Effects of biopesticides in Tetragonisca angustula latreille (Hymenoptera: Meliponinae) pollinators / Efeitos de biopesticidas em polinizadores Tetragonisca angustula latreille (Hymenoptera: Meliponinae) / Efectos de los bioplaguicidas sobre los polinizadores Tetragonisca angustula latreille (Hymenoptera: Meliponinae)

Stingless bees Tetragonisca angustula (Latreille) (Hymenoptera: Meliponinae) are pollinators of native and cultivated plants and are therefore in contact with areas contaminated by pesticides. These native bees were evaluated for changes in gene expression of esterase isoenzymes (EST) and peptides after contamination by contact with growth regulators from insecticides Gallaxy® EC 100, Natuneem and Azamax after 48, 120, 168 hours, 30 and 60 days. EST-4 presented an increase in relative activity after contamination with Gallaxy® EC 100 at 6.2 × 10-2 g a.i./mL; Natuneem at 7.5 × 10-5 g a.i./mL; and Azamax at 1.2 × 10-3 g a.i/mL after 60 days, 48 h, and 60 days, respectively. Inhibition of the relative activity of EST-4 was detected after contamination by Natuneem at 1.5 × 10-5 g a.i./mL and Azamax at 1.2 × 10-3 g a.i./mL after 48 h and 30 days, respectively. The insecticide growth regulators promoted changes in protein synthesis of T. angustula adult workers resulting in an increase or decrease in the relative intensity of bands, and the appearance of new peptides when compared with controls. Changes in protein synthesis have been identified mainly after long period of contamination, 120 and 168 h with the IGRs Gallaxy® EC 100 (at 0.78 and 1.25 g a.i./mL), Azamax (at 1.2 × 10-3 and 6 × 10-3 g a.i./mL), and Natuneem (at 7.5 × 10-5 and 3 × 10-3 g a.i./mL), and at 60 days with Natuneem (at 1.5 × 10-5 g a.i./mL).(AU)

Abelhas sem ferrão Tetragonisca angustula (Latreille) (Hymenoptera: Meliponinae) são polinizadores de plantas nativas e cultivadas e, portanto, estão em contato com áreas contaminadas por biopesticidas. Essas abelhas nativas foram avaliadas quanto a alterações na expressão gênica de isoenzimas esterases (EST) e peptídeos após contaminação por contato com reguladores de crescimento de inseticidas Gallaxy® EC 100, Natuneem e Azamax após 48, 120, 168 horas, 30 e 60 dias. A EST-4 apresentou um aumento na atividade relativa após a contaminação com Gallaxy® 100 EC em 6,2 × 10-2 g i.a./mL, Natuneem em 7,5 × 10-5 g i.a./mL e Azamax em 1,2 × 10-3 g i.a./mL após 60 dias, 48 h e 60 dias, respectivamente. A inibição da atividade relativa de EST-4 foi detectada após contaminação pelo Natuneem a 1,5 × 10-5 g i.a./mL e Azamax a 1,2 × 10-3 g i.a./mL após 48 he 30 dias, respectivamente. Os reguladores de crescimento de inseticidas promoveram alterações na síntese protéica de trabalhadores adultos de T. angustula, resultando em um aumento ou diminuição da intensidade relativa das bandas e no aparecimento de novos peptídeos em comparação com os controles. Alterações na síntese de proteínas foram identificadas principalmente após um longo período de contaminação, 120 e 168 h com o IGRs Gallaxy® EC 100 (0,78 e 1,25 g i.a./mL), Azamax (1,2 × 10-3 e 6 × 10-3 g i.a./mL) e Natuneem (7,5 × 10-5 e 3 × 10-3 g i.a./mL) e 60 dias com Natuneem (1,5 × 10-5 g i.a./mL).(AU)

