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Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.

The present study tested the effects of a newly identified indolin-3-one compound (compound 1), produced by Pseudomonas aeruginosa, on HepG2 cells. The MTT assays demonstrated decreased metabolic activities in HepG2 cells treated with compound 1, with dose- and time-dependent intensifying effect, starting at a concentration of 40 µM. The IC50 after 24, 48, 72, and 96 h treatments were 41.35, 52.7, 92.79 and 66.65 μM of compound 1, respectively. Below 80 µM, no significative damage on erythrocytes membranes was observed by the hemolytic assays. The RT-qPCR revealed that the compound modulated key genes involved in carcinogenesis process, indicating possible indolin-3-one mechanisms of action. The data showed that gene expression alterations promoted by compound 1, in concentrations up to 60 μM after 48 h, led to a decrease in cellular progression and there was no direct cellular damage. In addition, non-cytotoxic concentrations of compound 1 halved the concentration of the chemotherapeutic doxorubicin, maintaining similar therapeutic effect against HepG2 cells. The novelty of the molecule and the biological activities observed in the present study emphasize the potential of the compound 1 in cancer therapy research.

The goal of this work was to design specific cyp1a primers for the fish Prochilodus lineatus to study the expression of this gene and its relation to the activity of biotransformation phase I enzyme (ethoxyresorufin O-deethylase - EROD) and genotoxic damage after 6 and 24 h of benzo(a)pyrene (B(a)P) intraperitoneal injection. In comparison to fish injected only with canola oil (vehicle), the expression of cyp1a and EROD activity both in the liver and gills were significantly higher after 6 and 24 h of B(a)P injection. A significant increase in DNA damage was detected in liver and blood cells after 6 h of B(a)P injection and in the gill cells after both times, probably caused by intermediate metabolites of B(a)P. Thus, the expression of cyp1a and its relationship with the corresponding enzyme activity is a potential biomarker for evaluation P. lineatus exposure to organic compounds.

Made available in DSpace on 2018-12-11T17:16:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Bauhinia holophylla (Bong.) Steud. (Fabaceae) is a plant used in Brazilian folk medicine to treat diabetes and inflammation. This study evaluated the phytochemical properties, cytotoxic, apoptotic, mutagenic/antimutagenic effects and alterations in gene expression (RNAm) in HepG2 cells treated with the B. holophylla extract. The phytochemical profile highlight the presence of flavonoids isorhamentin and quercetin derivates. The MTT assay was used to evaluate the cytotoxicity of different concentrations for different treatment times. Three concentrations (7.5, 15, 30 µg/mL) were chosen for assessment of apoptosis (AO/EB), mutagenicity (micronucleus), and cell cycle kinetics (flow cytometry). Thereafter, the concentration of 7.5 µg/mL was chosen to evaluate the protective effects against DNA damage induced by benzo[a]pyrene (B[a]P). At concentrations higher than 7.5 µg/mL (between 10 and 50 µg/mL), the extract was cytotoxic, induced apoptosis, and caused antiproliferative effects. However, it did not induce micronucleus and a reduction of apoptotic and micronucleated cells was observed in treatments that included the extract and B[a]P. The protective effect is attributable to the presence of flavonoids, described as antioxidants, inhibitors of DNA adduct and activators of detoxifying enzymes. The results of the present study such as absence of cytotoxic and mutagenic effects and protective effects against known carcinogens suggest that B. holophylla has potential for use soon as herbal medicine. Department of General Biology Biological Sciences Center State University of Londrina - UEL, Rod Celso Garcia Cid PR 445, Km 380, University Campus, P.O. Box 6001 Department of Biological Sciences Faculty of Pharmaceutical Sciences of Araraquara São Paulo State University - UNESP Campus Litoral Paulista São Paulo State University - UNESP Department of Biological Sciences Faculty of Sciences of Bauru São Paulo State University – UNESP Department of Botany Biosciences Institute São Paulo State University - UNESP Institute of Biological Investigation Clemente Estable Department of Biological Sciences Faculty of Pharmaceutical Sciences of Araraquara São Paulo State University - UNESP Campus Litoral Paulista São Paulo State University - UNESP Department of Biological Sciences Faculty of Sciences of Bauru São Paulo State University – UNESP Department of Botany Biosciences Institute São Paulo State University - UNESP FAPESP: 2009/16147-5 FAPESP: 2009/52237-9 FAPESP: 2012/01996-0

There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-β-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor.

The β-glucans (β-G) are polysaccharides produced by various organisms, and sulfation of β-G renders them more soluble. With the objective to assess the effects of sulfated and non-sulfated β-G extracted from Agaricus brasiliensis in MCF-7 cells, assays were used to evaluate cytotoxicity, genotoxicity, cell proliferation and mRNA expression. The sulfated and non-sulfated β-G showed dose-dependent cytotoxicity at concentrations of 5 and 10 μg/mL, by the MTT assay. However, only cytotoxicity was observed after 24 h by the Red Neutral test for sulfated β-G, with no genotoxicity for either β-G in comet assay. Proliferation was decreased only at 72 h at a concentration of 100 μg/mL of sulfated β-G. Treatment with 5 μg/mL of sulfated β-G for 6 h reduced the expression of pro-apoptotic genes and stress signaling genes, cell cycle arrest, damage and cell migration. The 5 μg/mL of non-sulfated β-G for 6 h reduced the expression of the stress response gene and signaling damage. These results indicate that the cytotoxicity in the MTT is not cell death, and that, in general, sulfated β-G have greater cytotoxicity compared to non-sulfated β-G.

Made available in DSpace on 2018-11-26T17:39:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-25 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Context: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood. Objective: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN-and ISA-induced expression of ERCC1 or BAX genes. Materials and methods: HeLa and/or CHO-K1 cell lines were tested (3 or 24 h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72 h) tests after treatment with IRN (0.1 to 200 mu M) or ISA (0.5 to 50 mu M). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24 h) by gavage (50, 100 and 150 mg/kg determined from the LD50 - 1 g/kg b.w.) and submitted to comet assay in vivo. Results: IRN reduced the viability of CHO-K1 (24 h; 5 to 200 mu M) and HeLa cells (10 to 200 mu M), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 mu M; HeLa: 5 and 10 mu M). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24 h) at all doses tested. IRN (100 and 150 mg/kg) also induced genotoxicity in vivo (4 h). Conclusion: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations. Univ Estadual Londrina, Ctr Biol Sci, Dept Gen Biol, Celso Garcia Cid Rd,PR 445 Km 380, BR-86057970 Londrina, Parana, Brazil Sao Paulo State Univ, Araraquara Inst Chem, Araraquara, Brazil Sao Paulo State Univ, Araraquara Fac Pharmaceut Sci, Dept Biol Sci, Araraquara, Brazil Sao Paulo State Univ, Expt Campus Paulista Coast, Sao Vicente, Brazil Yale Sch Med, Abraham Ribicoff Res Facil, New Haven, CT USA Sao Paulo State Univ, Araraquara Inst Chem, Araraquara, Brazil Sao Paulo State Univ, Araraquara Fac Pharmaceut Sci, Dept Biol Sci, Araraquara, Brazil Sao Paulo State Univ, Expt Campus Paulista Coast, Sao Vicente, Brazil

