Effect of entomopathogenic fungi on the control of the coffee berry borer in the laboratory

Among the management and control tactics of the coffee berry borer Hypothenemus hampei (Ferrari), there is the use of entomopathogenic fungi. Due to the importance of prospecting isolates of entomopathogenic fungi for the control of the coffee berry borer, the objective of this study was to evaluate the efficiency of 26 isolates of entomopathogenic fungi in the control of this insect pest in the laboratory. The coffee berry borers were immersed in a solution adjusted to the concentration of 1 to 3 × 108 conidia/mL of each isolate and the control treatment (sterilized water). After seven days total mortality and confirmed mortality were evaluated. The isolates that caused the highest mortality and two commercial isolates were selected for evaluation of lethal concentration (LC50 and LC90) and lethal time (LT50 and LT90). Coffee berry borers were treated at different conidia concentrations for lethal concentration to assess total and confirmed mortality. For a lethal time, the coffee berry borers were treated at the concentration of 108 conidia/mL of the selected isolates, and, after two days and every 24 hours until the eighth day, the number of dead individuals was verified. Among the 26 isolates evaluated, 24 presented mortality higher than the control treatment, and three presented mortality higher than 85%. In the LC50 and LC90 assays, the IBCB 353 and IBCB 364 isolates were more lethal to H. hampei. In the LT50 and LT90 assays, the IBCB 66 and IBCB 353 isolates caused lethality in a shorter time.

Anti-hyperglycemic fraction from Alternanthera sessilis L. leaves gets elucidated following bioassay-guided isolation and mass spectrometry

Abstract The anecdotal use of Alternanthera sessilis L. as a relief for diabetes has been known in the Philippines for generations, and antidiabetic activity of similar varieties in other countries is likewise documented. However, the compounds responsible for this activity remain unclear. This study aims to isolate the anti-hyperglycemic fraction of local A. sessilis leaves and identify the compounds in this fraction. Methanol extract of A. sessilis leaves and its hexane, ethyl acetate (ASE), and water fractions were administered to alloxan-induced diabetic mice. ASE (250mg/kg) had the highest anti-hyperglycemic activity at 6-h post-treatment (25.81%±12.72%), with almost similar blood glucose reduction rate as metformin (30.13±3.75%, p=0.767). Repeated fractionation employing chromatographic separation techniques followed by in vivo anti-hyperglycemic assay yielded partially purified subfractions. A. sessilis ethyl acetate subfraction 4-2 (100mg/kg) displayed remarkable suppression of blood glucose rise in diabetic mice at 6-h post-treatment (26.45±3.75%, p<0.0001), with comparable activity with metformin (100mg/kg, 27.87±5.65%, p=0.652). Liquid chromatography/mass spectrometry showed eight distinct peaks, with four peaks annotated via the Traditional Chinese Medicine library and custom library for A. sessilis. Among these, luteolin, apigenin, ononin, and sophorabioside were identified as putative compounds responsible for the anti-hyperglycemic activity. This result provided basis for the reported anecdotal claims and potential utility of the local variety of A. sessilis leaves as sources of anti-hyperglycemic agents

A new approach to mark termites (Cornitermes cumulans (Kollar) Blattodea: Isoptera) for laboratory bioassays / Uma nova abordagem de marcador tópico em cupins para bioensaios de laboratório

Behavioral lab bioassays involving termites must be promptly performed to allow intended observations prior to death from dissecation, typical of these soft-bodied insects. To this end, topic markers have been proposed as an alternative to histological stains which, while not always toxic are inevitably lengthy to apply. Among recommended topic markers, gouache is easy to apply, dries out quickly, but it is known affect termites in the long run, being suitable only to short-term bioassays. Its alternative, colored glue, is also easy to apply, but it takes long to dry and it is too dense and heavy, being thus prone to affect termite walking patterns. Here we tested a mix of gouache and colored glue aiming to combine the qualities of both into a suitable topical marker for Cornitermes cumulans termites. Similar patterns of survival presented by marked and unmarked termites ruled out concerns about toxicity of this mixture. Such results were consistent across distinct group densities evidencing that the mixture does not interfere with, nor it is affected by, crowding effects. Because crowding regulates interindividual interactions and these underlie most behaviors, the mixture can be thought to be suitable to behavioral studies. We argue that this 1:2 glue:gouache mixture is an excellent alternative to mark termites for lab bioassays. Being atoxic, cheap, easy to apply, and non-invasive, this mixture may happen to be useful not only for termites but also in bioassaying other similarly soft-bodied insects.