Las abejas sin aguijón Tetragonisca angustula (Latreille) (Hymenoptera: Meliponinae) son polinizadores de plantas nativas y cultivadas y, por lo tanto, están en contacto con áreas contaminadas por bioplaguicidas. Estas abejas nativas fueron evaluadas para detectar cambios en la expresión génica de isoenzimas esterasa (EST) y péptidos después de la contaminación por contacto con los reguladores del crecimiento insecticidas Gallaxy® EC 100, Natuneem y Azamax después de 48, 120, 168 horas, 30 y 60 días. EST-4 mostró un aumento en la actividad relativa después de la contaminación con Gallaxy® 100 EC a 6.2 × 10-2 g i.a./mL, Natuneem a 7.5 × 10-5 g i.a./mL y Azamax a 1.2 × 10-3 g i.a./mL después de 60 días, 48 hy 60 días, respectivamente. La inhibición de la actividad relativa de EST-4 se detectó después de la contaminación por Natuneem a 1.5 × 10-5 g i.a./mL y Azamax a 1.2 × 10-3 g i.a./mL después de 48 hy 30 días. respectivamente. Los insecticidas reguladores del crecimiento promovieron cambios en la síntesis de proteínas de trabajadores adultos de T. angustula, resultando en un aumento o disminución de la intensidad relativa de las bandas y en la aparición de nuevos péptidos en relación a los controles. Los cambios en la síntesis de proteínas se identificaron principalmente después de un largo período de contaminación, 120 y 168 h con IGRs Gallaxy® EC 100 (0.78 y 1.25 g i.a./mL), Azamax (1.2 × 10-3 y 6 × 10-3 g i.a./mL) y Natuneem (7.5 × 10-5 y 3 × 10-3 g i.a./mL) y 60 días con Natuneem (1.5 × 10-5 g i.a./mL).(AU)

Determination of insecticides' lethal concentrations and metabolic enzyme levels in Triatoma dimidiata / Determinación de concentraciones letales y niveles de enzimas metabólicas de insecticidas en Triatoma dimidiata

Abstract: Objective: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata. Materials and methods: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays. Results: Respectively the LC50 and LC99 µg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and β-eterase levels. Conclusion: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata.

Resumen: Objetivo: La factibilidad de usar los papeles impregnados y ensayos bioquímicos según la OMS para determinar concentraciones letales (CL50 y CL99) y niveles enzimáticos en la resistencia a insecticidas en Triatoma dimidiata. Material y métodos: Se calcularon la CL50 y CL99 con papeles impregnados según la OMS a diferentes concentraciones de malatión, propoxur y deltametrina; el porcentaje de acetilcolinesterasa insensible (iAChE); y los niveles de esterasas, glutatión S-transferasas, y monooxigenasas en ninfas de laboratorio del estadio I (5-7 días) se determinaron usando los ensayos bioquímicos según la OMS. Resultados: Se obtuvieron las CL50 y CL99 µg / cm2 respectivamente para malatión 43.83 y 114.38, propoxur 4.71 y 19.29, y deltametrina 5.80 y 40.46. Un 30% de las chinches tuvo iAChE, y sólo pocos individuos tuvieron niveles superiores de P450 y β-eterasas. Conclusión: Los papeles impregnados y ensayos bioquímicos que describe la OMS para otros insectos demostraron ser métodos factibles para monitorear la resistencia a insecticidas y las enzimas metabólicas involucradas en T. dimidiata.

Cometabolic biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of QPEH1 esterase

BACKGROUND: Quizalofop-p-ethyl (QPE), a unitary R configuration aromatic oxyphenoxypropionic acid ester (AOPP) herbicide, was widely used and had led to detrimental environmental effects. For finding the QPEdegrading bacteria and promoting the biodegradation of QPE, a series of studies were carried out. RESULTS: A QPE-degrading bacterial strain YC-XJ1 was isolated from desert soil and identified as Methylobacterium populi, which could degrade QPE with methanol by cometabolism. Ninety-seven percent of QPE (50 mg/L) could be degraded within 72 h under optimum biodegradation condition of 35°C and pH 8.0. The maximum degradation rate of QPE was 1.4 mg/L/h, and the strain YC-XJ1 exhibited some certain salinity tolerance. Two novel metabolites, 2-hydroxy-6-chloroquinoxaline and quinoxaline, were found by high-performance liquid chromatography/mass spectroscopy analysis. The metabolic pathway of QPE was predicted. The catalytic efficiency of strain YC-XJ1 toward different AOPPs herbicides in descending order was as follows: haloxyfop-pmethyl ≈ diclofop-methyl ≈ fluazifop-p-butyl N clodinafop-propargyl N cyhalofop-butyl N quizalofop-p-ethyl N fenoxaprop-p-ethyl N propaquizafop N quizalofop-p-tefuryl. The genome of strain YC-XJ1 was sequenced using a combination of PacBio RS II and Illumina platforms. According to the annotation result, one α/ß hydrolase gene was selected and named qpeh1, for which QPE-degrading function has obtained validation. Based on the phylogenetic analysis and multiple sequence alignment with other QPE-degrading esterases reported previously, the QPEH1 was clustered with esterase family V. CONCLUSION: M. populi YC-XJ1 could degrade QPE with a novel pathway, and the qpeh1 gene was identified as one of QPE-degrading esterase gene.