There is a lack of consensus on whether the role of folate in cancer cells is protective or harmful. The use of folates in combination with cancer-targeting therapeutic regimens requires detailed information to ensure their safe and proper use. Therefore, we evaluated the effects of folic acid (FA) in combination with the chemotherapeutic compounds doxorubicin (DXR), camptothecin (CPT) and methyl methanesulfonate (MMS) on the growth of MCF-7 cells. The data generated from the RTCA assays demonstrated that FA did not affect proliferation in MCF-7 cells treated with DXR and CPT; however, FA reduced the efficacy of MMS treatment. RTCA data also confirmed that DXR and CPT exert their cytotoxic effects in a time-dependent manner and that CPT induced a significantly greater decrease in MCF-7 cell proliferation compared with DXR. The MTT assay failed to detect a reduction in cell proliferation in cells treated with MMS. We quantified the mRNA expression levels of genes associated with cellular stress response, cell cycle and apoptosis pathways using RT-qPCR. The addition of FA to DXR or CPT promoted a similar shift in the gene expression profile of MCF-7 cells compared with cells treated with DXR or CPT without FA; however, this shift did not alter the bioactivity of these drugs. Rather, it indicated that these drugs promoted cell death by alternative mechanisms. In contrast, the addition of FA to MMS reduced the efficacy of the drug without changing the gene expression profile. None of the genes encoding folate receptors that were analyzed were differentially expressed in cells treated with or without FA. In conclusion, supplementation with 450 μM FA was not cytotoxic to MCF-7 cells. However, the addition of FA to anti-cancer drugs must be performed cautiously as the properties of FA might lead to a reduction in drug efficacy. Keywords: Folate, Breast cancer, Cytotoxicity, RTCA, Impedance, qPCR

Candida parapsilosis is found frequently as commensal organism on epithelial tissues, and is also an increasing cause of nosocomial infection. Scanning electron microscope (SEM) observations were used to analyse the capability of C. parapsilosis cells to adhere and grow as biofilm on human natural substrates and to compare the adherence pattern of isolates exhibiting distinct phenotypes. Cells from the crepe phenotype are predominantly elongated and form pseudohyphae whereas cells from the smooth phenotype are yeast-shaped, either in liquid cultures or on human nail and hair surfaces. The electron micrographs revealed that C. parapsilosis cells from the smooth phenotype adhered in higher number to both surfaces compared to the observed for the crepe phenotype. SEM analysis of human hair surface revealed that cells from the smooth phenotype appear as clumped blastoconidia of uniform morphology embedded in a flocculent extracellular material forming biofilm. The extracellular material and biofilm were seeing in a less extension in the crepe phenotype. A distinct adherence pattern was observed when human nail was used as substrate. Here C. parapsilosis cells seem to be linked to surface structures of human nail plate. Fibrillar extracellular material was observed connecting neighbouring cells as well as nail surface.

INTRODUÇÃO: Leveduras do gênero Candida são responsáveis pela maioria das infecções fúngicas em humanos. Candida tropicalis tem sido uma das mais comumente isoladas dentre as espécies não-albicans. O objetivo foi analisar a hemólise in vitro promovida por isolados clínicos de C. tropicalis provenientes de sangue e outras amostras clínicas de pacientes internados no Hospital Universitário da UEL, PR-Brasil. MÉTODOS: Foi avaliada a hemólise promovida por 28 isolados clínicos de C. tropicalis, sendo os isolados agrupados em classes de acordo com os níveis de hemólise. RESULTADOS: A maioria dos isolados de sangue apresentou hemólise fraca (+), enquanto as classes de hemólise forte (+++) e muito forte (++++) foram as predominantes nos isolados de outras amostras clínicas como urina, lesão de unha e secreção traqueal, embora não tenham sido detectadas diferenças estatísticas (p>0,05). CONCLUSÕES: Isolados de C. tropicalis, obtidos de diferentes amostras clínicas, apresentam capacidade de promover hemólise in vitro.