Bioensaios comportamentais em laboratório com cupins devem ser realizados rapidamente a fim de garantir observações antes da morte por dissecação, típico desses insetos de corpo mole. Para este fim, marcadores tópicos têm sido propostos como uma alternativa para marcadores histológicos que, embora nem sempre tóxico, possuem uma aplicação demorada. Entre os marcadores tópicos recomendados, tinta guache é de fácil aplicação, rápida secagem, porém afeta os cupins em bioensaios longos, sendo adequado apenas para bioensaios curtos. Sua alternativa, cola colorida, também é de fácil aplicação mas leva muito tempo para secar e é muito denso e pesado, afetando os padrões de caminhamento dos cupins. No presente estudo, nós testamos uma mistura de tinta guache e cola colorida objetivando combinar as qualidades de ambos os marcadores tópicos em um marcador tópico adequado para Cornitermes cumulans. Padrões similares de sobrevivência entre cupins marcados e controle indicam a ausência de toxicidade na mistura de tinta guache e cola colorida. Tais resultados são consistentes em grupos de densidades distintas, o que comprova que a mistura não interfere, nem sofre efeitos de aglomeração. Uma vez que a aglomeração regula as interações inter-individuais e afetam a maioria dos comportamentos, a mistura pode ser adequada para estudos comportamentais. Nós argumentamos que a mistura de tinta guache e cola (1:2) é uma excelente alternativa como marcador tópico em cupins para bioensaios em laboratório. Sendo atóxico, barato, fácil de aplicar e não invasivo, esta mistura pode ser útil não só para os cupins, mas também em bioensaios com outros insetos de corpo mole.

Comparative application of biological and ninhydrin- derivatized spectrophotometric assays in the evaluation and validation of amikacin sulfate injection

Abstract Instrumental techniques are preferred over bioassay methods for antibiotic quantification mainly due to speed and ability to quantify metabolites in biological samples; however, the potency and biological activity of these drugs cannot be assessed. Two methods - agar well diffusion (bio-assay) and spectrophotometric methods were used to evaluate amikacin sulfate injection. Agar plates were inoculated with S. aureus inoculum; zones of inhibition from its susceptibility to amikacin were obtained, while spectrophotometric absorption at 650 nm of ninhydrin- derivatized amikacin in phosphate buffer (pH 8) was measured. Methods performance showed linearity from 1 - 16 µgmL-1 (bioassay, r = 0.9994) and 10-50 µgmL-1 (spectrophotometric, r = 0.9998). Molar absorptivity was 2.595 x 104 Lmol-1cm-1. Limits of detection and quantification were 1.07 and 3.24 µgmL-1 respectively for bioassay method, while corresponding values for spectrophotometric method were 0.98 and 2.97 µg mL-1. Relative standard deviations were ≤ 2.0% for both methods, with recoveries from 95.93 - 100.25%. Amikacin in brands ranged from 97.53 ± 2.68 to 100.84 ± 1.82%, student's t-test was ≤ 2.78 (n = 4) with respect to label claim for both methods. Experimental paired t-test (t = 2.07; n = 4) and F-test (F = 3.94; n = 4) values indicated no significant difference between both methods, hence comparable and can jointly be used in quality control assessment of antibiotics

LncRNA-599554 sponges miR-15a-5p to contribute inductive ability of dermal papilla cells through positive regulation of the expression of Wnt3a in cashmere goat

BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.

Cost analysis of smear microscopy and the Xpert assay for tuberculosis diagnosis: average turnaround time

Abstract INTRODUCTION: Rapid and accurate tuberculosis detection is critical for improving patient diagnosis and decreasing tuberculosis transmission. Molecular assays can significantly increase laboratory costs; therefore, the average time and economic impact should be evaluated before implementing a new technology. The aim of this study was to evaluate the cost and average turnaround time of smear microscopy and Xpert assay at a university hospital. METHODS: The turnaround time and cost of the laboratory diagnosis of tuberculosis were calculated based on the mean cost and activity based costing (ABC). RESULTS: The average turnaround time for smear microscopy was 16.6 hours while that for Xpert was 24.1 hours. The Xpert had a mean cost of USD 17.37 with an ABC of USD 10.86, while smear microscopy had a mean cost of USD 13.31 with an ABC of USD 6.01. The sensitivity of smear microscopy was 42.9% and its specificity was 99.1%, while the Xpert assay had a sensitivity of 100% and a specificity of 96.7%. CONCLUSIONS: The Xpert assay has high accuracy; however, the turnaround time and cost of smear microscopy were lower than those of Xpert.

Sistema microbiológico de cinco bioensayos para la detección de residuos de antibióticos / Five-assay microbiological system for the screeningf antibiotic residues

Abstract A novel microbiological system in microtiter plates consisting of five bioassays is presented for the detection and classification of antibiotic residues in milk. The bioassays were optimized for the detection of beta-lactams (Bioassay B: Geobacillus stearothermophilus), macrolides (Bioassay M: Bacillus megaterium with fusidic acid), tetracyclines (Bioassay T: B. megaterium with chloramphenicol), quinolones (Bioassay Q: Bacillus licheniformis) and sulfamides (Bioassay QS: B. licheniformis with trimethoprim) at levels near the maximum residue limits (MRL). The response of each bioassay was interpreted visually (positive or negative) after 4-5.5h of incubation. The system detects and classifies beta-lactams (5 pg/l of amoxicillin, 4 pg/l of ampicillin, 36 pg/l of cloxacillin, 22 pg/l of amoxicillin, 3 pg/l of penicillin, 114 pg/l of cephalexin, 89pg/l of cefoperazone and 116 pg/l of ceftiofur), tetracyclines (98 pg/l of chlortetracycline, 92 pg/l of oxytetracycline and 88 pg/l of tetracycline), macrolides (33 pg/l of erythromycin, 44 pg/l of tilmicosin and 50 pg/l of tylosin), sulfonamides (76 pg/l of sulfadiazine, 85 pg/l of sulfadimethoxine, 77 pg/l of sulfamethoxazole and 87pg/l of sulfathiazole) and quinolones (94 pg/l of ciprofloxacin, 98 pg/l of enrofloxacin and 79 pg/l marbofloxacin). In addition, the specificity values were high for B, T, Q (99.4%), M (98.8%) and QS (98.1%) bioassays. The control of antibiotics through this system can contribute to improving the quality and safety of dairy products.

Resumen Se presenta un novedoso sistema microbiológico en placas de microtitulación compuesto por 5 bioensayos para la detección y clasificación de residuos de antibióticos en leche. Los bioensayos fueron optimizados para la detección de betalactámicos (bioensayo B: Geobacillus stearothermophilus), macrólidos (bioensayo M: Bacillus megaterium con ácido fusídico), tetraciclinas (bioensayo T: Bacillus megaterium con cloranfenicol), quinolonas (bioensayo Q: Bacillus licheniformis) y sulfamidas (bioensayo QS: Bacillus licheniformis con trimetoprima), a niveles cercanos a los límites máximos de residuos (LMR). La respuesta de cada bioensayo se interpretó visualmente (positiva o negativa) después de 4 a 5,5 h de incubación. El sistema detecta y clasifica betalactámicos (5 pg/l de amoxicilina, 4 pg/l de ampicilina, 36 pg/l de cloxacilina, 22 pg/l de amoxicilina, 3 pg/l de penicilina, 114 pg/l de cefalexina, 89 pg/l de cefoperazona y 116 pg/l de ceftiofur), tetraciclinas (98 pg/l de clortetraciclina, 92 pg/l de oxitetraciclina y 88 pg/l de tetraciclina), macrólidos (33 pg/l de eritromicina, 44 pg/l de tilmi-cosina y 50 pg/l de tilosina), sulfamidas (76 pg/l de sulfadiacina, 85 pg/l de sulfadimetoxina, 77 pg/l de sulfametoxazol y 87 pg/l de sulfatiazol) y quinolonas (94 pg/l de ciprofloxacina, 98 pg/l de enrofloxacina y 79pg/l de marbofloxacina). Además, los valores de especificidad fueron altos para los bioensayos B, T, Q (99,4%), M (98,8%) y QS (98,1%). El control de residuos de antibióticos mediante este sistema puede contribuir a mejorar la calidad e inocuidad de los productos lácteos.

Use of erythrocytes in cytotoxicity and toxicity assays of medicinal plant extracts: analysis of their application and bibliometric study / Utilización de eritrocitos en ensayos de citotoxicidad y toxidez de extractos de plantas medicinales: análisis de su aplicación y estudio bibliométrico

Plant species have been used for therapeutic purposes since ancient times and are still in use today since these products represent a source of raw material for the production of phytotherapeutic formulations. Screening and investigation of plants with pharmacological potential require the evaluation of characteristics related to their action, efficacy and safety in different steps. Among these steps, pre- clinical trials are used to evaluate the properties of the test product in in vitro experiments, such as cytotoxicity assays. Within this context, this study consists of a bibliometric analysis of some in vitro cytotoxicity and toxicity assays in erythrocytes used during bioprospecting of medicinal plants. The results demonstrated the wide application of erythrocytes to evaluate the biological effects of medicinal plant extracts. The methods were found to be valid and effective for the preliminary investigation of the in vitro cytotoxicity and toxicity of plant products.

El uso de especies vegetales para fines terapéuticos es una práctica histórica y todavía bastante actual, ya que estos productos pueden representar una fuente de materia prima para la producción de formulaciones fitoterápicas. En investigación de plantas con potencial farmacológico requiere la evaluación de su acción, eficacia y seguridad, a través de diferentes etapas. Entre estas, en los ensayos preclínicos se evalúan las propiedades del producto-prueba en experimentos in vitro, tales como ensayos de citotoxicidad, entre otros. En este aspecto, el presente estudio consiste en un análisis bibliométrico acerca de algunas pruebas de citotoxicidad y toxicidad in vitro en eritrocitos realizados en los ensayos de bioprospección de plantas medicinales. Los resultados evidencian la amplia utilización de eritrocitos para la evaluación de los efectos biológicos de extractos de plantas medicinales, apuntándolos como métodos válidos y eficaces para la investigación preliminar de la citotoxicidad y toxicidad in vitro de productos vegetales.

Evaluation of recombinant human interferon beta 1b by liquid chromatography methods and bioassay

Recombinant human interferon beta 1b (rhIFNß-1b) is clinically used to treat multiple sclerosis. A reversed-phase liquid chromatography (RP-LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE-LC) method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 µg/mL (r2 = 0.9998) and 0.50-200 µg/mL (r2 = 0.9999), respectively, for RP-LC and SE-LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNß-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic

Uso do bioensaio com Allium cepa L. e análises físico-químicas e microbiológicas para avaliação da qualidade do Rio da Ilha, RS, Brasil / Allium cepa L. bioassay and physicochemical and microbiological analysis to evaluate the water quality of the Ilha River, RS, Brazil

O estudo avalia a toxicidade, citotoxicidade, genotoxicidade e análises físico-químicas e microbiológicas de amostras de águas coletadas em dois pontos (nascente e foz) do Rio da Ilha - um dos principais afluentes do Rio dos Sinos, RS, Brasil - em dois períodos: inverno (2014) e verão (2015), através do bioensaio com Allium cepa que fornece esses dados através da mensuração das raízes dos bulbos, índice mitótico e presença de aberrações cromossômicas. Os resultados demonstraram níveis de citotoxicidade principalmente na foz do rio, e alguns parâmetros (DBO5, fósforo, alumínio, chumbo, ferro, níquel e coliformes termotolerantes) acima da legislação estabelecida, mesmo a região sofrendo pouco impacto de origem antrópica.

This study evaluates the toxicity, cytotoxicity, genotoxicity and physicochemical and microbiological analysis of water samples collected at sites (source and mouth) of the Ilha River -one of the main tributaries of the Sinos River, RS, Brazil - in winter (2014) and summer (2015), by Allium cepa bioassay which provided the data by measuring the roots of the bulbs, mitotic index and presence of chromosomal aberrations. The results show levels of cytotoxicity especially at the mouth of the river, and some parameters (DBO5, phosphorus, aluminum, lead, iron, nickel and fecal coliforms) above the limits established by the Brazilian legislation, despite the localization of the region in an area under minor anthropic impact.

Evaluación de la resistencia a insecticidas en cepas de Aedes aegypti (Diptera: Culicidae) de la Región Caribe de Costa Rica / Evaluation of insecticide resistance in strains of Aedes aegypti (Diptera: Culicidae) from the Caribbean region of Costa Rica

Introducción: el dengue y chikungunya son virosis antroponóticas transmitidas por Aedes aegypti que afectan extensas áreas del continente americano incluyendo Costa Rica. La reciente introducción del virus Zika representa un nuevo reto para los sistemas de salud. Dada la ausencia de tratamiento antiviral y vacunas, el control del vector Ae. aegypti, representa la única alternativa para minimizar el impacto de estas virosis. En Costa Rica, el control químico del vector se hace mediante la aplicación de piretroides (cipermetrina y deltametrina) y del organofosforado temefós; de ahí la importancia de detectar la aparición de resistencia a estos insecticidas. Objetivo: determinar el nivel de resistencia a temefós, cipermetrina y deltametrina en tres cepas de Ae. aegypti de la Región Caribe de Costa Rica, así como los mecanismos de detoxificación enzimática correspondientes. Métodos: la resistencia a temefós, cipermetrina y deltametrina se evaluó mediante bioensayos larvarios. Grupos de 20 larvas se expusieron por 24 h a 5 concentraciones de insecticidas que generaron una mortalidad entre el 2 y el 100 por ciento. Cada concentración se evaluó mediante cinco réplicas y se calculó la concentración que causa el 50 por ciento de letalidad (CL50). Como control susceptible se empleó la cepa Rockefeller. Con cada cepa se calculó un factor de resistencia 50 por ciento (FR50) para cada insecticida. Cuando se observó resistencia, se repitieron los bioensayos mediante exposición previa de las larvas a butóxido de piperonilo (PB) y S,S,S, tributilfosforotritioato (DEF) para evaluar el mecanismo detoxificante relacionado. Resultados: ninguna de las cepas evaluadas fue resistente al temefós. En las cepas Guápiles y Limón se determinó una resistencia incipiente a cipermetrina (CL50= 0,01022, FR50= 7,35 y CL 50= 0,01016, FR50= 7,30, respectivamente), mientras que en la cepa Siquirres se detectó resistencia a deltametrina (CL50= 0,01973 mg/L, FR5= 12,64). En los casos referidos hubo una disminución de la resistencia cuando se dio el pretratamiento con PB, lo que indica una detoxificación mediada por el sistema Cit P450 monooxigenasa. Conclusiones: los resultados en el presente estudio demuestran que el temefós sigue siendo efectivo para el control larvario de Ae. aegypti en las principales localidades de la región Caribe de Costa Rica. Con respecto a los piretroides se alerta ante la aparición de resistencia, lo que conlleva la necesidad de optimizar los procesos de monitoreo y la implementación de otras alternativas de control químico(AU)

Introduction: dengue and chikungunya are anthroponotic virus infections transmitted by Aedes aegypti mosquitoes. These conditions affect large areas of the American continent, including Costa Rica. The recent introduction of Zika virus infection is a new challenge for health systems. Given the absence of antiviral treatment and vaccines, Aedes aegypti control is the only alternative to minimize the impact of these viral diseases. In Costa Rica chemical control of the vector is based on the use of pyrethroids (cypermethrin and deltamethrin) and the organophosphate larvicide temephos, hence the importance of detecting the emergence of resistance to these insecticides. Objective: determine the level of resistance to temephos, cypermethrin and deltamethrin in three Aedes aegypti strains from the Caribbean region of Costa Rica, as well as the corresponding enzymatic detoxification mechanisms. Methods: resistance to temephos, cypermethrin and deltamethrin was evaluated with larval bioassays. Groups of 20 larvae were exposed to 5 insecticide concentrations for 24 h. Mortality ranged between 2 and 100 percent. Each concentration was evaluated by means of five replications, and estimation was performed of the concentration causing 50 percent lethality (CL50). The Rockefeller strain was used as susceptible control. Each strain underwent estimation of a 50 percent resistance factor (RF50) for each insecticide. Whenever resistance was observed, the bioaasays were repeated with prior exposure of the larvae to piperonyl butoxide (PBO) and S.S.S. phosphotrithiate tributyl (DEF) to evaluate the corresponding detoxification mechanism. Results: none of the strains evaluated was resistant to temephos. Incipient resistance to cypermethrin was detected in strains Guápiles and Limón (CL50= 0.01022, RF50= 7.35 and CL 50= 0.01016, RF50= 7.30, respectively), whereas resistance to deltamethrin was detected in the Siquirres strain (CL50= 0.01973 mg/L, RF50= 12.64). In the above-mentioned cases resistance decreased when pre-treatment with PBO was provided, indicating the presence of detoxification mediated by the Cyt P450 monooxygenase system. Conclusions: results show that temephos continues to be effective for larval control of Aedes aegypti in the main areas of the Caribbean region of Costa Rica. A warning is hereby given about the emergence of pyrethroid resistance, leading to the need to optimize monitoring processes and the implementation of other chemical control alternatives(AU)

Resistencia a insecticidas y sus mecanismos bioquímicos en Aedes aegypti del municipio Boyeros en los años 2010 y 2012 / Insecticide resistance and its biochemical mechanisms in Aedes aegypti mosquitoes from the municipality of Boyeros in the years 2010 and 2012

Introducción: el programa de control de Aedes aegypti (L.) (Diptera: Culicidae) en Cuba utiliza fundamentalmente temefos (ABATE) como larvicida y piretroides como adulticidas, y se ha utilizado, esporádicamente, el organofosforado (OF) clorpirifos. El monitoreo de la resistencia a estos insecticidas es esencial para lograr un control efectivo de esta especie. Objetivo: determinar la resistencia a temefos en larvas y sus mecanismos, y evaluar el nivel de resistencia a los insecticidas más utilizados como adulticidasen cinco áreas de salud del municipio Boyeros, La Habana, colectadas indistintamente en los años 2010-2012. Métodos: se evaluó la resistencia a temefos y la eficacia del mismo, en su formulación comercial (ABATEX G1), en larvas, a través de los bioensayos recomendados por la Organización Mundial de la Salud. Los mecanismos de resistencia se realizaron a través de ensayos bioquímicos. La resistencia en el estado adulto se determinó a través del método de las botellas impregnadas. Resultados: la resistencia a temefos en larvas disminuyó del año 2010 al 2012. El producto comercial de temefos mostró 100 por ciento de mortalidad entre 5 y 12 días. Se demostró que las esterasas y monooxigenasas desempeñaron un papel importante en la resistencia a temefos en larvas. En el estado adulto, se observó resistencia a piretroides y a clorpirifos en algunas cepas. Conclusiones: estos resultados corroboran la necesidad de establecer estrategias de control integrado para preservar la vida útil de los insecticidas disponibles para el control de Ae. aegypti en el municipio Boyeros(AU)

Introduction: the program for control of Aedes aegypti (L.) (Diptera: Culicidae) in Cuba is mainly based on the use of temephos (Abate) as larvicide and pyrethroids as adulticides. Organophosphate (OP) insecticide Chlorpyrifos has also been used on occasion. Monitoring resistance to these insecticides is essential to achieve effective control of the species. Objective: determine temephos resistance in larvae and its mechanisms, and evaluate the level of resistance to the insecticides most commonly used as adulticides in five health areas from the municipality of Boyeros, Havana, surveyed in the years 2010 and 2012. Methods: an evaluation was conducted of resistance to and effectiveness of temephos in its commercial formulation (Abatex G1) in larvae, using tests recommended by the World Health Organization. Resistance mechanisms were assessed with biochemical assays. The impregnated bottle bioassay was used to determine resistance in the adult stage. Results: larval resistance to temephos decreased from 2010 to 2012. The commercial product temephos showed 100 percent mortality between 5 and 12 days. Esterases and monooxygenases were found to play an important role in larval resistance to temephos. Some strains showed resistance to pyrethroids and Chlorpyrifos in the adult stage. Conclusions: these results corroborate the need to set up integrated control strategies to preserve the useful life of the insecticides available to control Aedes aegypti in the municipality of Boyeros(AU)

Bioprospecting of Cellulase Producing Extremophilic Bacterial Isolates from India.