Partial purification and biochemical characterization of an alkaline esterase from Sorghumbicolor

Esterases are enzymes that present good potential for industrial applications since they catalyze the formation or cleavage of ester bonds in water-soluble substrates, and sorghumseeds can represent an alternative source of this enzyme. The extraction of esterase from sorghumseeds is an economical alternative to obtain an enzyme of great interest. Esterases may improve the quality or accelerate the maturation of cheeses, cured bacon and fermented sausages and may also resolve racemic mixtures. Recently, seed esterases have been the focus of much attention as biocatalysts. In some cases, these enzymes present advantages over animal and microbial lipases due to some quite interesting features such as specificity and low cost, being a great alternative for their commercial exploitation as industrial enzymes The esterase studied here was extracted from sorghumseeds and some of its biochemical properties determined using synthetic substrates (p-nitrophenyl butyrate, caprylate, laurate and palmitate). The enzyme presented optimum activity at pH 8.0 and was stable in all the pH ranges studied. The optimum temperature for its activity was 40ºC but it showed low stability at this temperature (40% relative activity). The values derived for Km and Vmax were 0.67mM and 125 U.mg-1, respectively, obtained using p-nitrophenyl butyrate as the substrate. The enzyme showed an increase in activity when K2HPO4was added to the reaction medium, but the ions Mn2+, CO+, Hg+and Fe2+strongly inhibited the enzyme activity. This enzyme showed a preference for the hydrolysis of short chain fatty acids. The characteristics of sorghumesterase are very similar to those of the microbial esterases used in detergent processing.

Germination temperatures affect the physiological quality of seeds of lettuce cultivars / Qualidade fisiológica de sementes de alface sob diferentes temperaturas na germinação

The objective of this study was to evaluate the physiological quality allied to biochemical quality of lettuce seeds by germination and enzymes expression at 20, 25, 30, 35, 40 and 42ºC. Germination speed index and percentage of germination were estimated. Isoenzyme expressions were assessed by alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), catalase (CAT), esterase (EST), pyruvate decarboxylase (PDC) and glutamate oxaloacetate transferase (GOT). The experiment consisted of a completely randomized design in a factorial scheme 4x6, with four cultivars and six different temperatures, with four replications. The highest germination and vigor were observed for cv. 'Everglades' at 35°C, which proved that this cultivar is thermotolerant. Catalase can be considered a genetic marker for the identification ofthermotolerant lettuce cultivars. Cultivar 'Everglades' has potential to be used in lettuce breeding programs aimed at cultivars tolerant to high temperatures during germination.

O objetivo deste estudo foi avaliar a qualidade fisiológica e bioquímica de sementes de alface por meio da germinação e expressão de enzimas a 20, 25, 30, 35, 40 e 42ºC. As variáveis velocidade de germinação e o índice de velocidade de germinação foram estimadas. As expressões das enzimas alcool desidrogenase (ADH), malato desidrogenase (MDH), catalase (CAT), esterase (EST), piruvate descarboxilase (PDC) e glutamato oxaloacetato transferase (GOT) foram avaliadas. Para análise dos genótipos foi empregado o delineamento inteiramente casualizado em esquema fatorial 4x6, testando quatro cultivares e seis diferentes temperaturas, com quatro repetições. A maior germinação e vigor foram observadas para a cv. 'Everglades' a 35°C, o que prova que esta cultivar é termotolerante. A catalase pode ser considerada um marcador para a identificação de cultivares de alface termotolerantes. A cultivar 'Everglades' tem potential para uso em programas de melhoramento visando tolerância à alta temperatura durante a germinação.

Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein

BACKGROUND: Biologically active peptides produced from fish wastes are gaining attention because their health benefits. Proteases produced by halophilic microorganisms are considered as a source of active enzymes in high salt systems like fish residues. Hence, the aim of this study was the bioprospection of halophilic microorganisms for the production of proteases to prove their application for peptide production. RESULTS: Halophilic microorganisms were isolated from saline soils of Mexico and Bolivia. An enzymatic screening was carried out for the detection of lipases, esterases, pHB depolymerases, chitinases, and proteases. Most of the strains were able to produce lipases, esterases, and proteases, and larger hydrolysis halos were detected for protease activity. Halobacillus andaensis was selected to be studied for proteolytic activity production; the microorganism was able to grow on gelatin, yeast extract, skim milk, casein, peptone, fish muscle (Cyprinus carpio), and soy flour as protein sources, and among these sources, fish muscle protein was the best inducer of proteolytic activity, achieving a protease production of 571 U/mL. The extracellular protease was active at 50°C, pH 8, and 1.4 M NaCl and was inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of H. andaensis was used to hydrolyze fish muscle protein for peptide production. The peptides obtained showed a MW of 5.3 kDa and a radical scavenging ability of 10 to 30% on 2,2-diphenyl-1-picrylhydrazyl and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and a ferric reducing ability of plasma. Conclusion: The use of noncommercial extracellular protease produced by H. andaensis for biologically active peptide production using fish muscle as the protein source presents a great opportunity for high-value peptide production.

Esterase activity in homogenates of Helicoverpa armigera (Hubner) (Lepidoptera: noctuidae) exposed to different insecticides and the behavioral effect / Atividade esterase em homogenatos de helicoverpa armigera (hubner) (lepidoptera: noctuidae) expostos a diferentes inseticidas e o efeito comportamental

The toxicity of some insecticides to Helicoverpa armigera was studied through sublethal effects, evaluating the enzymatic activity of esterase and the behavioral response. The commercial formulation of insecticides selected were chlorpyrifos, spinosad, indoxacarb, chlorantraniliprole, lambda-cyhalothrinand Bacillus thuringiensis, corresponding the most used by farmers to control of H. armigera. To determine the esterase activity, the larvae were fed with soybean leaf discs dipped in insecticide solution using the lethal concentration (LC50). For the behavioral response, filter paper were immersed in three concentrations of insecticides (LC50, LC95 and recommend dose.), then the behavior of the larvae observed in Videomex-One. The results for the enzymatic activity showed an increase in the activity of the esterase, with variation along the treatments and the time of exposure of the insects to the insecticides. With exception of spinosad, other insecticides showed an increase in EST-α activity, 6 and 24 hours after contact of caterpillars with insecticide.Different behavioral patterns of walking (walking distance, walking speed and resting time) were observed for H. armigera exposed to different insecticides. H. armigera exposed to chlorpyrifos, lambda-cyhalothrin and B. thuringiensis insecticides show greater esterase activity. Regarding walking behavior, the results confirms enzymatic activity, where H. armigera have behavioral alteration after exposure to insecticide.

A toxicidade de alguns inseticidas a Helicoverpa armigera foi estudada através de efeitos subletais, avaliando a atividade enzimática da esterase e a resposta comportamental. A formulação comercial dos inseticidas selecionados foram clorpirifos, espinosad, indoxacarb, clorantraniliprole, lambda-cialotrina eBacillus thuringiensis utilizados pelos agricultores para controle de H. armigera. Para determinar a atividade da esterase, as larvas foram alimentadas com discos de folha de soja mergulhados em solução de inseticida usando a concentração letal (CL50). Para os testes de características comportamentais, papel filtro foi imerso em três concentrações de inseticidas (CL50, CL95 e dose recomendada), posteriormente o comportamento das larvas foi observado em Videomex-One. Os resultados para a atividade enzimática mostraram aumento da atividade daesterase, com variação ao longo dos tratamentos e do tempo de exposição dos insetos aos inseticidas. Com exceção do spinosad, outros inseticidas mostraram um aumento na atividade EST-α, 6 e 24 horas após o contato das lagartas com inseticida. Diferentes padrões comportamentais de caminhada (distância percorrida, velocidade de caminhada e tempo de repouso) para H. armigera exposto a diferentes classes de inseticidas. H. armigera exposta aos inseticidas clorpirifos, lambda-cialotrina e B. thuringiensis mostraram maior atividade da esterase. Em relação ao comportamento ambulante, os resultados confirmam a atividade enzimática, onde H. armigera teve alteração comportamental após exposição ao inseticida.

Notum attenuates HBV-related liver fibrosis through inhibiting Wnt 5a mediated non-canonical pathways

BACKGROUND: Non-canonical Wnt pathways play important roles in liver fibrosis. Notum is a newly discovered inhibitor to Wnt proteins. This study was to investigate anti-fibrotic effects of Notum. METHODS: 53 patients with hepatitis B virus (HBV) infection as well as a cell co-culture system of LX-2 and Hep AD38 cells were engaged in this study. Clinical, biological and virological data of each patient were analyzed. Cell viability was detected at different time points. mRNA and protein levels of NFATc1 (Nuclear factor of activated T-cells), Jnk, α-SMA, Col1A1 and TIMP-1 were detected both in LX-2 and liver tissue. Protein levels of NFATc1 and Jnk in liver tissue and their correlations with fibrosis score were analyzed. RESULTS: Hepatitis B virus replication up-regulated Wnt5a induced NFATc1 and Jnk activity in Hep AD38. Notum suppressed NFATc1, Jnk and fibrosis genes expression, reduced cell viability in co-cultured LX-2 cells induced by HBV. Interestingly, Patients with HBV DNA > 5log copies/ml had higher mRNA levels of NFATc1 and fibrosis genes than patients with HBV DNA < 5log copies/ml. Most importantly, protein expressions of NFATc1 and pJnk have positive correlations with liver fibrosis scores in HBV-infected patients. CONCLUSIONS: Our data showed that Notum inhibited HBV-induced liver fibrosis through down-regulating Wnt 5a mediated non-canonical pathways. This study shed light on anti-fibrotic treatment.

A novel chlorpyrifos hydrolase CPD from Paracoccus sp. TRP: molecular cloning, characterization and catalytic mechanism

Background: Biodegradation is a reliable approach for efficiently eliminating persistent pollutants such as chlorpyrifos. Despite many bacteria or fungi isolated from contaminated environment and capable of degrading chlorpyrifos, limited enzymes responsible for its degradation have been identified, let alone the catalytic mechanism of the enzymes. Results: In present study, the gene cpd encoding a chlorpyrifos hydrolase was cloned by analysis of genomic sequence of Paracoccus sp. TRP. Phylogenetic analysis and BLAST indicated that CPD was a novel member of organophosphate hydrolases. The purified CPD enzyme, with conserved catalytic triad (Ser155-Asp251-His281) and motif Gly-Asp-Ser-Ala-Gly, was significantly inhibited by PMSF, a serine modifier. Molecular docking between CPD and chlorpyrifos showed that Ser155 was adjacent to chlorpyrifos, which indicated that Ser155 may be the active amino acid involved in chlorpyrifos degradation. This speculation was confirmed by site-directed mutagenesis of Ser155Ala accounting for the decreased activity of CPD towards chlorpyrifos. According to the key role of Ser155 in chlorpyrifos degradation and molecular docking conformation, the nucleophilic catalytic mechanism for chlorpyrifos degradation by CPD was proposed. Conclusion: The novel enzyme CPD was capable of hydrolyze chlorpyrifos and Ser155 played key role during degradation of chlorpyrifos.

Performance of the dipstick screening test as a predictor of negative urine culture / Desempenho da fita de urina como resultado presuntivo para cultura de urina negativa

ABSTRACT Objective To investigate whether the urine dipstick screening test can be used to predict urine culture results. Methods A retrospective study conducted between January and December 2014 based on data from 8,587 patients with a medical order for urine dipstick test, urine sediment analysis and urine culture. Sensitivity, specificity, positive and negative predictive values were determined and ROC curve analysis was performed. Results The percentage of positive cultures was 17.5%. Nitrite had 28% sensitivity and 99% specificity, with positive and negative predictive values of 89% and 87%, respectively. Leukocyte esterase had 79% sensitivity and 84% specificity, with positive and negative predictive values of 51% and 95%, respectively. The combination of positive nitrite or positive leukocyte esterase tests had 85% sensitivity and 84% specificity, with positive and negative predictive values of 53% and 96%, respectively. Positive urinary sediment (more than ten leukocytes per microliter) had 92% sensitivity and 71% specificity, with positive and negative predictive values of 40% and 98%, respectively. The combination of nitrite positive test and positive urinary sediment had 82% sensitivity and 99% specificity, with positive and negative predictive values of 91% and 98%, respectively. The combination of nitrite or leukocyte esterase positive tests and positive urinary sediment had the highest sensitivity (94%) and specificity (84%), with positive and negative predictive values of 58% and 99%, respectively. Based on ROC curve analysis, the best indicator of positive urine culture was the combination of positives leukocyte esterase or nitrite tests and positive urinary sediment, followed by positives leukocyte and nitrite tests, positive urinary sediment alone, positive leukocyte esterase test alone, positive nitrite test alone and finally association of positives nitrite and urinary sediment (AUC: 0.845, 0.844, 0.817, 0.814, 0.635 and 0.626, respectively). Conclusion A negative urine culture can be predicted by negative dipstick test results. Therefore, this test may be a reliable predictor of negative urine culture.

RESUMO Objetivo Verificar se a triagem de urina por fitas reativas é capaz de predizer a cultura de urina. Métodos Estudo retrospectivo realizado entre janeiro e dezembro de 2014 com 8.587 pacientes, com solicitação médica de triagem de urina (fita), sedimento urinário e cultura de urina. Foram analisados sensibilidade, especificidade, valor preditivo positivo, valor preditivo negativo e curva ROC. Resultados Foram positivas 17,5% das culturas. O nitrito apresentou sensibilidade de 28% e especificidade de 99%. O valor preditivo positivo foi de 89% e o valor preditivo negativo de 87%. Esterase apresentou sensibilidade de 79% e especificidade de 84%. Valor preditivo positivo e valor preditivo negativo foram de 51% e 95%, respectivamente. A combinação de nitrito ou esterase positivos apresentou sensibilidade de 85% e especificidade de 84%. Valor preditivo positivo e valor preditivo negativo foram, respectivamente, 53% e 96%. O sedimento positivo (mais de dez leucócitos por microlitro) apresentou sensibilidade de 92% e especificidade de 71%. O valor preditivo positivo foi 40% e o negativo, 98%. A combinação de nitrito e sedimento urinário positivos apresentou sensibilidade de 82% e especificidade de 99%. Os valores preditivos positivo e negativo foram 91% e 98%, respectivamente. Para o nitrito ou esterase positivos mais os leucócitos positivos, a sensibilidade foi de 94% e a especificidade de 84%. O valor preditivo positivo foi de 58% e o negativo foi de 99%. Com base na curva ROC, o melhor indicador de urocultura positiva foi a associação entre a esterase ou nitrito positivos na fita mais os leucócitos positivos no sedimento, seguido por nitrito e esterase positivos, sedimento urinário positivo isolado, esterase positiva isolada, nitrito positivo isolado e, finalmente, pela associação entre nitrito e sedimento urinário positivos (AUC: 0,845, 0,844, 0,817, 0,814, 0,635 e 0,626, respectivamente). Conclusão Uma urocultura negativa pode ser prevista com resultados negativos na fita. Portanto, este teste pode ser um preditor confiável de urocultura negativa.

Enzymatic potential of heterotrophic bacteria from a neutral copper mine drainage

