenizing the visceral organs from 50 specimens in methanol, followed by filtration and concentration in a rotary vacuum evaporator. Finally, the concentrate was partitioned with chloroform to remove lipids. The resulting extract possessed antimitotic and hemolytic activity. The former was demonstrated as a delay in the development of sea urchin eggs by partially inhibiting the process of cleavage (first cleavage, EC50 +/- SEM = 3.44 +/- 0.84 mg/ml). The < 500 molecular fraction of the extract obtained by ultrafiltration also inhibited cell proliferation (the number of viable cells was decreased by 68% with 500 micrograms/ml) and DNA synthesis of T47D cells derived from human breast carcinoma as measured by [3H]-thymidine incorporation (66% of the control value after 24-h incubation with 100 micrograms/ml). Dose-dependent hemolysis obtained with P. nigra extract on mouse erythrocytes had an EC50 +/- SEM = 1.12 +/- 0.02 mg/ml for a 0.5% erythrocyte suspension. Hemolysis could be reduced by pre-incubating the cells with choline-containing phospholipid. Sphingomyelin (40 micrograms/ml) increased the EC50 by two-fold to 2.86 +/- 0.04 mg/ml, but phosphatidylcholine (80 micrograms/ml) did not modify hemolysis.

In this study, the ovary morphology of newly emerged ant queens ofAtta sexdens rubropilosa was studied in whole mount preparations by confocal microscopy. The ovaries are composed of approximately 40 ovarioles, showing non-synchronic oocyte maturation. The terminal filament with clusters of undifferentiated cells was found at the distal end of the ovarioles. Next to this region is the germarium, composed of several elongated cystocytes interconnected by cytoplasmic bridges. The nurse cells (23-28 cells) result from asymmetric mitosis. Cytoskeleton analysis showed F-actin concentrated at the muscle cells of the external tunica and in fusomes inside the ovarioles. Microtubules were concentrated around the nuclei of the nurse and follicular cells. In contrast, the oocytes and the external tunica showed faint staining for tubulin.

Abstract Brazilian flora is considered one of the most diverse in the world and represents a source of new molecules with bioactivity against several diseases. 7-epiclusianone, a prenylated benzophenone was isolated from Garcinia brasiliensis, a plant named bacupari in folk medicine. Previous studies showed that this compound has dose-dependent cytotoxic effect in several cell lines derived from human cancers and antiproliferative effect by inducing cell cycle arrest in G1/S and apoptosis in A549 lung cancer cell line. The present study aimed to analyze the cytotoxic and/or antiproliferative potential of 7-epiclusianone in human breast cancer cell lines cultured in monolayer and as spheroids. Monolayer cell cultures are commonly used for testing drug effects largely because of their easy maintenance, but they do not represent the spatial interactions of cells within a tumor. Spheroids in 3D cell cultures can overcome some of those limitations thus mimicking the architecture of solid tumors. Initially the 3D conditions were established for both cell lines MCF7 and Hs578T. Spheroids were morphologically characterized by light and transmission electron microscopy. MCF-7 spheroids showed typical epithelial organization with cohesive cells, in accordance with higher expression levels of E-cadherin compared to monolayer. In Hs578T spheroids, cells assumed fibroblast-like morphology concentrically organized, low E-cadherin expression and synthesis of extracellular matrix components. The IC50 values of 7-epiclusianone were 20 μM for Hs 578T cells and 6 μM for MCF-7 monolayers cell cultures. At this concentration, the compound treatment arrested monolayer cell cultures in G0/G1. 7-epiclusianone reduced the mRNA levels for cyclins D1 and E in line MCF-7, while only cyclin E mRNA in Hs 578T. The compound did not change the microfilaments organization or the nuclear integrity, as observed by laser scanning confocal microscopy. Interestingly, 7-epiclusianone treated cultures exhibit higher senescence indexes while apoptotic cells were not detected. Altogether, these data suggest that 7-epiclusianone is a promising molecule against breast cancer cells. The three-dimensional culture was more resistant to treatment with the compound than the monolayer, therefore more comprehensive studies are needed to understand better the effects of 7-epiclusianone on this type of culture. (Supported by FAPESP and CNPq) Citation Format: Bianca Rocha-Sales, Paula Rezende-Teixeira, Evandro Luís Oliveira Niero, Camila Lauand, Marisa Ionta, Simone SL Hanemann, Glaucia M. Machado-Santelli. Cell senescence and antitumor potential of 7-epiclusianone in human breast cancer cell lines cultured in monolayer and as spheroids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2844.

1. Collagen biosynthesis and processing by primary cultures (2nd subculture) of guinea pig embryo fibroblasts were studied. Collagen represents about 15% of the protein synthesized and extruded by these cells under the culture conditions employed. 2. After incubating the cells with [3H]-proline for varying periods of time, the labelled products from the cell layer and the medium were analyzed by polyacrylamide gel electrophoresis. 3. Fluorography of the corresponding gels showed that the cell layer contained only mature alpha 1 (I) and alpha 2 chains, while the medium contained five extra bands; four with mobilities between beta-dimers and alpha 1 (I), and one migrating between alpha 1 (I) and alpha 2. These molecules were identified as predominantly type-I collagen precursors and represent about 96% of the collagen synthesized by the fibroblasts. 4. Kinetic studies of procollagen processing after both short- and long-term labelling periods showed that removal of amino- and carboxy-terminal peptide extensions occurred independently.

