Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Cruz, Deu John M.,Bonotto, Rafaela M.,Gomes, Rafael G. B.,da Silva, Camila T.,Taniguchi, Juliana B.,No, Joo Hwan,Lombardot, Benoit,Schwartz, Olivier,Hansen, Michael A. E.,Freitas-Junior, Lucio H. Public Library of Science 2013 PLoS neglected tropical diseases Vol.7 No.10

<▼1><P>Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC<SUB>50</SUB> values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.</P></▼1><▼2><P><B>Author Summary</B></P><P>Recent outbreaks and expanding global distribution of Chikungunya virus (CHIKV) in different regions of Asia, Africa and Europe necessitates the development of effective therapeutic interventions. At present, only two antiviral compounds (chloroquine and ribavirin) that inhibit viral infection <I>in vitro</I> have been used in clinical cases of chikungunya infections. However, neither of these compounds have shown strong efficacy in vivo. Recent attempts to identify new antiviral candidates for CHIKV using cell-based phenotypic approach have been reported. In this study, we developed a simple cell-based high-throughput assay using resazurin to identify potential anti-CHIKV compounds. This high-throughput assay is based on the metabolic reduction of resazurin to the highly fluorescent resorufin by viable cells as an indicator of activity against CHIKV-induced CPE. We screened 4,000 small molecules belonging to the BioFocus kinase inhibitor chemical library and found a cluster of related molecules with antiviral activity against CHIKV. Finally, we characterized the putative mode of action of these active compounds using an image-based high content assay and conventional virological methods (<I>i.e.</I>, virus yield reduction assay, microneutralization assay).</P></▼2>

Susceptibility of Mice to Trypanosoma evansi Treated with Human Plasma Containing Different Concentrations of Apolipoprotein L-1

Aleksandro S. Da Silva,Vinicius R. Fanfa,Mateus A. Otto,Lucas T. Gressler,Kaio C.S. Tavares,Cicera R. Lazzarotto,Alexandre A. Tonin,Luiz C. Miletti,Marta M. M. F. Duarte,Silvia G. Monteiro 대한기생충학열대의학회 2011 The Korean Journal of Parasitology Vol.49 No.4

The aim of this study was to test the susceptibility of mice to Trypanosoma evansi treated with human plasma containing different concentrations of apolipoprotein L-1 (APOL1). For this experiment, a strain of T. evansi and human plasma (plasmas 1, 2, and 3) from 3 adult males clinically healthy were used. In vivo test used 50 mice divided in 5 groups (A to E) with 10 animals in each group. Animals of groups B to E were infected, and then treated with 0.2 ml of human plasma in the following outline: negative control (A), positive control (B), treatment with plasma 1 (C), treatment with plasma 2 (D), and treatment with plasma 3 (E). Mice treated with human plasma showed an increase in longevity of 40.9±0.3 (C), 20±9.0 (D) and 35.6±9.3 (E) days compared to the control group (B) which was 4.3±0.5 days. The number of surviving mice and free of the parasite (blood smear and PCR negative) at the end of the experiment was 90%, 0%, and 60% for groups C, D, and E, respectively. The quantification of APOL1 was performed due to the large difference in the treatments that differed in the source plasma. In plasmas 1, 2, and 3 was detected the concentration of 194, 99, and 115 mg/dl of APOL1, respectively. However, we believe that this difference in the treatment efficiency is related to the level of APOL1 in plasmas.

Genotoxic Evaluation of Mikania laevigata Extract on DNA Damage Caused by Acute Coal Dust Exposure

Freitas, Tiago P.,Heuser, Vanina D.,Tavares, Priscila,Leffa, Daniela D.,Silva, Gabriela A. da,Citadini-Zanette, Vanilde,Romao, Pedro R.T.,Pinho, Ricardo A.,Streck, Emilio L.,Andrade, Vanessa M. The Korean Society of Food Science and Nutrition 2009 Journal of medicinal food Vol.12 No.3

In the present article, we report data on the possible antigenotoxic activity of Mikania laevigata extract (MLE) after acute intratracheal instillation of coal dust using the comet assay in peripheral blood, bone marrow, and liver cells and the micronucleus test in peripheral blood of Wistar rats. The animals were pretreated for 2 weeks with saline solution (groups 1 and 2) or MLE (100 mg/kg) (groups 3 and 4). On day 15, the animals were anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg), and gross mineral coal dust (3 mg/0.3mL saline) (groups 2 and 4) or saline solution (0.3 mL) (groups 1 and 3) was administered directly in the lung by intratracheal administration. Fifteen days after coal dust or saline instillation, the animals were sacrificed, and the femur, liver, and peripheral blood were removed. The results showed a general increase in the DNA damage values at 8 hours for all treatment groups, probably related to surgical procedures that had stressed the animals. Also, liver cells from rats treated with coal dust, pretreated or not with MLE, showed statistically higher comet assay values compared to the control group at 14 days after exposure. These results could be expected because the liver metabolizes a variety of organic compounds to more polar by-products. On the other hand, the micronucleus assay results did not show significant differences among groups. Therefore, our data do not support the antimutagenic activity of M. laevigata as a modulator of DNA damage after acute coal dust instillation.