Abstract Copper mine drainages are restricted environments that have been overlooked as sources of new biocatalysts for bioremediation and organic syntheses. Therefore, this study aimed to determine the enzymatic activities (esterase, epoxide hydrolase and monooxygenase) of 56 heterotrophic bacteria isolated from a neutral copper mine drainage (Sossego Mine, Canaã dos Carajás, Brazil). Hydrolase and monooxygenase activities were detected in 75% and 20% of the evaluated bacteria, respectively. Bacterial strains with good oxidative performance were also evaluated for biotransformation of organic sulfides. Fourteen strains with good enzymatic activity were identified by 16S rRNA gene sequencing, revealing the presence of three genera: Bacillus, Pseudomonas and Stenotrophomonas. The bacterial strains B. megaterium (SO5-4 and SO6-2) and Pseudomonas sp. (SO5-9) efficiently oxidized three different organic sulfides to their corresponding sulfoxides. In conclusion, this study revealed that neutral copper mine drainages are a promising source of biocatalysts for ester hydrolysis and sulfide oxidation/bioremediation. Furthermore, this is a novel biotechnological overview of the heterotrophic bacteria from a copper mine drainage, and this report may support further microbiological monitoring of this type of mine environment.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

Esterase isozymes patterns of grape vine (Vitis vinifera L) are altered in response to fungicide exposure / Padrões de isozimas Esterases de videira (Vitis vinifera L) alterados em resposta à exposição a fungicidas

Current analysis characterizes the effect of different fungicides often applied for pest control on α−and ß-esterase patterns of four economically important table-wine grape cultivars (Italia, Rubi, Benitaka and Brasil) of Vitis vinifera. The α- and ß-esterase patterns in bud leaves of the cultivars were assessed by native PAGE analysis. Cabrio Top® compound inhibited EST-2, EST-5, EST-6, EST-7, EST-8, EST-9 and EST-10 carboxylesterases, whereas EST-4, EST-11, EST-12, EST-13, EST-14 acetylesterases and EST-16 carboxylesterase were detected as weakly stained bands. Carboxylesterases and acetylesterases were also detected as weakly stained bands when exposed to fungicides Orthocide 500®, Positron Duo® and Folicur PM®. No changes in α- and ß-esterase patterns were reported when the vines were exposed to the fungicides Rovral SC®, Kumulus DF®, Curzate M®, Score® or Cuprogarb 500®. The evidence of functional changes in carboxylesterase and acetylesterase levels in current study is a warning to grape producers on the dangers inherent in the indiscriminate use of potent and modern fungicides extensively used in agriculture. The inhibition effect of fungicides on esterase isozyme molecules seems to be independent of the fungicide chemical.

O presente estudo caracterizou o efeito de diferentes fungicidas comumente aplicados como medidas de controle de pragas sobre padrões de α- e ß-esterases de quatro importantes cultivares de uva de mesa (Itália, Rubi, Benitaka e Brasil) de Vitis vinifera. Os padrões de α- e ß-esterases de brotos foliares das cultivares foram avaliados por PAGE. O composto Cabrio Top® inibiu as carboxilesterases EST-2, EST-5, EST-6, EST-7, EST-8, EST-9 e EST-10, enquanto as acetilesterases EST-4, EST-11, EST-12, EST-13, EST-14 e a carboxilesterase EST-16 foram detectadas como bandas fracamente coradas. As carboxilesterases e acetilesterases também foram detectadas como bandas fracamente coradas quando expostas aos fungicidas Orthocide 500®, Positron Duo® e Folicur PM®. Não foram observadas alterações nos padrões de α- e ß-esterases quando as videiras foram expostas aos fungicidas Rovral SC®, Kumulus DF®, Curzate M®, Score® ou Cuprogarb 500®. A evidência de alterações em nível funcional em carboxilesterases e acetilesterases, apresentada neste estudo, pode servir como um alerta aos produtores de uva dos perigos inerentes ao uso indiscriminado de fungicidas potentes e modernos amplamente utilizados hoje na agricultura. O efeito dos fungicidas sobre as enzimas esterases parece ser independente do grupo químico ao qual pertence o fungicida.

Effects of piperonyl butoxide on the toxicity of the organophosphate temephos and the role of esterases in the insecticide resistance of Aedes aegypti

Introduction The effects of piperonyl butoxide (PBO) on the toxicity of the organophosphate temephos (TE) and the role of esterases in the resistance of Aedes aegypti to this insecticide were evaluated. Methods A. aegypti L4 larvae susceptible and resistant to TE were pre-treated with PBO solutions in acetone at concentrations of 0.125, 0.25, 0.5, 1, and 2% for 24h and subsequently exposed to a diagnostic concentration of 0.02mg/L aqueous TE solution. The esterase activity of the larvae extracts pre-treated with varying PBO concentrations and exposed to TE for three time periods was determined. Results At concentrations of 0.25, 0.5, 1, and 2%, PBO showed a significant synergistic effect with TE toxicity. High levels of esterase activity were associated with the survival of A. aegypti L4 larvae exposed to TE only. Conclusions The results of the biochemical assays suggest that PBO has a significant inhibitory effect on the total esterase activity in A. aegypti larvae. .