Summary The aim of this study was to determine in vitro haemolytic and protease activities of Candida parapsilosis and Candida tropicalis isolates, obtained from anatomically distinct sites. Analysis of haemolytic activity of C. parapsilosis and C. tropicalis isolates obtained from the same anatomic site revealed that C. tropicalis isolates from blood had statistically higher activity (P

A candidíase, principal infecção fúngica do ser humano, é provocada por leveduras do gênero Candida, que fazem parte da microbiota endógena e exógena do corpo humano. O objetivo deste trabalho foi avaliar a presença das espécies de Candida spp, em diversos sítios anatômicos. Um total de 90 amostras clínicas foram isoladas em ágar Sabouraud contendo antibióticos e identificadas em meio cromogênico CHROMagar Candida ®. Deste total, 58 amostras foram provenientes de secreção vaginal, 17 de raspado de unha, 8 de escarro e 7 de raspado de pele. Das amostras de secreção vaginal, 89% corresponderam a espécie C. albicans, seguido de espécies de Candida não-albicans, sendo 5,1% de C. krusei e 1,7% de C. glabrata, C. tropicalis e Cândida sp. , individualmente. Para as amostras isoladas de raspado de pele não foi observada prevalência de C. albicans (28%). As espécies Candida não-albicans corresponderam a 70% das amostras, sendo distribuídas em C. glabrata (14%), C. krusei (28%) e demais espécies de Candida (28%). A prevalência de espécies Candida não-albicans foi também observada para as amostras isoladas de raspado de unha, sendo que 29% foram caracterizadas como C. glabrata, 23% C. krusei e Cândida sp. , individualmente e 11% de C. tropicalis. Amostras de C. albicans, isoladas neste sítio anatômico, representaram somente 11%. Em amostras obtidas de escarro foi observada somente duas espécies, com prevalência de C. albicans (75%) seguida de C. tropicalis (25%). As amostras identificadas como C. albicans em meio CHROMagar Candida ® , foram confirmadas quanto ao crescimento a 42ºC e pelo emprego da técnica de seminested PCRCandidiasis is the main human fungal infection caused by yeasts of the genus Candida which are part of endogenous microflora of the human body. The aim of this study was to analyze the incidence of Candida species obtained from different infection processes. A total of 90 clinical samples were isolated in Sabouraud agar supplemented with antibiotics, and identified by CHROMagar Candida ®. Among them, 58 samples were isolated from vaginal secretion, 17 samples were from nail, 8 were from sputum and 7 were from skin. From the former, 89% were identified as C. albicans, followed by non albicans species, being 5.1% C. krusei, 1.7% C. glabrata, 1.7% C. tropicalis and 1.7% Candida sp. For samples isolated from nail it was not observed prevalence of C. albicans (28%); species nonalbicans corresponded to 70% of the samples, being 14% C. glabrata, 528% C. krusei and 28% Candida sp. The prevalence of non albicans species was also observed from samples isolated from nail, being 29% C. glabrata, 23% C. krusei, 23% Candida sp and 11% C. tropicalis. C. albicans in this infection site represented only 11%. Only two species were present in samples from sputum, being 75% C. albicans and 25% C. tropicalis. Samples identified as C. albicans in CHROMagar Candida ® agar were confirmed by growth at 42ºC and by seminested PCR approach.

There have been reports of genetic effects affecting the metabolism of Hg and Pb individually, and thus modulating their toxicities. However, there is still a knowledge gap with respect to how genetics may influence the toxicities of these toxic metals during a co-exposure scenario. This present study is therefore aimed at investigating the effects of polymorphisms in genes (GSTM1, GSTT1, GSTP1, GCLM, GCLC, GPx1, ALAD, VDR and MDR1) that have been implicated in Hg and Pb metabolisms affects the kinetics of these metals, as well as various blood antioxidant status parameters: MDA and GSH, and the activities of CAT, GPx and ALAD among populations that have been co-exposed to both Hg and Pb. Study subjects (207 men; 188 women) were from an Amazonian population in Brazil, exposed to Hg and Pb from diet. The blood levels of Hg and Pb were determined by ICP-MS while genotyping were performed by PCR assays. The median values of Hg and Pb in blood were 39.8µg/L and 11.0µg/dL, respectively. GSTM1, ALAD and VDR polymorphisms influenced Hg in blood (β=0.17; 0.37 and 0.17; respectively, p

