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Las macrófitas acuáticas han sido utilizadas en procesos de biorremediación, ya que pueden absorber algunas sustancias disueltas y producir oxígeno mediante fotosíntesis. Con base en los estudios de remoción de compuestos tóxicos por parte de plantas acuáticas que permitan la depuración de aguas residuales, la presente investigación tuvo como propósito principal buscar la disminución de la contaminación por cianuro y la toxicidad de las aguas residuales del lavado de arena del proceso de cianuración de oro de la mina La Coqueta, mediante la utilización de macrófitas acuáticas flotantes (Eichhornia crassipes,Pistia stratiotesSalvinia auriculata); además, de acuerdo con los resultados de pruebas cualitativas, encontrar el destino final del cianuro en las plantas al ser absorbidos por éstas. El tratamiento de agua cianurada se realizó mediante un sistema de acuarios, los cuales contenían el agua proveniente de la mina, en ellos se depositaron las plantas para el tratamiento, se tomaron parámetros fisicoquímicos de control (pH, porcentaje de saturación de oxígeno y conductividad), además de la determinación de cianuro y toxicidad en el agua, pruebas cualitativas de compuestos nitrogenados en las plantas y bioensayos conDaphnia pulexen las plantas y en el agua tratada. Con base en los resultados de las pruebas cualitativas, se evidenció que las macrófitas pueden cambiar su metabolismo en condiciones de estrés y este cambio de metabolismo induce también a que se produzcan otro tipo de metabolitos secundarios no presentes naturalmente (e. g. glicósidos cianogénicos y alcaloides) en las plantas sometidas al tratamiento.La remoción de cianuro por parte de estas especies fue hasta del 86% al final del tratamiento, demostrando gran eficiencia para remover compuestos tóxicos del agua cianurada proveniente de la mina La Coqueta, ya que en los bioensayos realizados conDaphnia pulexindicaron que estos organismos podían resistir a las características del agua después del tratamiento con las plantas. Señalando que se puede realizar un tratamiento natural, ambientalmente seguro y económicamente viable que permita descontaminar el agua cianurada proveniente del lavado de las arenas del beneficio del oro a través del proceso de cianuración. Aquatic macrophytes have been used in bioremediation processes, as they can absorb some dissolved substances and produce oxygen through photosynthesis. Based on the studies of toxic compounds removal by aquatic plants that allow wastewater treatment, this research had as main purpose to search the reduction of cyanide contamination and toxicity of waste water from sand washing in the cyanidation process inLa Coquetagold mine by means of the use of floating aquatic macrophytes (Eichhornia crassipes, Salvinia auriculata, Pistiastratiotes). Similarly, according to the results of qualitative tests, a goal was to find the final destiny of cyanide afterplants had absorbed it. Treatment of cyanide water was conducted through a system of tanks containing water from the mine in which plants for treatment were included and physicochemical control parameters (pH, oxygen saturation percentage and conductivity) were taken. Besides determination of cyanide and water toxicity, qualitative testsof nitrogenous compounds in plants and bioassays withDaphnia pulexin plants and in the treated water were determined. Based on the results of qualitative tests ti was evidenced that macrophytes can change their metabolism under stress conditions and this change in metabolism also induces the production of other types of secondary metabolites not present naturally (e.g. cyanogenic glycosides and alkaloids) in plants subjected to treatment. The removal of cyanide by these species was up to 86% at end of treatment, showing high efficiency for removal of toxic compounds of cyanide water fromLa Coquetamine, as bioassays conducted usingDaphnia pulexindicated that these organism could resist water characteristics after treatment with plants, noting that natural treatment environmentally safe and economically feasible can be made to decontaminate cyanide water from the washing of sands resulting from cyanide gold processing.

Acute myeloid leukaemia (AML) is associated with exposure to benzene and treatment with chemotherapeutic agents. It is thought to arise from damage to specific regions of DNA, resulting in chromosome rearrangements or loss. For instance, a deletion on the long arm of chromosome 5 [e.g. del(5q31)] is common in AML patients previously treated with alkylating agents, such as melphalan, or exposed to benzene. Translocations of the MLL gene at 11q23 are frequently observed in AML arising from treatment with topoisomerase II inhibitors, such as etoposide. Our goal was to determine whether or not breakage at 5q31 and 11q23 is selectively induced by these chemical agents. To address this question, the comet assay combined with fluorescence in situ hybridization (comet-FISH) was used to detect DNA breakage in the specific chromosomal regions in an in vitro model. TK6 lymphoblastoid cells were exposed to melphalan, etoposide or the benzene metabolite, hydroquinone (HQ), at various concentrations. HQ, melphalan and etoposide induced DNA breaks at both 5q31 and 11q23 chromosome regions in a dose-dependant manner. However, HQ produced significantly more DNA damage at 5q31 than at 11q23. Etoposide produce slightly more DNA damage at 11q23 and melphalan had a somewhat greater effect at 5q31, but not significantly so. Thus, HQ and melphalan act similarly, perhaps explaining some similarities between benzene- and alkylating agent-induced AML. Comet-FISH also appears to be a useful approach for detecting and comparing damage to specific chromosome regions of significance in leukaemogenesis.