Perfil de suscetibilidade da população de Aedes aegypti (Diptera: Culicidae) da ilha de Santiago, Cabo Verde, a inseticidas / Susceptibility profile of Aedes aegypti from Santiago Island, Cape Verde, to insecticides

Em Cabo Verde, arquipélago situado na Costa Ocidental Africana, os primeiros casos de dengue ocorreram em 2009, com a notificação de mais de 21.000 casos, a maioria desses registrados na Ilha de Santiago. O mosquito Aedes aegypti foi identificado como vetor, e ações para seu controle, usando os inseticidas temephos (larvicida) e a deltametrina (adulticida), têm sido implementadas. Objetiva-se com esse trabalho avaliar o atual status de suscetibilidade a inseticidas e caracterizar os mecanismos de resistência nessa população. Amostras de A. aegypti da ilha de Santiago foram coletadas através de armadilhas de oviposição, para o estabelecimento de uma população a ser analisada. Foram realizados bioensaios do tipo dose diagnóstica, usando garrafas impregnadas com doses únicas dos adulticidas malathion (organofosforado), deltametrina (piretróide) e cipermetrina (piretróide), e bioensaios do tipo dose resposta, usando múltiplas concentrações dos inseticidas temephos (organofosforado), Bacillus thuringiensis sorovariedade israelensis (bactéria entomopatogênica) e diflubenzuron (inibidor de síntese de quitina). Para a investigação dos mecanismos de resistências, foram realizados testes bioquímicos com substratos específicos para quantificar a atividade das enzimas glutationa S-transferases, esterases (alfa, beta e PNPA) e oxidases de função mista, ligadas a detoxificação de xenobióticos, e a taxa de inibição da acetilcolinesterase ligada a insensibilidade do sítio alvo...

Cape Verde, an archipelago located on the West African Coast, recorded the first cases of dengue in 2009 in an epidemic with more than 21,000 reportedcases. The worst affected area was Santiago Island...

Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia) and from domiciliary areas, including El Palmar (Bolivia) and La Pista (Argentina). Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL) were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR) value (44.90) compared to the susceptible reference strain (NFS), whereas the Veinte de Octubre samples had the lowest value (0.50). Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP) on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein) and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively). The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP).

Endophytic fungi producing of esterases: evaluation in vitro of the enzymatic activity using pH indicator

A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS). The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project "Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest". The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1 -carboxylesterases) for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue), changing the color of the reaction medium (from blue to yellow), that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea asapotential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms.

Spatial distribution and esterase activity in populations of Aedes (Stegomyia) aegypti (Linnaeus) (Diptera: Culicidae) resistant to temephos

INTRODUCTION: The need for studies that describe the resistance patterns in populations of Aedes aegypti (Linnaeus) in function of their region of origin justified this research, which aimed to characterize the resistance to temephos and to obtain information on esterase activity in populations of Aedes aegypti collected in municipalities of the State of Paraíba. METHODS: Resistance to temephos was evaluated and characterized from the diagnostic dose of 0.352mg i.a./L and multiple concentrations that caused mortalities between 5% and 99%. Electrophoresis of isoenzymes was used to verify the patterns of esterase activity among populations of the vector. RESULTS: All populations of Aedes aegypti were resistant to temephos, presenting a resistance rate (RR) greater than 20. The greatest lethal dose 50% of the sample (CL50) was found for the municipality of Lagoa Seca, approximately forty-one times the value of CL50 for the Rockefeller population. The populations characterized as resistant showed two to six regions of α and β-esterase, called EST-1 to EST-6, while the susceptible population was only seen in one region of activity. CONCLUSIONS: Aedes aegypti is widely distributed and shows a high degree of resistance to temephos in all municipalities studied. In all cases, esterases are involved in the metabolism and, consequently, in the resistance to temephos. .

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.