This study aims to evaluate the effects of polymorphisms in glutathione (GSH-) related genes (GSTM1,GSTT1,GSTP1,GCLM, andGCLC) in the distribution of Hg in the blood compartments in humans exposed to methylmercury (MeHg). Subjects (n=88), exposed to MeHg from fish consumption, were enrolled in the study. Hg species in the plasma compartment were determined by LC-ICP-MS, whereas genotyping was performed by PCR assays. Mean total Hg levels in plasma (THgP) and whole blood (THgB) were10±4.2and37±21, whereas mean evels of plasmatic MeHg (MeHgP), inorganic Hg (IHgP), and HgP/HgB were4.3±2.9,5.8±2.3 µg/L, and0.33±0.15, respectively.GSTM1andGCLCpolymorphisms influence THgP and MeHgP (multivariate analyses,P<0.050). Null homozygotes forGSTM1showed higher THgP and MeHgP levels compared to subjects withGSTM1(THgPβ=0.22,P=0.035; MeHgPβ=0.30,P=0.050) and persons carrying at least one T allele forGCLChad significant higher MeHgP (β=0.59,P=0.046). Also, polymorphicGCLMsubjects had lower THgP/THgB than those with the nonvariant genotype. Taken together, data of this study suggest that GSH-related polymorphisms may change the metabolism of MeHg by modifying the distribution of mercury species iin plasma compartment and the HgP/HgB partitioning.

Leveduras do gênero Candida estão entre os principais patógenos causadores de infecções nosocomiais em todo o mundo. Desta forma, este trabalho teve como objetivo estabelecer o perfil epidemiológico da candidúria no HU de Londrina/PR entre os anos de 2006 e 2007, classificar a extensão da candidúria em diferentes níveis e identificar fatores de risco específicos para o isolamento de espécies de Candida não-albicans. Foram incluídos no estudo isolados clínicos de Candida spp obtidos de indivíduos urocultura positivos cuja detecção foi ≥ 102 UFC mL-1. Também foi realizada a análise comparativa ultraestrutural da interação de diferentes morfotipos de C. parapsilosis com a superficie de unha e cabelo humanos pelo emprego da microscopia eletrônica de varredura (MEV) e a avaliação da expressão dos genes SAPs de 15 isolados clínicos de C. tropicalis em resposta aos substratos BSA e hemoglobina pela técnica de PCR em tempo real. Um total de 157 isolados de Candida foi obtido, incluindo C. albicans (n=72), C. tropicalis (n=54), C. glabrata (n=20), C. parapsilosis (n=8), C. krusei (n=2) e C. dubliniensis (n=1). A extensão da candidúria foi classificada em tres diferentes níveis: nível 1 (candidúria leve), nível 2 (candidúria de risco) e nível 3 (candidúria pesada). Houve diferença significativa entre a freqüência de isolamento de C. albicans e espécies não-albicans em relação aos diferentes níveis de candidúria estabelecidos neste estudo, evidenciando a candidúria nível 3 (candidúria pesada) como um fator de risco independente para o isolamento de espécies não-albicans. A MEV revelou que as células do fenótipo ―smooth‖ de C. parapsilosis aderem em maior número em ambas as superfícies em relação aos observados para o fenótipo ―crepe‖. Diferentes perfis na qualidade e quantidade de produção de matriz extracelular entre os dois morfotipos de C. parapsilosis também foram observados. A avaliação da expressão dos genes SAPs de C. tropicalis mostrou que as condições de cultivo testadas proporcionaram a indução significativa dos genes SAPT1, SAPT2 e SAPT3 em relação à situação controle (ausência de substrato), cujo perfil de expressão variou qualitativa e quantitativamente de acordo com o substrato utilizado e os diferentes isolados. Pode-se concluir que a contagem de UFC mL-1 de urina e subseqüente classificação da extensão da candidúria em diferentes níveis pode ser utilizada como marcador de isolamento de espécies de Candida não-albicans. Conclui-se também que diferentes substratos influenciam a expressão diferencial de genes relacionados à morfologia, adesão e produção de matriz extracelular de C. parapsilosis, assim como genes de virulência (SAPs) de C. tropicalis, respostas estas que também se mostraram linhagem-dependente, não relacionadas ao sítio de isolamento. Candida species are the major pathogens causing nosocomial infections worldwide. Thus, this study aimed to establish the epidemiological profile of candidúria at UH of Londrina/PR between the years 2006 and 2007, to classify the extent of candiduria at different levels and to identify specific risk factors for the isolation of Candida non-albicans species. The study included clinical isolates of Candida obtained from patients positive urine cultures whose detection was ≥ 102 CFU mL-1. We also performed comparative ultrastructural analysis of the interaction of two different morphotypes of C. parapsilosis with the surface of human hair and nail by the use of scanning electron microscopy (SEM) and the evaluation of SAPs gene expression of 15 clinical isolates of C. tropicalis in response to BSA and hemoglobin substrates by real time PCR. A total of 157 Candida isolates were obtained, including C. albicans (n = 72), C. tropicalis (n = 54), C. glabrata (n = 20), C. parapsilosis (n = 8), C. krusei (n = 2) and C. dubliniensis (n = 1). The extent of candiduria was classified into three different levels: Level 1 (light candiduria), level 2 (risk candiduria) and Level 3 (heavy candiduria). There was a significant difference between the frequency of isolation of C. albicans and non-albicans species in relation to different levels of candiduria established in this study, showing candiduria level 3 (heavy candidúria) as an independent risk factor for the isolation of non-albicans species. The electron micrographs revealed that cells of the"smooth" phenotype of C. parapsilosis adhered in greater numbers in both areas compared to those observed for the phenotype "crepe." Different profiles in quality and quantity of extracellular matrix production between the two morphotypes of C. parapsilosis was also observed. The assessment of the SAPs genes expression in C. tropicalis showed that growing conditions tested resulted in significant induction of genes SAPT1, SAPT2 and SAPT3 in relation to the control situation (absence of substrate), whose expression profiles varied qualitatively and quantitatively according to the substrate and the different isolates. It can be concluded that the number of CFU mL-1 of urine and subsequent classification of the extent of candiduria at different levels can be used as an isolation marker of Candida non-albicans species. We also conclude that different substrates influence the differential expression of genes related to morphology, adhesion and extracellular matrix production of C. parapsilosis, as well as virulence genes (SAPs) of C. tropicalis, and that these responses were also strain dependent, not related to the anatomic site of isolation.

Candida tropicalis has been identified as one of the most prevalent pathogenic yeast species of the Candida-non-albicans (CNA) group. Study of switching in C. tropicalis has not been the subject of extensive research. Therefore, we investigated switching event and characterized the ultrastructural architecture of different phenotypes and biofilm produced in a C. tropicalis clinical strain. Cells switched heritably, reversibly, and at a high frequency between four phenotypes readily distinguishable by the shape of colonies formed on agar at 25°C. SEM analysis was used to verify the architecture of whole Candida colonies at ultrastructural level. The smooth phenotype (parental phenotype) colony showed a hemispherical shape character, while the semi-smooth was characterized by the presence of shallow marginal depressions. The ring and rough phenotypes exhibited more complex architecture and were characterized by the presence of deep central and peripheral depressions areas. The biofilm-forming ability varied among the switch phenotypes. After 12h incubation, the smooth phenotype formed less biofilm compared to the other phenotypes (P0.05). The electron microscopy analysis revealed that filamentation (pseudohyphae) was associated with ring and rough colonies. The ultrastructural analysis allowed the observation of the arrangement of individual cells within the colonies. At the deep central and peripheral depressions areas of the ring and rough colonies extracellular material was seen in different arrangements. The data presented here open new avenues to study a possible role for extracellular material in the formation and maintenance of the architecture of switch phenotypes in C. tropicalis. It is therefore essential that more strains be investigated to determine the biological significance of extracellular material in C. tropicalis phenotypic switching phenomenon.