As part of the Fourth International Workshop on Genotoxicity Testing (IWGT), held 9-10 September 2005 in San Francisco, California, an expert working group on the Comet assay was convened to review and discuss some of the procedures and methods recommended in previous documents. Particular attention was directed at the in vivo rodent, alkaline (pH >13) version of the assay. The aim was to review those protocol areas which were unclear or which required more detail in order to produce a standardized protocol with maximum acceptability by international regulatory agencies. The areas covered were: number of dose levels required, cell isolation techniques, measures of cytotoxicity, scoring of comets (i.e., manually or by image analysis), and the need for historical negative/positive control data. It was decided that a single limit dose was not sufficient although the required number of dose levels was not stipulated. The method of isolating cells was thought not to have a qualitative effect on the assay but more data were needed before a conclusion could be drawn. Concurrent measures of cytotoxicity were required with histopathological examination of tissues for necrosis or apoptosis as the "Gold Standard". As for analysing the comets, the consensus was that image analysis was preferred but not required. Finally, the minimal number of studies required to generate a historical positive or negative control database was not defined; rather the emphasis was placed on demonstrating the stability of the negative/positive control data. It was also agreed that a minimum reporting standard would be developed which would be consistent with OECD in vivo genotoxicity test method guidelines.

The genotoxicity of zidovudine (AZT) based treatments was investigated in human H9 lymphoblastoid cells in an in vitro study and in red blood cells (RBCs) from perinatally exposed HIV-1-infected mothers and their infants in an observational cohort study. Exposure of H9 cells for 24 hr to AZT produced dose-dependent increases in Comet assay tail moment (TM) when electrophoresed at pH 13.0, but not at pH 12.1 or pH 8.0, suggesting that DNA damage was via alkali-labile lesions and not double-stranded DNA strand breaks. The TM dose response at pH 13.0 correlated directly with AZT-DNA incorporation determined by AZT-radioimmunoassay. Levels of DNA damage in utero, measured by Comet assay TM, were similar in cord blood mononuclear cells of nucleoside analog-exposed newborns (n = 43) and unexposed controls (n = 40). In contrast, the glycophorin A (GPA) somatic cell mutation assay (which screens for large-scale DNA damage in RBCs) showed clear evidence that GPA N/N variants, arising from chromosome loss and duplication, somatic recombination, and gene conversion, were significantly elevated in mother–child pairs receiving prepartum AZT plus lamivudine (3TC). Cord blood from newborns exposed to AZT-3TC had GPA N/N variant frequencies of 4.7 ± 0.7 (mean ± SE) × 10−6 RBCs (n = 26 infants) compared with 2.2 ± 0.3 × 10−6 RBCs for unexposed controls (n = 30 infants; P < 0.001). Elevations in GPA N/N variants generally persisted through 1 year of age in nucleoside analog-exposed children. Overall, the mutagenic effects found in mother–child pairs receiving AZT-based treatments justify their surveillance for long-term genotoxic consequences. Environ. Mol. Mutagen., 2007. © 2007 Wiley-Liss, Inc.

Cuando hablamos de las “rebeldías de los maestros”, lo hacemos desde el proceso interior del ser educador, nos referimos a aquellas posturas crí-ticas y reveladoras, exigiéndonos transitar por varios escenarios: nuestras autobiografías que han develado las huellas epocales y rebeldías que de una u otra forma se conectan con diferentes historias de maestros; y de acontecimientos transcurridos a partir de la década de los 60, en donde se manifiesta una realidad política, económica, educativa, y religiosa, trans-versalidas por el desarrollo y las experiencias como maestros; el exilio, el silencio, las rebeldías, el revelarse, la desesperanza y el poder, aspectos que marcan el camino recorrido y por recorrer, levantando códigos y ca-tegorías que influyen en el quehacer docente; la innovación, pertinencia y utilidad de la investigación, partiendo del sujeto en relación con los otros, transformándose a sí mismo y a los demás, pero sobre todo, donde surgen nuevas posturas que rompen el silencio, el aquietamiento y la tranquilidad; permitiéndonos a través de nuevos códigos, rebelarnos.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