Aim: To isolate and identify the potential extremophilic cellulase producing strain viz., psychrophiles, halophiles, thermophiles and to compare the Cellulase activity from samples collected from different geographical regions of India. Place and Duration of Study: Bharathiar University, Department of Biotechnology, Molecular Microbiology Lab, Coimbatore, Tamilnadu, India, between January to April 2011. Methodology: Cellulase-producing extremophilic bacteria viz., psychrophiles, halophiles, and thermophiles have been isolated from soil samples. According to morphology and pigmentation, 138 distinct bacteria were isolated and screened for cellulase activity by Gram’s iodine–carboxymethylcellulose plate (CMC) assay. On the basis of the cellulase activity, six potent cellulase-producing isolates from each cluster viz., P14, P36, H6, H13, T2 and T3 were selected for 16S rRNA gene based identification. The strains were optimized for maximum cellulase activity at various temperature and pH range. Results: The phylogenetic relationship revealed that P14 and P36 psychrophilic isolates possessed maximum identity with Bacillus simplex (100%) and Arthrobactercitreus (99%), with a cellulase activity of 14.10± 1.73 and 18.27± 0.71 UmL-1 respectively. Likewise, among halophiles, H6 and H13 were identified as Bacillus subtilis and Bacillus endophyticus (99%), with a cellulase activity of 14.87 ± 0.55 and 16.83 ± 0.44 U mL-1, correspondingly. In thermophiles, T2 and T3 showed close proximity with Bacillus amyloliquefaciens and Bacillus megaterium (99%), with a cellulase activity of 21.53 ± 1.30 and 19.93 ± 0.38 U mL-1 respectively. Conclusion: In the present study, the thermophilic isolates showed promising Cellulase activity compared to psychrophiles and halophiles.

Preliminary phytochemical and antileishmanial studies of the ethanolic extracts of Pterodon pudescens / Estudos preliminares sobre a fitoquimica e a atividade anti-leishmania de extratos etanólicos de Pterodon pudescens

Pentavalent antimonials are the first choice for the treatment of human leishmaniasis. However in rural areas the traditional plants may be preferred for the treatment of lesions. In recent years a number of papers are published related to the natural products especially plant derivative with infectious diseases. The present work was undertaken to evaluate the antileishmanial activity of Pterodon pubescens which is a native tree widely distributed over the central region of Brazil and used in folk medicine as wine infusions to treat inflammatory disease. The phytochemical screening and the biological essay of ethanolic extract of Pterodon pudescens (PPE) leaves at the concentrations of 150, 300, 450, 600 µg/ml were tested in vitro in Leishmania amazonensis-infected macrophages to support its traditional medicinal use as a leishmaniasis remedy. Phytochemical screening of PPE has shown the presence of catechemical tannins, steroids, triterpenoids and flavonoids. The biological test suggests that PPE were found to control parasite burden of cell cultures in dose-dependent manner. These findings highlight the fact that the apparent potency of Pterodon pudescens compounds, together with their widely distribution over Latin America and Brazil, may represent a promising antileishmanial agent.

Antimoniais pentavalentes são a primeira escolha para o tratamento das leishmanioses humanas. No entanto, no interior brasileiro plantas tradicionais são usadas para o tratamento dessas lesões. De fato, recentes trabalhos tem relatado o potencial terapêutico de produtos naturais, especialmente derivados de plantas. Neste estudo avaliamos a atividade leishmanicida de Pterodon pubescens, uma árvore nativa, distribuída pela região central brasileira e usada em infusões para tratamento de inflamações. Foi realizada a análise fitoquímica e o ensaio in vitro em macrófagos infectados com Leishmania amazonensis em concentrações de 150, 300, 450, 600 µg/ml do extrato etanólico de folhas de Pterodon pudescens (PPE) para comprovar o uso tradicional desta planta como terapia para as leishmanioses. Os testes fitoquímicos indicaram a presença de taninos catequímicos, flavonas, esteroides, triterpenoides, flavonoides e xantonas. Os ensaios biológicos revelaram que o PPE foi capaz de controlar a carga parasitária em macrófagos de maneira dose dependente. Estes resultados corroboram com o potencial terapêutico de compostos de Pterodon pudescens e, junto com sua ampla distribuição no Brasil, podem representar promissor agente leishmanicida.