Genotoxic Evaluation of Mikania laevigata Extract on DNA Damage Caused by Acute Coal Dust Exposure

Tiago P. Freitas,Vanina D. Heuser,Priscila Tavares,Daniela D. Leffa,Gabriela A. da Silva,Vanilde Citadini-Zanette,Pedro R.T. Romão,Ricardo A. Pinho,Emilio L. Streck,Vanessa M. Andrade 한국식품영양과학회 2009 Journal of medicinal food Vol.12 No.3

In the present article, we report data on the possible antigenotoxic activity of Mikania laevigata extract (MLE) after acute intratracheal instillation of coal dust using the comet assay in peripheral blood, bone marrow, and liver cells and the micronucleus test in peripheral blood of Wistar rats. The animals were pretreated for 2 weeks with saline solution (groups 1 and 2) or MLE (100mg/kg) (groups 3 and 4). On day 15, the animals were anesthetized with ketamine (80mg/kg) and xylazine (20mg/kg), and gross mineral coal dust (3mg/0.3mL saline) (groups 2 and 4) or saline solution (0.3mL) (groups 1 and 3) was administered directly in the lung by intratracheal administration. Fifteen days after coal dust or saline instillation, the animals were sacrificed, and the femur, liver, and peripheral blood were removed. The results showed a general increase in the DNA damage values at 8 hours for all treatment groups, probably related to surgical procedures that had stressed the animals. Also, liver cells from rats treated with coal dust, pretreated or not with MLE, showed statistically higher comet assay values compared to the control group at 14 days after exposure. These results could be expected because the liver metabolizes a variety of organic compounds to more polar by-products. On the other hand, the micronucleus assay results did not show significant differences among groups. Therefore, our data do not support the antimutagenic activity of M. laevigata as a modulator of DNA damage after acute coal dust instillation.

Thickness effects on the optical properties of layer-by-layer poly(p-phenylene vinylene) thin films and their use in energy-modulated structures

E. Laureto,M.A.T. da Silva,R.V. Fernandes,J.L. Duarte,I.F.L. Dias,A. Marletta 한국물리학회 2012 Current Applied Physics Vol.12 No.3

Poly(p-phenylene vinylene) (PPV) thin films were produced by layer-by-layer (LbL) method, using soluble PPV-precursor and dodecylbenzenesulfonate salt (DBS). The amount of deposited layers strongly influences the optical properties of the thermally converted PPV film. The absorbance and luminescence spectra of ultra-thin films (consisting of only two or three PPV layers) are shifted to smaller wavelengths with respect to spectra of thicker films. This is related to the smaller average conjugation length of polymer chains, resulting in a higher HOMOeLUMO gap energy of the material. However, if a thick film is produced by repeating the deposition process and thermal conversion of ultra-thin layers, the optical spectra are still displaced to higher energies in comparison with those of thicker films produced by the conventional continuous deposition of layers. This result enabled the production of multilayered polymeric films with modulated energy profile, taking the number of deposited layers as the only variable in the manufacturing process of the structure. The aim is to guide the excitation to specific regions of the material through the Förster-type energy transfer processes. Such systems can be used at interfaces electrode/polymer and/or electrode/polymeric active layers in order to improve the performance of organic optoelectronic devices.

Increased Cytokine and Nitric Oxide Levels in Serum of Dogs Experimentally Infected with Rangelia vitalii

Francine C. Paim,Aleksandro S. Da Silva,Carlos Breno V. Paim,Raqueli T. Franca,Marcio M. Costa,Marta M. M. F. Duarte,Manuela B. Sangoi,Rafael N. Moresco,Silvia G. Monteiro,Sonia Terezinha A. Lopes 대한기생충학열대의학회 2013 The Korean Journal of Parasitology Vol.51 No.1

This study aimed to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and nitrite/nitrate (NOx) in serum of dogs experimentally infected with Rangelia vitalii. Twelve female mongrel dogs were divided into 2 groups; group A (uninfected controls) composed by healthy dogs (n=5) and group B consisting of dogs inoculated with R. vitalii (n=7). Animals were monitored by blood smear examinations, which showed intraerythrocytic forms of the parasite on day 5 post-infection (PI). Blood samples were collected through the jugular vein on days 0, 10, and 20 PI to determine the serum levels of IFN-γ, TNF-α, IL-1, IL-6, and NOx. Cytokines were assessed by ELISA quantitative sandwich technique, and NOx was measured by the modified Griess method. Cytokine levels (IFN-γ, TNF-α, IL-1, and IL-6) were increased (P<0.01) in serum of infected animals. Serum levels of NOx were also increased on days 10 PI (P<0.01) and 20 PI (P<0.05) in infected animals. Therefore, the infection with R. vitalii causes an increase in proinflammatory cytokines and nitric oxide content. These alterations may be associated with host immune protection against the parasite.

LEGACY EXTRAGALACTIC UV SURVEY (LEGUS) WITH THE<i>HUBBLE SPACE TELESCOPE</i>. I. SURVEY DESCRIPTION

Calzetti, D.,Lee, J. C.,Sabbi, E.,Adamo, A.,Smith, L. J.,Andrews, J. E.,Ubeda, L.,Bright, S. N.,Thilker, D.,Aloisi, A.,Brown, T. M.,Chandar, R.,Christian, C.,Cignoni, M.,Clayton, G. C.,Silva, R. da,Mi IOP Publishing 2015 The Astronomical journal Vol.149 No.2