Alterations in matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have been implicated in adverse left ventricular (LV) remodeling after myocardial infarction (MI). However, the direct mechanistic role of TIMPs in the post-MI remodeling process has not been completely established. The goal of this project was to define the effects of altering endogenous MMP inhibitory control through combined genetic and pharmacological approaches on post-MI remodeling in mice. This study examined the effects of MMP inhibition (MMPi) with PD-166793 (30 mg·kg−1·day−1) on LV geometry and function (conductance volumetry) after MI in wild-type (WT) mice and mice deficient in the TIMP-1 gene [TIMP-1 knockout (TIMP1-KO)]. At 3 days after MI (coronary ligation), mice were randomized into four groups: WT-MI/MMPi ( n = 10), TIMP1-KO-MI/MMPi ( n = 10), WT-MI ( n = 22), and TIMP1-KO-MI ( n = 23). LV end-diastolic volume (EDV) and ejection fraction were determined 14 days after MI. Age-matched WT ( n = 20) and TIMP1-KO ( n = 28) mice served as reference controls. LVEDV was similar under control conditions in WT and TIMP1-KO mice (36 ± 2 and 40 ± 2 μl, respectively) but was greater in TIMP1-KO-MI than in WT-MI mice (48 ± 2 vs. 61 ± 5 μl, P < 0.05). LVEDV was reduced from MI-only values in WT-MI/MMPi and TIMP1-KO-MI/MMPi mice (42 ± 2 and 36 ± 2 μl, respectively, P < 0.05) but was reduced to the greatest degree in TIMP1-KO mice ( P < 0.05). LV ejection fraction was reduced in both groups after MI and increased in TIMP1-KO-MI/MMPi, but not in WT-MI/MMPi, mice. These unique results demonstrated that myocardial TIMP-1 plays a regulatory role in post-MI remodeling and that the accelerated myocardial remodeling induced by TIMP-1 gene deletion can be pharmacologically “rescued” by MMP inhibition. These results define the importance of local endogenous control of MMP activity with respect to regulating LV structure and function after MI.

Recent studies have been directed at modulating the heart failure process through inhibition of activated matrix metalloproteinases (MMPs). We hypothesized that a loss of MMP inhibitory control by tissue inhibitor of MMP (TIMP)-1 deficiency alters the course of postinfarction chamber remodeling and induced chronic myocardial infarction (MI) in wild-type (WT) and TIMP-1−/− mice. Left ventricular (LV) pressure-volume loops obtained from WT and TIMP-1−/− mice demonstrated that LV end-diastolic volume [52 ± 4 (WT) vs. 71 ± 6 (TIMP-1−/−) μl] and LV end-diastolic pressure [9.0 ± 1.2 (WT) vs. 12.7 ± 1.4 (TIMP-1−/−) mmHg] were significantly increased in the TIMP-1−/− mice 2 wk after MI. LV contractility was reduced to a similar degree in the WT and TIMP-1−/− groups after MI, as indicated by a significant fall in the LV end-systolic pressure-volume relationship. Ventricular weight and cross-sectional areas of LV myocytes were significantly increased in TIMP-1−/− mice, indicating that the hypertrophic response was more pronounced. The observed significant loss of fibrillar collagen in the TIMP-1−/−controls may have been an important contributory factor for the observed LV alterations in the TIMP-1−/− mice after MI. These findings demonstrate that TIMP-1 deficiency amplifies adverse LV remodeling after MI in mice and emphasizes the importance of local endogenous control of cardiac MMP activity by TIMP-1.

Genetic toxicology tests are conducted to determine if a compound (drug) has the potential to cause mutations or chromosomal damage, data that has value in predicting its carcinogenic potential. For new pharmaceuticals, test data from a standard test battery are required prior to administering the compound to humans. This test battery includes in vivo studies of chromosomal damage in the rodent hematopoietic system, traditionally performed in the bone marrow, but in recent years, the in vivo micronucleus assay has been validated for use in peripheral blood. In addition, new regulatory guidelines, like ICH S2R1, allow the use of a second in vivo assay as part of the genotoxicity assessment, and having different in vivo assays to use will benefit understanding the in vivo genotoxicity profile of candidate pharmaceuticals. Three ‘new’ in vivo tests are described—the in vivo Comet assay, the in vivo Pig-a gene mutation assay, and the liver micronucleus (MN) assay. An established test, the transgenic rodent (TGR) gene mutation assay, is presented as a test whose use for regulatory decision-making may increase due to the recent introduction of robust testing protocols and new test guidelines. The chapter also discusses refinements in how the traditional genotoxicity tests are conducted and interpreted, including new ways of following up on their findings. Finally, the chapter discusses what has been described as a paradigm shift—that is, a move away from qualitative categorizations of genotoxic vs. non-genotoxic to the use of quantitative genotoxicity data as an adverse outcome on which regulatory decisions may be made.

La sistemática, la ciencia que estudia y clasifica la diversidad biológica, tiene como meta el inventariar todas las especies que habitan este planeta. Esta labor es complicada, pues a la fecha han sido formalmente descritas y nombradas cerca de un millón y medio de especies, que representan un bajo porcentaje de las 30 millones de especies que se estima que existen. Determinar a qué grupo taxonómico (familia, género y especie) pertenece un organismo puede ser difícil y, en muchos casos, sólo puede hacerlo de manera confiable un taxónomo experto en ese grupo particular. Generalmente la identificación requiere del uso de claves dicotómicas y de la comparación de las características morfológicas del organismo. Sin embargo, las características distintivas pueden ser difíciles de observar (por ejemplo, atributos microscópicos o de la anatomía interna), estar presentes solamente en un estadio particular del ciclo de vida (generalmente en los individuos adultos), o en uno de los dos sexos. Por otra parte, cuando se tiene un ejemplar incompleto, como una pluma de ave o una semilla, la identificación de la muestra no puede llevarse a cabo.

Oculoauriculofrontonasal syndrome was the subset of patients with oculo-auriculo-vertebral spectrum and frontonasal malformation. Radiographic evidence of tracheal duplication was documented in a male infant with oculoauriculofrontonasal syndrome. Although previously unreported in oculoauriculofrontonasal syndrome, airway anomalies in our case can be attributed to the oculo-auriculo-vertebral component of the oculoauriculofrontonasal syndrome.

Aim To compare in a laboratory study two negative pressure systems and syringe irrigation, regarding the delivery of a contrast solution (CS) to working length (WL) and into simulated lateral canals and the effective volume of irrigant aspirated during negative pressure irrigation. Methodology Twenty single-canaled incisor training models were constructed with six simulated lateral canals each (2, 4 and 6 mm to WL) and a size 40, 0.04 taper apical size canal. Each model underwent all irrigation procedures (EndoVac at WL (EndoVac-0) and WL–2 mm (EndoVac-2), iNP needle with negative pressure (iNPn) and syringe irrigation with the iNP needle (iNPs) and a 30-G side-slot needle placed at WL (SI0) and WL–2 (SI2) mm in a crossover design. CS was delivered at 4 mL min−1 for 60 s with a peristaltic pump and a recovery device collected the volume (in mL) of irrigant suctioned by the negative pressure groups. The irrigation procedures were digitally recorded, and a still image of the 60-s time-point of irrigation was evaluated for CS distance to WL (in millimetres) after irrigation and penetration into lateral canals (3-point scale). Statistical tests used were Kruskal–Wallis and Dunn's test. Results EndoVac-0, iNPn and iNPs had median distances of CS to WL of 0 mm, followed by SI0 (0.2 mm), SI2 (0.7 mm) and EndoVac-2 (1.7 mm). There were no significant differences between EndoVac-0, iNPn, iNPs and SI0, but these were significantly different to SI2 and EndoVac-2 (P

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed.

Introduction Subcutaneous mastectomy is the first surgical procedure to be completed by female to male transsexuals after appropriate mental health and endocrine therapy. Objectives of subcutaneous mastectomy in this group are to masculinize the chest by the removal of breast tissue and skin excess, reduction and proper positioning of the nipple‐areola complex, obliteration of the infra‐mammary fold, and ideally with a minimal of chest wall scars. In this study, the largest series of subcutaneous mastectomies in female‐to‐male transsexuals to date is presented. Aims Our aim was to determine relations between surgical technique, risk factors, complications, reoperations and secondary corrections in female‐to‐male transsexuals. Methods We performed a retrospective survey study on 404 mastectomies in 202 female‐to‐male transsexuals during the period of 2000–2011. Main Outcome Measures Primary outcomes for this study were complication rate, acute reoperations, secondary corrections, surgical time, and length of hospital stay in relation to the surgical technique used. Results The average age of these patients at time of the intervention was 31 years (±10) with an average BMI of 25 kg/m 2 (±4). The chosen technique depended strongly on breast volume, which, in turn, was strongly related to BMI and age. The number of acute reoperations and secondary corrections depended on the surgical technique. The total rate of acute complications was 5.0%. This percentage was highest in surgeries without skin resection (10.5%). To improve overall aesthetic results, the following secondary corrections were performed: nipple and/or areola corrections (8.9%), scar revisions (12.6%), and chest contouring (17.8%). Conclusion This study shows a correlation between the surgical technique, complication rate, and length of hospital stay. In general, the larger the breast, the larger the scars that remain after the operation. On the other hand, the smaller the scars resulting from the operation, the higher the risk of hematoma. Cregten‐Escobar P, Bouman MB, Buncamper ME, and Mullender MG. Subcutaneous mastectomy in female‐to‐male transsexuals: A retrospective cohort‐analysis of 202 patients. J Sex Med 2012;9:3148–3153.

Background Callitropsis guadalupensis (Guadalupe cypress) is endemic to Guadalupe Island, Mexico, where it is the dominant species of the only forest. The species has suffered declining numbers following the introduction of goats to the island over 150 years ago. Callitropsis guadalupensis is closely related to Callitropsis forbesii (Tecate cypress), distributed in small isolated populations in mainland Baja California and southern California. The objective of the present study was to compare the genetic diversity of the island endemic to the continental species. Methodology/Principal Findings We measured genetic diversity in Callitropsis guadalupensis (n = 54) from Guadalupe Island and in Callitropsis forbesii (n = 100) from five populations in mainland Baja California. The plastid DNA trnS-trnG spacer and the trnL-trnF region were chosen for characterization. Thirty-four haplotypes were observed, of which six were shared between both species. One of these haplotypes was also shared with three other species, Callitropsis lusitanica, Callitropsis montana, and Callitropsis stephensonii. Haplotype diversity (h) and nucleotide diversity (π) were significantly higher for Callitropsis guadalupensis (h = 0.698, π = 0.00071) than for Callitropsis forbesii (h = 0.337, π = 0.00024). Conclusions/Significance Callitropsis guadalupensis shows no evidence of a founder effect or of a genetic bottleneck, and can be added to a growing list of insular species with higher genetic diversity than their mainland relatives.

In vitro genotoxicity testing has played an important role in the safety assessment of chemicals, pharmaceuticals, cosmetic ingredients and environmental pollutants since the mid-1970s. This testing allows for an early look into the possible genetic hazards of a material before expensive animal testing is needed or prior to clinical trials. In vitro genetic toxicology can identify mutations and chromosome damage which may be progenitors of carcinogenesis. Standard regulatory assays are low-throughput, have a high compound requirements and are time consuming. In recent years, industry has identified the need to support early, rapid safety assessment of most chemicals to reduce attrition, costs and to comply with the initiatives to reduce, refine or replace animal testing (3Rs). To improve such screening, higher throughput assays have been developed to help with chemical compound selection and to build up structure–activity relationships. In parallel, these assays have been developed with a better understanding of the nature of the genotoxicity observed and to complement the risk assessment process. Finally, 30 years later, new technologies and initiatives are helping researchers further understand the nature of genetic damage, the role of epigenetic changes in carcinogenesis and, through multi-disciplinary high-throughput collaborations, how genotoxicity fits into toxicity ‘signatures’ or ‘fingerprints’ of thousands of compounds to create better risk prediction models.

There is considerable discussion within the scientific community as to the appropriate measures of cytotoxicity to use when deciding on the maximum concentration of a substance to test in vitro for its ability to induce DNA damage using the Comet assay. Conventional cytotoxicity assessment methods, such as trypan blue dye exclusion or relative cell number (cell counts) may not be the most biologically relevant measurement for cytotoxicity in this assay. Thus, we evaluated for decreased levels of adenosine triphosphate (ATP) and activation of Caspase-3/7 as well as relative cell number and trypan blue exclusion in order to understand the correlation among test compound concentration, cytotoxicity and genotoxicity outcomes in the Comet assay. We tested two non-genotoxic and non-cytotoxic compounds (d-glucose and ethanol), two non-genotoxic but cytotoxic compounds (2,4-dichlorophenol and tunicamycin) and four genotoxic and cytotoxic compounds (methyl methanesulfonate, ethyl methanesulfonate, etoposide and 4-nitroquinoline-N-oxide) in TK6 human lymphoblast cells. Our data show that measuring ATP and Caspase-3/7 levels provides more rapid and perhaps more biologically relevant measures of cytotoxicity compared with trypan blue dye exclusion and relative cell number. Furthermore, incorporating these two assays into the Comet assay also provided insight on the cytotoxic mode of action of the chemicals tested. By extrapolation, such assays may also be useful in other in vitro genotoxicity assays.

A structural event in the progression of left ventricular (LV) failure is myocardial extracellular matrix (ECM) remodeling. The myocardial fibroblast is a major cell type influencing the ECM, but whether and to what degree specific phenotypic differences in myocardial fibroblasts can be demonstrated to occur in culture with the development of LV failure remains unclear. Adult pigs (25 kg) were used for control myocardial fibroblast preparations (N=5) or following pacing-induced LV failure (N=5; 240 bpm, 3 weeks). LV remodeling occurred with pacing as evidenced by increased LV end diastolic volume (132+/-11 vs. 60+/-4 mL for control; P

P.A. Escobar *, R.A. Kemper , J. Tarca , J. Nicolette , M. Kenyon , S. Glowienke , S.G. Sawant , J. Christensen , T.E. Johnson , C. McKnight , G. Ward , S.M. Galloway , L. Custer , E. Gocke , M.R. O’Donovan , K. Braun , R.D. Snyder , B. Mahadevan b,1,2 Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877-0368, USA Merck Research Laboratories West Point, PA 19486, USA AbbVie, Abbott Park, IL 60064-3500, USA d Pfizer Global Research and Development, Groton, CT, USA Novartis Pharma AG, Werk Klybeck Klybeckstrasse 141, Basel CH-4057, Switzerland Amgen Inc., Thousand Oaks, CA 91320-1799, USA Bristol Myers-Squib, New Brunswick, NJ 08903, USA Hoffman-La Roche Ltd., Basel, Switzerland AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK j Sanofi-Aventis Deutschland GmbH, D-65926 Frankfurt, Germany RDS Consulting Services, Maineville, OH 45039, USA

O Trypanosoma cruzi e considerado um dos eucariotos mais resistentes a Radiação ionizante, suportando doses sub-letais de ate 1,0 kGy. A radiação ionizante do tipo gama promove a quebra das fitas simples e duplas do DNA, além de gerar espécies reativas de oxigênio (ROS). Apos a irradiação, os parasitos cessam o seu crescimento por ate 96 horas, retomando-o em seguida e atingindo a fase estacionaria em ate 240 horas apos irradiação. O padrão de bandas cromossomais, que são extensamente fragmentadas pela radiação, e restabelecido cerca de 48 horas apos o estresse. Para tolerar tais danos, e necessário um eficiente aparato de reconhecimento e reparo de lesões. Neste trabalho, investigamos a radioresistencia em T.cruzi, mediante a análise do seu perfil de expressão protéica e da sua atividade mitocondrial em diferentes tempos apos uma dose de 500 Gy de radiação gama. Utilizando a metodologia 2D-DIGE, foram encontrados 543 spots protéicos diferencialmente expressos apos a exposição do T. cruzi a radiação. A partir destes spots, 53 proteínas foram identificadas por espectrometria de massas (MS/MS) e classificadas de acordo com sua função biológica. Analisando a anotação funcional das 53 proteínas, observamos uma tendência para a superexpressão de proteínas com peso molecular abaixo do predito. Esta tendência indica a possível existência de modificações pos-traducionais e/ou de mecanismos de processamento das proteínas identificadas, que geraria polipeptídios mais curtos do que o predito em resposta a radiação gama. Outras tendências observadas em nossas analises de proteômica incluem alterações nos seguintes processos biológicos: regulação positiva do processo de síntese de proteínas, repressão de proteínas envolvidas no processo de enovelamento (exceto pela indução de duas chaperonas do retículo endoplasmático), repressão das vias de geração de ATP por fosforilação oxidativa, da glicolise, e do metabolismo de aminoácidos. Nossos resultados também indicam que a síntese protéica de#novo e essencial para a recuperação do T. cruzi apos o estresse por exposição a radiação gama, pois o tratamento com inibidores de tradução interfere drasticamente no retorno ao crescimento dos parasitos irradiados. Ao avaliarmos a atividade mitocondrial de T. cruzi, xvi! verificamos que parasitos irradiados produzem mais ATP do que parasitos não irradiados, alem de consumirem mais O2. Adicionalmente, a análise da expressão gênica de T. cruzi irradiado por qRT-PCR mostrou que os genes do maxicírculo mitocondrial foram induzidos apos a irradiação. Ambos os resultados indicam uma maior atividade da mitocôndria apos o estresse. Entretanto, observamos uma menor produção de H2O2 nos parasitos irradiados, indicando assim a existência de um mecanismo eficiente de eliminação de radicais livres. O presente estudo revela a resposta peculiar de T. cruzi a radiação ionizante e suscita questionamentos pertinentes sobre como este organismo e capaz de modificar seu perfil de expressão protéica e sua atividade mitocondrial para possibilitar a sobrevivência em condições tão adversas. The Trypanosoma cruzi is considered as one of the eukaryotes most resistant to ionizing radiation, enduring sub-lethal doses of up to 1.0 kGy. The ionizing radiation by gamma rays promotes double-strand and single-strand breaks on the DNA and, additionally, generates reactive oxygen species (ROS). After irradiation the parasites cease growing for as long as 96 hours, further resuming growth and reaching the stationary phase no later than 240 hours after irradiation. The pattern of chromosomal bands which are extremely fragmented by radiation exposure is restored approximately 48 hours after the stress. To enable the parasite to tolerate such damage condition, an efficient apparatus to recognize and repair lesions is necessary. In the present work we investigate T. cruzi radioresistance by analyzing its protein expression patterns and mitochondrial activity in different time points after a dose of 500 Gy of gamma rays. Using the 2DTDIGE methodology, 543 spots were identified as significantly differentially expressed after T. cruzi exposure to radiation. From these 543 spots, 53 different proteins were identified by mass spectrometry (MS/MS) and classified according to biological function. When the functional annotation of these 53 proteins was analyzed, a tendency of overexpression of proteins with molecular weight lower than predicted was observed. This tendency suggests the presence of post-translational modifications and/or processing mechanisms acting upon the identified proteins, leading to the emergence of peptides which are shorter than predicted in response to gamma radiation exposure. Other tendencies observed on proteomic analysis include alterations on the following biological processes: positive regulation of the protein synthesis process, repression of proteins involved with folding processes (except for two endoplasmic reticulum chaperones, which are induced), repression of ATP generation through oxidative phosphorylation, of glycolysis, and of the amino acids metabolism. Our results also indicate that the de novo protein synthesis is essential for T. cruzi recovery from gamma radiation exposure, since the treatment with translation inhibitors drastically interferes with the growth resumption on irradiated parasites. When the mitochondrial activity of T. cruzi xviii was evaluated, we verified that irradiated parasites generate higher levels of ATP than their non-irradiated counterparts, with an associated higher O2 consumption by the former. Additionally, the gene expression analysis of irradiated T. cruzi using qRT-PCR showed that genes from the mitochondrial maxicircle were induced after radiation exposure. Both of these results point to a higher mitochondrial activity after irradiation-caused stress. Nevertheless, we have also observed a lower H2O2 production by irradiated parasites, thus indicating the existence of an efficient mechanism for free radicals clearance in the organism. The present study unravels the peculiar response of T. cruzi to ionizing radiation and evokes questions about the manner in which this organism is capable of performing drastic changes on its protein expression profile and mitochondrial activity to enable its survival in such adverse conditions.