Comparison of three larval bioassays to evaluate susceptibility of Rhipicephalus (Boophilus) microplus to amitraz / Comparação de três bioensaios larvais para avaliar a suscetibilidade de Rhipicephalus (Boophilus) microplus ao amitraz

Field samples of Rhipicephalus (Boophilus) microplus from the state of Rio Grande do Sul, Brazil, were assessed using the following methods: larval packet test (LPT), larval immersion test (LIT) and syringe immersion test (SIT). The following parameters were determined for each population and for the Mozo susceptible reference strain: lethal concentration for 50% (LC50) with its 95% confidence interval (95% CI), regression line slope and resistance ratio (RR). Using the LPT, only one population was susceptible to amitraz, presenting a RR of 1.9. Using the same technique, the other populations presented RRs of between 92.9 and 3445.8 and were considered resistant. The LC50 of the Mozo strain calculated using the LPT, LIT and SIT was 2.9, 27.3, and 52.7 µg/mL, respectively. In general, a good fit to the probit statistical model was only achieved using the LPT. The results obtained in this study impair recommendations for using the LIT and SIT to diagnose amitraz resistance in R. (B.) microplus populations. Additional studies are required to improve the sensitivity of these tests in relation to the LPT.

Amostras de Rhipicephalus (Boophilus) microplus coletadas à campo no Rio Grande do Sul, Brasil, foram analisadas pelos seguintes métodos: teste do pacote de larvas (TPL), teste de imersão de larvas (TIL) e teste de imersão em seringas (TIS). Os seguintes parâmetros foram determinados para cada população e para a amostra referência suscetível Mozo: concentração letal para 50% (CL50) e seu intervalo de confiança de 95% (IC95%), inclinação da reta de regressão e os fatores de resistência (FR). Pelo TPL, apenas uma população foi sensível ao amitraz, com FR de 1,9. Utilizando a mesma técnica, as outras amostras apresentaram FR entre 92,9 e 3445,8 sendo consideradas resistentes. As CL50 da cepa Mozo calculadas por meio do TPL, TIL e TIS foram 2,9, 27,3 e 52,7 µg/mL, respectivamente. De forma geral, a adequação ao modelo estatístico de probitos só foi alcançada com o uso do TPL. Os resultados obtidos no presente estudo limitam a recomendação de uso do TIL e TIS para diagnóstico de resistência ao amitraz em populações de R. (B.) microplus. Estudos adicionais são necessários para aprimorar a sensibilidade destes testes em relação ao LPT.

Avaliação in situ da genotoxicidade de triazinas utilizando o bioensaio Trad-SHM de Tradescantia clone 4430 / In situ genotoxity evaluation of triazines using Tradescantia clone 4430 Trad-SHM bioassay

O bioensaio da mutação do pelo estaminal de Tradescantia clone 4430 (Trad-SHM) foi utilizado para avaliar a genotoxicidade de um herbicida composto por triazinas (atrazina e simazina) após exposição in situ. Trinta vasos da planta foram expostos durante a aplicação do herbicida (grupo teste) mantendo-se um grupo controle em casa de vegetação. A genotoxicidade foi expressa em termos de eventos mutantes pink (EMP) e a análise dos dados foi realizada por meio do teste t de Student em oito dias de avaliação (C8D = controle 8 dias; T8D = teste 8 dias) e no dia de pico (CPD = controle dia de pico; TPD = teste dia de pico). A exposição ao herbicida causou um número significativamente maior de EMP no grupo teste (T8D = 2,27; TPD = 4,69) do que no controle (C8D = 0,71; CPD = 0,62), demonstrando a existência de risco genotóxico associado ao uso das triazinas, sendo o bioensaio Trad-SHM uma eficiente ferramenta para avaliar o poten­cial genotóxico destes contaminantes ambientais causadores de efeitos adversos à saúde humana.

Tradescantia 4430 clone stamen hair mutation (Trad-SHM) bioassay was used to evaluate the genotoxicity of a herbicide composed of triazines (atrazine and simazine) after in situ exposure. Thirty plant pots were exposed during the herbicide application (test group) and a control group was maintained in a greenhouse (control group). Genotoxicity was expressed in terms of pink mutation events (EMPs) and the data analysis was performed by Student's t test comparing the control and contaminated group after eight-days (C8D = 8-day control; T8D = 8-day test) and peak day (CPD = peak day control; TPD = peak day test). Exposure to the herbicide caused a significantly higher number of EMPs in the test group (T8D = 2.27; TPD = 4.69) than in the control group (C8D = 0.71; CPD = 0.62). This demonstrates that the Trad-SHM bioassay is sensitive and efficient and can be used as tool to assess the genotoxic potential of environmental contaminants like triazines associated with adverse effects on human health.