Yeasts belonging to the genus Candida are responsible for the majority of fungal infections in humans. Candida tropicalis has been one of most commonly isolated non-albicans species. To analyze in vitro hemolysis promoted by clinical isolates of C. tropicalis obtained from blood and other clinical samples from hospitalized patients at the University Hospital of Londrina State University, Paraná, Brazil.The hemolysis promoted by 28 clinical isolates of C. tropicalis was evaluated, and the isolates were grouped into classes according to the hemolysis levels.The majority of the blood isolates showed weak hemolysis (+), while the classes of strong hemolysis (+++) and very strong hemolysis (++++) predominated among isolates from other clinical samples such as urine, nail lesions and tracheal secretions. However, no statistical differences were detected (p0.05).Isolates of C. tropicalis obtained from different clinical samples showed a capacity to promote in vitro hemolysis.

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

As indolinonas são moléculas farmacologicamente importantes, conhecidas por sua atividade inibidora de proteínas quinases, exibindo efeitos promissores no tratamento do câncer e outras doenças. Apesar das diferentes abordagens para a terapia e prevenção do câncer, esta doença continua a ser uma das principais causas de morte no mundo. Seus tratamentos atuais apresentam efeitos indesejados, fazendo com que novos compostos e terapias sejam necessários. A fim de contribuir para a solução deste problema, o presente estudo visou avaliar o potencial citotóxico, antiproliferativo e genotóxico de uma nova indolin-3-ona, assim como seu efeito na expressão de genes relacionados ao câncer. O composto apresentou efeito citotóxico sobre eritrócitos humanos apenas a partir da concentração de 32 µg/mL nos tempos de 24, 48, 72 e 96 horas de tratamento. Foi observado que os efeitos citotóxicos e antiproliferativo da indolin-3-ona sobre a linhagem celular HepG2, mensurados pelo ensaio do MTT, foram dose-tempo-dependentes, se intensificando de acordo com a variável em questão. O ensaio do cometa mostrou que a indolin-3-ona possui atividade genotóxica nas concentrações de 16 e 24 µg/mL, contudo, este ensaio detecta danos primários ao DNA que ainda podem ser reparados pelo sistema de reparo de DNA da célula. A partir dos ensaios de RT-qPCR foi possível observar que a indolin-3-ona modula a expressão de genes envolvidos no processo de carcinogênese, reprimindo a expressão gênica, principalmente de proto-oncogenes, também de forma dose-tempo-dependente. Estes resultados apontam para a indicação desta indolinona como uma nova molécula com potencial utilização na terapia do câncer. The indolinones are pharmacologically important molecules known for their inhibitory activity of protein kinases, showing promising effects in treatment of cancer and other diseases. Despite the different approaches for therapy and prevention of cancer, it remains a major cause of death worldwide. Its current treatments have unwanted effects, making new compounds and therapies needed. In order to solve this problem, the present study aimed to evaluate the cytotoxic, antiproliferative and genotoxic potential of a new indolin-3-one and its effect on expression of genes related to cancer. The compound showed cytotoxic effect on human erythrocytes only at concentrations above 32 µg/mL at times of 24, 48, 72 and 96 hours of treatment. The indolinone cytotoxic and antiproliferative effects on HepG2 cells, measured by the MTT assay, were dose-time-dependent, intensifying according to the variable in question. The comet assay revealed that indolinone has genotoxic activity at concentrations of 16 and 24 µg/mL, however, this assay detects only primary DNA damages, which can still be repaired by the DNA repair system of the cell. In RT-qPCR assays we could observe that indolinone modulates the expression of genes involved in the carcinogenesis process, suppressing the gene expression, specially of proto-oncogenes, also in a dose-time-dependent manner. These results point to the indication of this indolinone as a new molecule with potential use in cancer therapy.

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