Brucella abortus é considerada o principal agente etiológico da brucelose bovina, uma zoonose que provoca grandes perdas econômicas em todo o mundo. O objetivo desse estudo foi determinar o perfil diferencial de expressão proteica de células trofoblásticas bovinas nas fases iniciais da infecção por B. abortus. Foi usado o modelo de explantes confeccionados a partir de membrana cório-alantóidea de fetos no último trimestre de gestação infectados por suspensão deB. abortus 2308 (1.0 x 108 bactérias - MOI 1:1000). No estudo da cinética da infecção foram avaliados os tempos de 0,5, 2, 4 e 8 h pós-infecção no qual foi obtida reprodutibilidade alta entre triplicatas dos géis comparados (Match > 84%; IC > 0,77). O perfil de expressão deproteínas foi similar entre os tempos avaliados (Match > 75%). Os tempos de 0,5 e 4 h pósinfecção apresentaram as maiores diferenças qualitativas entre os géis e foram escolhidos para análise diferencial em gel (DIGE). Nessa análise não foram identificados spots com diferençasquantitativas significativas entre os géis comparados. Por outro lado, 103 spots presentes apenas em um grupo experimental foram selecionados para identificação por espectrometria de massa. As proteínas BLVRA, LGALS7, RPLP1, SCAMP2, TOLLIP, GALM, AHCY, PSAPL1/PSAP, OAT, C1QBP, NDUFS8, PRCII, RAB11A, CALM1, MDH1, PRDX3, ABHD14B, KRT14, KRT7, HMGB1, HEXB, AKR1B1, PDIA3 e TPM4 foram identificadas apenas nos extratos proteicos de células trofoblásticas infectadas. As proteínas identificadas nesse estudo servirão como alvos para novas pesquisas relacionadas à interação patógeno-hospedeiro na infecção por B. abortus. Brucella abortus is considered the main etiological agent of bovine brucellosis, a zoonotic disease that causes great economic losses worldwide. The aim of this study was to determine the differential profile of proteins expression of bovine trophoblast cells in the early stages ofinfection by B. abortus. Was used the explants model prepared from chorio-alantóidea of fetuses in the last trimester of pregnancy infected by suspending the B. abortus 2308 containing 1.0 x 108 bacteria (MOI 1:1000). In the study of the kinetics of infection was evaluated the timesof 0.5, 2, 4 and 8 h post infection with B. abortus. Was obtained high reproducibility between triplicate of gels compared (Match> 84%; CI > 0.77) and the proteins expression profile was similar among the times evaluated (Match > 75%). Times of 0.5 and 4 h post infection showedthe highest qualitative differences between the gels and were used for the differential in gel electrophoresis (DIGE). In this analysis have not been identified spots with significant quantitative differences between the compared gels. Moreover, 103 spots are present only in an experimental group were selected for identification by mass spectrometry. The proteins BLVRA, LGALS7, RPLP1, SCAMP2, TOLLIP, GALM, AHCY, PSAPL1/PSAP, OAT, C1QBP, NDUFS8, PRCII, RAB11A, CALM1, MDH1, PRDX3, ABHD14B, KRT14, KRT7, HMGB1, HEXB, AKR1B1, PDIA3 and TPM4 were identified only in protein extracts of trophoblastic cells infected by B. abortus. The proteins identified in this study will serve as targets for further research related to host-pathogen interaction in infection by B. abortus.