Antibacterial evaluation of Styrax pohlii and isolated compounds

The antibacterial activity of the compounds egonol (1) and homoegonol (2), of the crude ethanolic extract of Styrax pohlii (Styracaceae) aerial parts (EE), and of its n-hexane (HF), EtOAc (EF), n-BuOH (BF), and hydromethanolic (HMF) fractions was evaluated against the following microorganisms: Streptococcus pneumoniae (ATCC 6305), S. pyogenes (ATCC 19615), Haemophilus influenzae (ATCC 10211), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 10031). The broth microdilution method was used for determination of the minimum inhibitory concentration (MIC) during preliminary evaluation of antibacterial activity. The EE yielded MIC values of 400 µg/mL for S. pneumoniae and P. aeruginosa and 300 µg/mL for H. influenzae. The HF and EF fractions exhibited enhanced antibacterial activity, with MIC values of 200 µg/mL against S. pneumoniae, but only EF displayed activity against H. influenzae (MIC 200 µg/mL). The best MIC value with compounds 1 and 2 (400 µg/mL) was obtained for (1) against S. pneumoniae and P. aeruginosa. Therefore, 1 exhibited weak antibacterial activity against these standard strains.

As atividades antimicrobianas das substâncias egonol (1) e homoegonol (2), do extrato etanólico das partes aéreas de Styrax pohlii (Styracaceae) (EE), bem como das frações n-hexano (HF), AcOEt (EF), n-BuOH (BF) e hidrometanólica (HMF) foram avaliadas frente aos seguintes microorganismos: Streptococcus pneumoniae (ATCC 6305), S. pyogenes (ATCC 19615), Haemophilus influenzae (ATCC 10211), Pseudomonas aeruginosa (ATCC 27853) e Klebsiella pneumoniae (ATCC 10031). O método de microdiluição em caldo foi utilizado para a determinação da concentração inibitória mínima (CIM) na avaliação preliminar da atividade antimicrobiana. EE mostrou valores de CIM de 400 µg/mL para S. pneumoniae e P. aeruginosa, e 300 µg/mL para H. influenzae. As frações HF e EF apresentaram melhora na atividade antimicrobiana, com valores de CIM de 200 µg/mL frente S. pneumoniae, mas apenas EF apresentou ação contra H. influenzae (200 µg/mL). Em relação às substâncias 1 e 2, o melhor valor de CIM (400 µg/mL) foi obtido por 1 frente a S. pneumoniae e P. aeruginosa, que exibiu fraca atividade antimicrobiana contra estas cepas padrões.

Estrés oxidativo en cefalópodos: I. Determinación de TBARS / Oxidative stress in cephalopods: I. TBARS determination

El estrés oxidativo se produce cuando se genera un desbalance desfavorable entre las especies reactivas del oxígeno y las defensas antioxidantes, provocando daño oxidativo a macromoléculas. Varios estudios han resaltado la importancia del estrés oxidativo en el campo de la ecotoxicología, particularmente su relación con el impacto que generan los contaminantes que alcanzan los cuerpos de agua. El cuantifcar los parámetros de estrés oxidativo ha permitido el uso de los mismos como herramienta de diagnóstico (biomarcadores), con capacidad predictiva del impacto de los contaminantes sobre los organismos. Uno de los índices más frecuentemente utilizados para estimar el daño oxidativo a lípidos es la determinación de sustancias reactivas al ácido tiobarbitúrico (TBARS), producto fnal de la peroxidación lipídica. Octopus tehuelchus es un importante recurso pesquero en la costa patagónica, expuesto en algunas áreas a contaminación antrópica. Dado que el estudio de parámetros de estrés oxidativo aún no ha sido abordado en esta Clase de moluscos y que en muchos modelos biológicos, los contaminantes ambientales actúan generando estrés oxidativo, es clave encontrar sus blancos de acción, para empezar a caracterizar las alteraciones metabólicas y fsiológicas asociadas a su mecanismo de acción. El objetivo de este trabajo fue la puesta a punto del método de determinación de daño oxidativo a lípidos en distintos tejidos del pulpo Octopus tehuelchus desde modelos previamente ensayados en el laboratorio.

Oxidative stress occurs when there is an unfavorable imbalance between reactive oxygen species and antioxidant defenses, causing oxidative damage to macromolecules. Several studies have highlighted the importance of oxidative stress in the ecology feld related to the impact generated by pollutants reaching water bodies. The quantifcation of oxidative stress parameters led to their use as diagnostic tools (biomarkers) with predictive capability of showing the impact of pollutants on organisms. One of the most frequently used indexes to estimate the oxidative damage to lipids is the determination of reactive thiobarbituric acid substances (TBARs) (fnal product of lipid peroxidation). Octopus tehuelchus is an important fshery resource in the Patagonian coast exposed to anthropogenic pollution. The study of oxidative stress parameters has not been yet tackled in this class of molluscs. Taking into account that, in many biological models, environmental pollutants generate oxidative stress, it is important to fnd their targets of action, to start to characterize metabolic and physiological alterations associated to their mechanisms of action. The aim of this work was to adjust the method of determination of oxidative damage to lipids in various tissues of the octopus, Octopus tehuelchus, from models previously tested in the laboratory.