Abstract Chrysotile accounts for more than 90% of the asbestos used worldwide. This type of fiber is considered the least oncogenic among the asbestos fibers, mainly based on the fiber dimension and biopersistence inside the lung. It is associated with respiratory diseases and lung cancer and identified as human carcinogen by the International Agency for Research on Cancer. However the mechanism and potential of different fibers to cause cancer is still a matter of debate. The present study focuses the interaction of fibers with different cell types, analyzing their ability and mechanism of fiber internalization. After short chrysotile small fibers exposure time (4-8hrs) both non-small cell lung carcinoma and monocytic cell lines were able of fiber internalization. Transmission electron microscope (TEM) analyses showed fibers involved or not by membrane into the cytoplasm and sometimes seemed to be associated with the chromatin. Cytoskeleton elements were observed to be concentrated around the fibers and they were identified as intermediate filaments according to their diameter. Immunofluorescent preparations imaged by laser scanning confocal microscope and submitted to localization analyses showed that both vimentin and cytokeratin may participate in this process. The data suggest that the internalization of fibers may occur through endocytosis and/or by physical perforation of the plasma membrane. Since the scavenger receptors are responsible for recognition and internalization of dust particles by macrophages in lung, and some of them are linked to silica-containing particles recognition we evaluated their expression level of mRNA by using quantitative RT-PCR. The scavenger receptors appear to interfere with the internalization of chrysotile fibers process. SR-A and SCARA5 change their relative expression of mRNA after cell exposure to chrysotile fibers, such as SR-A and SCARA5. Furthermore, our study was the first to find the relationship between the SCARA5 receptor and chrysotile fibers. To check if features of the chrysotile internalization by epithelial and monocytic cells would be related with fibers shape or chemical composition, the cells were exposed to single walled carbon nanotubes in similar conditions. Financial support: FAPESP, CAPES and CNPq) Citation Format: Luana R. Ricardi, Paula Rezende-Teixeira, Marcelo Medina de Souza, Glaucia M. Machado-Santelli. Morphological and molecular study of cell-chrysotile interaction in two different cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3189. doi:10.1158/1538-7445.AM2014-3189

Abstract Due to their general cytotoxic effects, anticancer drugs pose a great risk to aquatic species, yet they have been among the least studied groups of drugs regarding their environmental impact. The present study aimed to identify the effects of the antileukemia drug cytarabine, also known as cytosine arabinoside (ara-C), on the development and neurobehavior of zebrafish larvae. Zebrafish eggs were exposed to cytarabine to assess survival, embryonic malformations, locomotor activity, DNA damage, and biochemical alterations. No mortality, teratogenicity, or genotoxicity was detected in zebrafish larvae up to 100 mg L-1. However, cytarabine decreased the total distance traveled by zebrafish under dark conditions, suggesting depression-like behavior in treated larvae. Furthermore, it was demonstrated that cytarabine exposure increased lipid peroxidation (LPO) and acetylcholinesterase (AChE) activity, while it inhibited catalase (CAT) activity. Multivariate analysis suggested that both AChE and LPO correlated to behavioral changes, while molecular docking and dynamics simulations suggested a direct binding and stability of cytarabine to AChE. Further analyses of the effects of this anticancer compound in the cholinergic transmission are in progress to allow a better understanding of its neurotoxicity.

Abstract This work presents the first study showing how photobiomodulation (PBM) significantly increases cellular and tissue repair and elucidating the role of PBM with low-level laser as a possible new therapy in pathologies in COVID-19-associated cytokine storm syndrome from a zebrafish model. Our results demonstrate new strategies for treating SARS-COV-2 using PBM to modulate the expression of the genes and metabolites involved in inflammatory processes. These metabolic alterations show that the r-Spike led to disturbance in the energetic and inflammatory system, corroborating with the severe clinical conditions of human patients. Furthermore, PBM decreased the gene expression levels of pro-inflammatory cytokines such as il1b , il6 , tnfa , and nfkbiab , and of factors involved in oxidative stress ( romo1 ) and energy metabolism ( slc2a1a, coa1 ), in various tissues, promoting an anti-inflammatory response. In summary, our study suggests that PBM may have a positive role in treating cytokine storm syndrome associated with COVID-19. PBM can significantly regulate the inflammatory response promoting cellular and tissue repair of injured tissues. This work suggests that PBM may have a positive role in treating COVID-19-associated cytokine storm syndrome. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials.

Abstract Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.

Shortly after the onset of implantation, polar mouse trophoblast cells proliferate and give rise to the ectoplacental cone, constituted by two distinct cell populations: undifferentiated, diploid cells and giant cells. Giant cells characteristically exhibit exaggerated dimensions and polyploid nuclei. In this study, we employ ectoplacental cones as a dynamic source of trophoblast giant cells to analyze cell proliferation, cell death, and ploidy under in vitro conditions. Our results show that DNA synthesis and the increase in the cell number are relevant only during the first 24 h of culture. Subsequently, DNA synthesis still occurs, mainly in the giant cell compartment, while the number of cells gradually decreases. Cell death by injury and apoptosis was also observed in the non-giant cell compartment of the ectoplacental cone. These findings suggest that the first 24 h of culture are crucial to the mitotic activity of the ectoplacental cone cells that gradually ceases, favoring the endoreduplication process. The DNA synthesis index during the subsequent experimental intervals emphasizes accumulation of DNA for the polyploidization. There was clear correlation between DNA content and nuclear dimension. The ploidy values for the trophoblast giant cells varied from 2C up to 368C in the giant cells, but were not as expressive as those known from in vivo conditions, probably due to the absence of regulatory factors specific to the embryonic-maternal interface. In situ hybridization and histochemistry for the nucleolus-organizing region showed that trophoblast nuclei have only two marker signals, indicative of a typical polytenic process. This present study elucidates important aspects of trophoblast behavior and provides new information on trophoblast physiology in vivo and in vitro.