A leishmaniose visceral (LV) é uma doença infecciosa grave causada por protozoários do gênero Leishmania. Nas Américas, o cão é o principal reservatório da doença, principalmente nas áreas urbanas, onde encontra-se em grande expansão. A LV é fatal se não for tratada adequadamente e/ou diagnosticada precocemente, porém, a eficácia dos métodos diagnósticos e vacinas disponíveis permanece parcial, sendo necessário o desenvolvimento de novas estratégias. Vários estudos demonstraram o potencial da proteína A2, específica do estágio amastigota de Leishmania, como antígeno vacinal e diagnóstico. No presente trabalho, inicialmente, testamos este antígeno, administrado na forma de vacina de DNA e observamos proteção em camundongos contra a infecção por L. (L.) chagasi e L. (L.) amazonensis. A seguir, descrevemos a proteção, em estudos de fase II, com cães beagles vacinados com a proteína A2 associada ao adjuvante saponina (constituindo a vacina Leish-tec.) contra a infecção por L. (L.) chagasi. Com relação ao diagnóstico utilizando este antígeno, observamos que peptídeos provenientes de análises in silico da proteína A2, associados a peptídeos de outras proteínas como NH, LACK e K39 apresentaram elevada sensibilidade e especificidade em testes com soros humanos ecaninos. No entanto, a busca por antígenos vacinais e diagnóstico na LV é incessante, e o atual estágio de desenvolvimento tecnológico com a possibilidade de utilizar abordagem proteômica para identificação de centenas de antígenos imunogênicos simultaneamente é atraente. Dessa forma, optamos por uma abordagem proteômica associada a análises in silico para identificar novos antígenos de L. (L.) chagasi. Nossa análise proteômica foi a primeira a utilizar amastigotas purificadas de baço de hamster e, além disso, é o primeiro estudo a mapear a antigenicidade da infecção por L. (L.) chagasi utilizando soros de cães de fase aguda e crônica. Todas as proteínas selecionadas porwestern blot-2DE (WB-2DE) foram mapeadas para epítopos de células B pelo software BepiPred e submetidas `a imuno-ensaios em membranas de celulose resultando na seleção de 25 peptídeos com elevado potencial para o diagnóstico sorológico da LV. Paralelamente, foi utilizada a técnica de DIGE para comparar a expressão diferencial deproteínas entre as formas amastigota e promastigota de L. (L.) chagasi e 40 proteínas foram identificadas, sendo 25 aumentadas em promastigotas, 5 aumentadas em amastigotas e 10 com expressão similar em ambas as formas. Além disso, todas as proteínas identificadas no trabalho, tanto por DIGE como por WB-2DE, forammapeadas para epítopos de células T utilizando o software NetCTL que permitiu a seleção de potenciais candidatos para o desenvolvimento de vacinas. Por fim, para complementar a análise de predição para células B, as mesmas proteínas imunogênicas identificadas por WB-2DE forma mapeadas utilizando outros dois programas de predição de epítopos (ABCPred e BCPred) e após análise adicional de imuno-ensaios mais 23 peptídeos foram identificados como potenciais antígenos diagnóstico na LVC. Esses novos peptídeos, juntamente com os 25 previamente identificados, foram submetidos a ensaios imuno-enzimáticos que resultaram em 10 antígenos com elevada sensibilidade e especificidade, inclusive maior do que a encontrada pelo kit EIE-LVC, que é o recomendado pelo Ministério da Saúde, que também falhou em detectar cães assintomáticos. Portanto, uma análise em grande escala de L. (L.) chagasi através do proteoma foi realizada para identificar epítopos de células B e T com potencial para utilização no desenvolvimento de testes diagnósticos e vacinas. Visceral leishmaniasis (VL) is a severe infectious disease caused by a protozoan of the genus Leishmania. In the Americas, the dog is the main reservoir of disease, mainly in urban areas where the disease is increasing. VL is fatal if it is not adequately treated and/or if it is not diagnosed early, however the efficacy of diagnostic methods andvaccines available remain partial, therefore it is necessary to develop new strategies. Several studies have demonstrated the potential of A2 protein, an amastigote stagespecific protein family in Leishmania, as vaccine and diagnosis. In our studies, using this antigen administered as DNA vaccine, we observed protection in mice against infection by L. (L.) chagasi and L. (L.) amazonensis. A2 antigen also induces protectionin phase II studies with beagle dogs vaccinated with A2 protein associated with the saponin adjuvant (constituting the vaccine Leish-tec.) against infection by L. (L.) chagasi. The partial protection induced by A2 antigen in vaccine studies led us to search for new proteins for further vaccine tests and future associations with the A2 antigen. Regarding the diagnosis, we found that the peptides derived from in silico analysis of the A2 protein, associated with peptides from other proteins such as NH, LACK and K39 showed high sensitivity and specificity in tests with human and canine sera. However, the search for vaccine and diagnosis antigens of VL is incessant, and the current stage of technological development with the possibility of using proteomic approach to identify hundreds of immunogenic antigens simultaneously is attractive. Thus, we chose aproteomic approach combined with in silico analyzes to identify new antigens of L. (L.) chagasi. Our proteomic analysis were the first to use amastigotes purified from hamster spleen and, furthermore, is the first study to map the antigenicity of infection by L. (L.) chagasi using sera from dogs of acute and chronic phase. All the selected protein bywestern blot-2DE (WB-2DE) were mapped to B cell epitopes by the BepiPred software and subjected to immunoassay through cellulose membranes resulting in the selection of 25 peptide with high potential for serological diagnosis of VL. In parallel, we used the technique of DIGE to compare the differential expression of proteins between the amastigote and promastigote forms of L. (L.) chagasi and 40 proteins were identified, 25 increased in promastigotes, 5 increased in amastigotes and 10 with similar expression in both forms. Moreover, all the proteins identified in the work by both DIGE as by WB 2DE have been mapped to T cell epitopes using the software NetCTL allowing the selection of potential candidates for vaccine development. Finally, to complete the analysis prediction for B cells, the same immunogenic proteins identified by WB-2DE were mapped using others two programs to predict epitopes (ABCPred and BCPred) and after additional analysis of immunoassays, more 23 peptides were identified as potential diagnostic antigens in the LVC. These new peptides, together with 25 previously identified, were subjected to immuno-enzymatic assays that resulted in 10 antigens withhigh sensitivity and specificity, even greater than that found by the EIE-LVC kit, which is recommended by the Ministry of Health, which also failed to detect asymptomatic dogs. Therefore, a large-scale analysis of L. (L.) chagasi through proteome was performed to identify B and T cell epitopes potentially useful in developing diagnostic tests and vaccines.