Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

INSEL: an in silico method for optimizing and exploring biorecognition assays

A practical in silico method for optimizing and exploring biointeraction-based events is developed.

Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Redox-tagged peptide for capacitive diagnostic assays

Early detection assays play a key role in the successful treatment of most diseases. Redox capacitive biosensors were recently introduced as a potential electroanalytical assay platform for point-of-care applications but alternative surfaces (besides a mixed layer containing ferrocene and antibody receptive component) for recruiting important clinical biomarkers are still needed. Aiming to develop alternative receptive surfaces for this novel electrochemical biosensing platform, we synthesized a ferrocene redoxtagged peptide capable of self-assembly into metallic interfaces, a potentially useful biological surface functionalization for bedside diagnostic assays. As a proof of concept we used C-reactive protein (CRP), as a model biomarker, and compared the obtained results to those of previously reported capacitive assays. The redox-tagged peptide approach shows a limit of detection of 0.8 nmol L 1 (same as 94 ng mL 1 ) and a linear range (R2 ∼98%) with the logarithm of the concentration of the analyte comprising 0.5–10.0 nmol L 1 , within a clinical relevant range for CRP.

Using the comet and micronucleus assays for genotoxicity studies: a review

Physical, chemical and biological agents can act in the DNA, resulting in mutation involved in cancer. Thus, genotoxic tests are required by regulatory agencies in order to evaluate potential risk of cancer. Among these tests, the comet assay (CA) and micronucleus assay (MNA) are the most commonly used. However, there are different protocols and recommendations already published. This is the first review, after the inclusion of CA in S2R1 guidance and OECD 489, which summarizes the main technical recommendations of both CA and MNA.

Evaluation of Blood Assays for Detection of Mycobacterium bovis in White-Tailed Deer (Odocoileus virginianus) in Michigan

Surveillance and control activities related to bovine tuberculosis (TB) in free-ranging, Michigan white-tailed deer (Odocoileus virginianus) have been underway for over a decade, with significant progress. However, foci of higher TB prevalence on private lands and limited agency ability to eliminate them using broad control strategies have led to development and trial of new control strategies, such as live trapping, testing, and culling or release. Such strategies require a prompt, accurate live animal test, which has thus far been lacking. We report here the ability of seven candidate blood assays to determine the TB infection status of Michigan deer. Our aims were twofold: to characterize the accuracy of the tests using field-collected samples and to evaluate the feasibility of the tests for use in a test-and-cull strategy. Samples were collected from 760 deer obtained via five different surveys conducted between 2004 and 2007. Blood samples were subjected to one or more of the candidate blood assays and evaluated against the results of mycobacterial culture of the cranial lymph nodes. Sensitivities of the tests ranged from 46% to 68%, whereas specificities and negative predictive values were all .92%. Positive predictive values were highly variable. An exploratory analysis of associations among several host and sampling-related factors and the agreement between blood assay and culture results suggested these assays were minimally affected. This study demonstrated the capabilities and limitations of several available blood tests for Mycobacterium bovis on specimens obtained through a variety of field surveillance methods. Although these blood assays cannot replace mass culling, information on their performance may prove useful as wildlife disease managers develop innovative methods of detecting infected animals where mass culling is publicly unacceptable and cannot be used as a control strategy.

Biomonitoring of Microcystin and Aflatoxin Co-Occurrence in Aquaculture Using Immunohistochemistry and Genotoxicity Assays

This work investigated the effects of co-occurring aflatoxin B-1 (AFB(1)) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB(1) by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB(1) was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB(1) showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI = 0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation. Published by Elsevier B.V.

Biomonitoring of microcystin and aflatoxin co-occurrence in aquaculture using immunohistochemistry and genotoxicity assays

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

The complement system of the goat: haemolytic assays and isolation of major proteins

[EN] Background: The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats (Capra aegagrus hircus) and to characterize the major goat complement system proteins. Results: The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC) plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone) was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H. Conclusions: Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

Development of screening assays to test novel integrin antagonists in allergic inflammation

Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action. Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils. Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

Quantitative multiparameter assays to measure the effect of adjuvants on human antigen-specific CD8 T-cell responses

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized.