In Vitro Metabolism of Montelukast by Cytochrome P450s and UDP-Glucuronosyltransferases.

Montelukast has been recommended as a selective in vitro and in vivo probe of cytochrome P450 (P450) CYP2C8 activity, but its selectivity toward this enzyme remains unclear. We performed detailed characterization of montelukast metabolism in vitro using human liver microsomes (HLMs), expressed P450s, and uridine 5'-diphospho-glucuronosyltransferases (UGTs). Kinetic and inhibition experiments performed at therapeutically relevant concentrations reveal that CYP2C8 and CYP2C9 are the principal enzymes responsible for montelukast 36-hydroxylation to 1,2-diol. CYP3A4 was the main catalyst of montelukast sulfoxidation and stereoselective 21-hydroxylation, and multiple P450s participated in montelukast 25-hydroxylation. We confirmed direct glucuronidation of montelukast to an acyl-glucuronide. We also identified a novel peak that appears consistent with an ether-glucuronide. Kinetic analysis in HLMs and experiments in expressed UGTs indicate that both metabolites were exclusively formed by UGT1A3. Comparison of in vitro intrinsic clearance in HLMs suggest that direct glucuronidation may play a greater role in the overall metabolism of montelukast than does P450-mediated oxidation, but the in vivo contribution of UGT1A3 needs further testing. In conclusion, our in vitro findings provide new insight toward montelukast metabolism. The utility of montelukast as a probe of CYP2C8 activity may be compromised owing to involvement of multiple P450s and UGT1A3 in its metabolism.

Expression profile and subcellular localization of HslV, the proteasome related protease from Trypanosoma cruzi.

Trypanosoma cruzi is a rare example of an eukaryote that has genes for two threonine proteases: HslVU complex and 20S proteasome. HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. In this study, we expressed and obtained specific antibodies to HslU and HslV recombinant proteins and demonstrated the interaction between HslU/HslV by coimmunoprecipitation. To evaluate the intracellular distribution of HslV in T. cruzi we used an immunofluorescence assay and ultrastructural localization by transmission electron microscopy. Both techniques demonstrated that HslV was localized in the kinetoplast of epimastigotes. We also analyzed the HslV/20S proteasome co-expression in Y, Berenice 62 (Be-62) and Berenice 78 (Be-78) T. cruzi strains. Our results showed that HslV and 20S proteasome are differently expressed in these strains. To investigate whether a proteasome inhibitor could modulate HslV and proteasome expressions, epimastigotes from T. cruzi were grown in the presence of PSI, a classical proteasome inhibitor. This result showed that while the level of expression of HslV/20S proteasome is not affected in Be-78 strain, in Y and Be-62 strains the presence of PSI induced a significantly increase in Hslv/20S proteasome expression. Together, these results suggest the coexistence of the protease HslVU and 20S proteasome in T. cruzi, reinforcing the hypothesis that non-lysosomal degradation pathways have an important role in T. cruzi biology.

Label-free aptasensor for p24-HIV protein detection based on graphene quantum dots as an electrochemical signal amplifier.

Human immunodeficiency virus (HIV) is still considered a pandemic, and the detection of p24-HIV protein has an important role in the early diagnosis of HIV in adults and newborns. The accessibility of these trials depends on the price and execution difficulty of the method, which can be reduced using electrochemical methods by using enzymeless approaches, disposable and accurate devices. In this work, graphene quantum dots were acquired by a simple synthesis and employed as an electrochemical signal amplifier and support for the aptamer immobilization through a feasible and stable modification of disposable screen-printed electrodes. The device has been easily assembled and used to detect p24-HIV protein without the interference of similar proteins or sample matrix. Using the best set of experimental conditions, a linear correlation between analytical signal and log of p24-HIV concentration from 0.93 ng mL-1 to 93 µg mL-1 and a limit of detection of 51.7 pg mL-1 were observed. The developed device was applied to p24 determination in spiked human serum and provided distinct levels of signal for positive and negative samples, successfully identifying real samples with the target protein. This sensor is a step towards the development of point-of-care devices and the popularization of electrochemical methods for trials and diagnostics of relevant diseases.

Design of experiments applied to stress testing of pharmaceutical products: A case study of Albendazole.

Forced degradation tests are studies used to assess the stability of active pharmaceutical ingredients (APIs) and their formulations. These tests are performed submitting the API under extreme conditions in order to know the main degradation products in a short period of time. The results of these studies are used to assess the degradation susceptibility of APIs and to validate chromatographic analytical methods. However, most of degradation studies are performed using one-factor-at-the-time (OFAT) which does not consider the interactions between degradation variables. This work proposes the use of Design of Experiment (DoE) approach in forced degradation of albendazole (ABZ). It was used a central composite design (CCD) to evaluate the forced degradation in a multivariate way. Experiments were performed taking into account the variables pH, temperature, oxidizing agent (H2O2) and UV radiation. It was verified the influence of the variables and their interactions on the ABZ degradation. The ABZ oxidation showed to be the main degradation route for ABZ, which is strongly influenced by the temperature. The hydrolysis was relevant at alkaline medium and high temperature. LC-IT-MSn was used to identify the degradation products. It was found three known degradation products (albendazole-2-amino, albendazole sulfoxide and albendazole sulfone) and a new derivate of albendazole molecule (albendazole sulfoxide with a chlorine). This last one was isolated and characterized by UPLC-QToF-MS and NMR analyses.

In vitro effects of citral on trypanosoma cruzi metacyclogenesis.

Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 µg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 µg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 µg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h) of about 31 µg/mL. Treatment with 30 µg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 µg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 µg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.

Study of the in vitro metabolic profile of a new α2-adrenergic agonist in rat and human liver microsomes by using liquid chromatography-multiple-stage mass spectrometry and nuclear magnetic resonance.

A potent synthetic α2-adrenergic agonist called PT-31, (3-(2-chloro-6-fluorobenzyl)-imidazolidine-2,4-dione), was recently detected as a potential drug to be used as an adjuvant drug to treat chronic pain. The excellent pharmacological property of PT-31 highlights the importance in elucidating its metabolism, which could provide valuable information about its metabolite profile for further pharmacokinetics studies and additionally to estimate the impact of its metabolites on the efficacy, safety and elimination of PT-31. In this work, the study of the in vitro metabolism of PT-31 was initially carried out by using a liquid chromatography coupled to ion trap multiple-stage mass spectrometer (LC-IT-MSn) and a hybrid triple quadrupole/linear ion trap mass spectrometer (LC-QTrap). The production of at least three unknown oxidative metabolites was observed. Structural identification of the unknown metabolites was carried out by combination of LC-MS experiments, including selected reaction monitoring (SRM) and multi-stage full scan experiments. Further analysis of 1H-NMR led to the structural confirmation of the major metabolite. The results indicated that PT-31 was metabolized by a hydroxylation reaction in the imidazolidine-2,4-dione ring in rat and human liver microsomes, producing the metabolite 3-(2-chloro-6-fluorobenzyl)-5-hydroxyimidazolidine-2,4-dione in rat liver microsomes. A carbon hydroxylation onto the benzyl ring, produced two other minor metabolites of the PT-31 in rat liver microsomes.

Aptamer-mediated Plasmodium-specific diagnosis of malaria.

There is a critical need for better malaria rapid diagnostic tests to discriminate Plasmodium falciparum and Plasmodium vivax infection given the recent observation of HRP2 deletions in P. falciparum parasites. We previously identified a DNA aptamer, 2008s, that targets P. falciparum lactate dehydrogenase (PfLDH) and developed a sensitive aptamer-tethered enzyme capture (APTEC) assay. Here, we characterise two different LDH-binding DNA aptamers in their species-specific activities, then integrate within biochemical diagnostic assays and test in clinical samples. An enzyme-linked oligonucleotide assay demonstrated that aptamer pL1 bound with high affinity to both PfLDH and P. vivax lactate dehydrogenase (PvLDH), whereas aptamer 2008s was specific to PfLDH. An aptamer-tethered enzyme capture (APTEC) assay confirmed the specificity of 2008s in binding and capturing the enzyme activity of PfLDH which could be observed colorimetrically. In malaria patient samples, the 2008s APTEC assay was specific for P. falciparum blood samples and could discriminate against P. vivax blood samples. An aptamer for specific detection of falciparum malaria holds promise as a new strategy for species-specific malaria diagnosis rather than the conventional HRP2 immuno-assay.

High Efficiency Binding Aptamers for a Wide Range of Bacterial Sepsis Agents.

Sepsis is a major health problem worldwide, with an extremely high rate of morbidity and mortality, partly due to delayed diagnosis during early disease. Currently, sepsis diagnosis requires bacterial culturing of blood samples over several days, whereas PCR-based molecular diagnosis methods are faster but lack sensitivity. The use of biosensors containing nucleic acid aptamers that bind targets with high affinity and specificity could accelerate sepsis diagnosis. Previously, we used the systematic evolution of ligands by exponential enrichment technique to develop the aptamers Antibac1 and Antibac2, targeting the ubiquitous bacterial peptidoglycan. Here, we show that these aptamers bind to four gram-positive and seven gram-negative bacterial sepsis agents with high binding efficiency. Thus, these aptamers could be used in combination as biological recognition elements in the development of biosensors that are an alternative to rapid bacteria detection, since they could provide culture and amplification-free tests for rapid clinical sepsis diagnosis.

Isolation of an Aptamer that Binds Specifically to E. coli.

Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers-called P12-31-is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies.

Development of a magnetic separation method to capture sepsis associated bacteria in blood.

Bloodstream infections are important public health problems, associated with high mortality due to the inability to detect the pathogen quickly in the early stages of infection. Such inability has led to a growing interest in the development of a rapid, sensitive, and specific assay to detect these pathogens. In an effort to improve diagnostic efficiency, we present here a magnetic separation method for bacteria that is based on mutated lysozyme (LysE35A) to capture S. aureus from whole blood. LysE35A-coated beads were able to bind different MSSA and MRSA isolates in the blood and also other six Gram-positive and two Gram-negative species in whole blood. This system was capable to bind bacteria at low concentrations (10CFU/ml) in spiked blood. Samples captured with the mutated lysozyme showed more responsive amplification of the 16S gene than whole blood at concentrations of 10(3)-10(5)CFU. These data demonstrate detection of S. aureus directly in blood samples, without in vitro cultivation. Our results show that capture with LysE35A-coated beads can be useful to develop a point of care diagnostic system for rapid and sensitive detection of pathogens in clinical settings.

The Effect of Protein Restriction in the In Vitro Metabolism of Albendazole in Rats.

This work presents an in vitro investigation of the effect of protein restriction on the metabolism of albendazole (ABZ). This study was conducted using liver microsomal fractions obtained from Wistar rats. For the quantitative analysis, a multidimensional High Performance Liquid Chromatography (2D HPLC) method was fully validated for the determination of the ABZ metabolites: albendazole sulfoxide, albendazole sulfone and albendazole 2-aminesulfone. The target compounds were directly extracted using a C8-RAM-BSA column (5.0x0.46 cm i.d.) and analyzed on a chromatographic chiral column containing amylose tris(3,5-dimethylphenylcarbamate) (150x4.6 mm i.d.). The in vitro biotransformation results showed that the protein restriction influenced the oxidative metabolism of ABZ. The production of R-(+)-ABZ-SO (1309 nmol/L) and S-(-)-ABZ-SO (1456 nmol/L) was higher in the control animals than in the animals fed with a diet containing 6% protein, which produced 778.7 nmol/L and 709.5 nmol/L for R-(+) and S-(-)-ABZ-SO enantiomers, respectively. These results were statistically inspected by Student´s t test and the results showed a significant difference between the two means (p<0.05). Moreover, the production of ABZ-SO enantiomers was enantioselective where the S-(-)-ABZ-SO was formed in greater amounts than the R-(+)-ABZ-SO in control animals (p=0.0231). However, the enantioselectivity was not observed when the in vitro biotransformation of ABZ was conducted using the microsomal fractions obtained from protein restriction animals (p>0.05). Furthermore, animal nutritional condition could affect the pattern of ABZ sulphoxidation indicating that the protein nutrition affect primarily the formation of R-(+)-ABZSO and S-(-)-ABZ-SO enantiomers.

Transtornos mentais comuns em estudantes universitários: abordagem epidemiológica sobre vulnerabilidades / Common mental disorders in university students: epidemiological approach about vulnerabilities / Transtornos mentales comunes en estudiantes universitários: abordaje epidemiologico acerca de las vulnerabilidades

OBJETIVO: identificar a ocorrência de transtornos mentais comuns em estudantes de uma instituição de Ensino Superior e associar com as características sociodemográficas e acadêmicas. MÉTODO: trata-se de uma pesquisa exploratória, descritiva e de abordagem quantitativa, de levantamento, realizada com 378 estudantes universitários de uma instituição do interior paulista, com a aplicação de questionário semiestruturado e do Self-Reporting Questionnaire. Os dados foram analisados com o uso de análise estatística descritiva e instruções respectivas para a análise do instrumento utilizado. RESULTADOS: 151 (39,9%) dos estudantes universitários entrevistados apresentaram escore de classificação para caso suspeito de transtornos de humor, de ansiedade e de somatização. Das variáveis sociodemográficas, os maiores índices foram para mulheres (43,7%), homossexuais (50,0%), cor de pele preta (42,9%) e em união estável (50,0%). Das variáveis acadêmicas, destacam-se maiores índices entre estudantes do curso de Administração (57,5%) e estudantes no período matutino (44,0%). CONCLUSÃO: a necessidade de planejamento de estratégias de prevenção e recuperação relacionadas à ocorrência de transtornos mentais comuns nessa população é clara, tendo em vista a vulnerabilidade a qual está exposta.

OBJECTIVE: to identify the occurrence of mental disorders, common in students of a higher education institution and associate it with the socio demographic and academic caracteristics. METHOD: it´s a data survey, exploratory, descriptive, quantitativeve approach, made with 378 university students from an institution in the inner city of São Paulo, applying the Self-Reporting Questionnaire. The data was analysed using the descriptive statistical analysis and instructions for the analysis of the instrument used. RESULTS: 151 (39,9%) of the interviewed university students show a suspicious classification score for humor disorder, anxiety and somatization. Regarding the sociodemographic variables, the highest index was for women (43,7%), homosexuals (50,0%), black colored people (42,9%), in a stable union (50,0%). Regarding the academic variables, the highest index was between business administration students (57,5%), and morning shift students (44,0%). CONCLUSION: the need to planning prevention and recovery strategies related to the occurency of common mental disorders in these people is clear, in view of the vulnerability they are exposed.

OBJETIVO: identificar la aparición de transtornos mentales comunes en estudiantes de uma institución de enseñanza superior y asociarla con las características sociodemograficos y académicos. MÉTODO: es uma investigación de levantamiento, exploratório, descriptivo, de abordaje cuantitativo, hecha con 378 estudiantes universitários de una institución del interior de San Pablo, con aplicación de cuestionário semiestructurado y Cuestionario de Autoinforme. Los datos fueron analizados con el análisis estadísticos descriptivos y instruciones respectivos para el análisis del instrumento utilizado. RESULTADOS: 151 (39,9%) de los estudiantes universitários entrevistados presentaron puntuación de clasificación para caso sospechoso de transtorno de humor, de ansiedad y de somatización. De los variables sociodemograficos, los mayores índices fueron para las mujeres (43,7%), homosexuales (50,0%), color de piel negra (42,9%), en unión estable (50,0%). De los variables académicos, los que más se distinguen con índices mayores están los estudiantes de la carrera de Administración (57,5%), y estudiantes del período matutino (44,0%). CONCLUSIÓN: la necesidad de planificación de estratégias de prevención y recuperación relacionadas a la aparición de transtornos mentales comunes en esta población es clara, teniendo en vista la vulnerabilidad en la que están expuestos.

Envolvimento com álcool, tabaco e outras substâncias por estudantes universitários / Undergraduate students and their entry into alcohol, tobacco and other drugs / Estudiantes universitarios y su incursión en el alcohol, tabaco y otras sustancias

Resumo Introdução O presente estudo teve por objetivo identificar o quantitativo de estudantes em uma instituição de ensino superior no interior paulista que vivenciam o envolvimento com o álcool, o tabaco e ou outras substâncias, e avaliar a ocorrência e nível de dependência nos mesmos. Materiais e Métodos Trata-se de extrato de uma pesquisa de levantamento, exploratória, descritiva, de abordagem quantitativa, realizada com 416 estudantes universitários. A coleta de dados se deu no decorrer do 3º trimestre de 2017, com aplicação de questionário semiestruturado, elaborado pelos autores e Questionário para Triagem do Uso de Álcool, Tabaco e Outras Substâncias. Os dados foram analisados com uso de análise estatística descritiva, teste de Qui-Quadrado de Pearson e de acordo com as instruções para aplicação do instrumento selecionado. A pesquisa foi submetida e aprovada pelo Comitê de Ética em Pesquisa. Resultados Os índices gerais de envolvimento e o tipo de drogas são: 140 (30%) para derivados do tabaco, 303 (66%) para bebidas alcoólicas, 89 (19%) para maconha, 32 (7%) para cocaína/crack, 24 (5%) para anfetaminas ou êxtase, 30 (7%) para inalantes, 45 (10%) para hipnóticos/sedativos, 36 (8%) para alucinógenos, e nove (2%) para opióides. Discussão Houve envolvimento com todos os tipos de substâncias, associado a romantização quanto ao uso das mesmas no ambiente universitário e facilidade de acesso. Conclusões O envolvimento de universitários com álcool, tabaco e outras substâncias é real e intenso. Além do envolvimento, o risco para dependência dessas substâncias se caracteriza como grande problema social e de saúde.

Abstract Introduction This study aimed to identify the number of students from a higher education institution into the interior of São Paulo state who experienced their entry into alcohol, tobacco and other substances in order to evaluate the occurrence and dependency level on them. Materials and Methods An extract of a compiling, exploratory, descriptive study under a quantitative approach was conducted in 416 undergraduate students. The data were collected over the third quarter of 2017 by applying a semi-structured questionnaire prepared by the authors in addition to the Triage Questionnaire for Alcohol, Tobacco and other Drugs Use. A descriptive statistical analysis was conducted for data analysis as well as a Pearson' chi-squared test, according to the instructions for the application of the selected instrument. This study was received and approved by the Research Ethics Committee. Results The general rates for entry and drug type are 140 (30%) for tobacco derivatives, 303 (66%) for alcohol beverages, 89 (19%) for marijuana, 32 (7%) for cocaine/crack, 24 (5%) for amphetamines or ecstasy, 30 (7%) for inhalant drugs, 45 (10%) for hypnotics/sedatives, 36 (8%) for hallucinogens and 9 (2%) for opioids. Discussion Students showed to be involved in all types of substances which is linked to flexibility in relation to their use in the university environment and their easy access. Conclusions Undergraduate students have a real and intense relationship with alcohol, tobacco and other drugs. In addition to this relationship, the risk of addiction to these substances is presented as a major social and healthcare problem.

Resumen Introducción El presente estudio tuvo como objetivo identificar la cantidad de estudiantes en una institución de educación superior en el interior paulista que experimentan su incursión en el alcohol, el tabaco y otras sustancias, y evaluar así la ocurrencia y el nivel de dependencia de los mismos. Materiales y Métodos Se trata de un extracto de un estudio de levantamiento, exploratorio, descriptivo, de enfoque cuantitativo, realizado con 416 estudiantes universitarios. Los datos fueron recolectados en el transcurso del 3º trimestre de 2017, mediante la aplicación de un cuestionario semi-estructurado, elaborado por los autores y del Cuestionario para Triaje de Uso de Alcohol, Tabaco y otras Sustancias. Los datos fueron analizados a través de un análisis estadístico descriptivo, la prueba de Qui-Cuadrado de Pearson y de acuerdo con las instrucciones para la aplicación del instrumento seleccionado. El estudio fue recibido y aprobado por el Comité de Ética en Investigación. Resultados Los índices generales de incursión y el tipo de drogas son: 140 (30%) para derivados del cigarrillo, 303 (66%) para bebidas alcohólicas, 89 (19%) para marihuana, 32 (7%) para cocaína/crack, 24 (5%) para anfetaminas o éxtasis, 30 (7%) para drogas inhaladas, 45 (10%) para hipnóticos/sedantes, 36 (8%) para alucinógenos, y nueve (2%) para opioides. Discusión Los estudiantes demostraron haber estado involucrados con todos los tipos de sustancias, lo que se asocia a la flexibilización con respecto al uso de las mismas en el ambiente universitario y a la facilidad de acceso. Conclusiones La relación de los universitarios con el alcohol, el cigarrillo y otras sustancias es real e intensa. Además de esa relación, el riesgo de adicción a estas sustancias se configura como un gran problema social y de salud.

Analysis of proteasomal proteolysis during the in vitro metacyclogenesis of Trypanosoma cruzi.

Proteasomes are large protein complexes, whose main function is to degrade unnecessary or damaged proteins. The inhibition of proteasome activity in Trypanosoma cruzi blocks parasite replication and cellular differentiation. We demonstrate that proteasome-dependent proteolysis occurs during the cellular differentiation of T. cruzi from replicative non-infectious epimastigotes to non-replicative and infectious trypomastigotes (metacyclogenesis). No peaks of ubiquitin-mediated degradation were observed and the profile of ubiquitinated conjugates was similar at all stages of differentiation. However, an analysis of carbonylated proteins showed significant variation in oxidized protein levels at the various stages of differentiation and the proteasome inhibition also increased oxidized protein levels. Our data suggest that different proteasome complexes coexist during metacyclogenesis. The 20S proteasome may be free or linked to regulatory particles (PA700, PA26 and PA200), at specific cell sites and the coordinated action of these complexes would make it possible for proteolysis of ubiquitin-tagged proteins and oxidized proteins, to coexist in the cell.

Inhibition of proteasome activity blocks Trypanosoma cruzi growth and metacyclogenesis.

Proteasomes are intracellular complexes that control protein degradation in organisms ranging from Archaebacteria to mammals. In some parasitic protozoa, the proteasome is involved in cell differentiation and replication. In this study, we have used proteasome inhibitors to determine the biological role of proteasomes during the replication and in vitro metacyclogenesis of Trypanosoma cruzi. We used light and transmission electron microscopy to analyze morphological data and flow cytometry to analyze changes in the cell cycle. The growth of T. cruzi epimastigote culture forms in liver infusion tryptose medium was inhibited by the presence of up to 10 microM lactacystin. Inhibition was dose-dependent, with IC50 (50% inhibitory concentration) of 4.35 microM after 24 or 72 h. The metacyclogenesis process in vitro was strongly (95%) inhibited by 5 microM lactacystin treatment. The adhesion phase was not affected, but the epimastigotes did not differentiate into metacyclic trypomastigotes. Most treated epimastigotes had replicated DNA, with swelling of the mitochondrion and an altered distribution of nuclear and kinetoplast DNA. Our findings suggest that inhibition of the ubiquitin-proteasome pathway in T. cruzi epimastigotes does not block adhesion, but disrupts cell division and affects factors triggering differentiation.

In vitro effects of citral on Trypanosoma cruzi metacyclogenesis

Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 μg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100 percent cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50 percent inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50 percent after 24 h (IC50/24 h) of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50 percent of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.

Sitobion graminis Takahashi, 1950 (Hemiptera, Aphididae): first record in Brazil, biological and morphometric parameters

A espécie Sitobion graminis Takahashi, 1950 (Hemiptera, Aphididae) foi detectada no Brasil pela primeira vez em 1998, em Curitiba, PR, associada às gramíneas Erianthus sp., Calamagrotis sp. e Paspalum urvilei. Os espécimes coletados e criados apresentavam uma notável variacão intrapopulacional no comprimento do corpo e apêndices e na esclerotinizacão dorso-abdominal. Esta espécie é reconhecida na Malásia, Nova Guiné, India, Filipinas e Africa, colonizando várias espécies de Poaceae. S. graminis diferencia-se das demais espécies do gênero Sitobion associadas a gramíneas no Brasil, por apresentar a cauda e sifúnculos negros e o último segmento rostral constrito na base. Dados de biologia foram obtidos em laboratório, onde ninfas recém-nascidas criadas sobre as inflorescências das gramíneas hospedeiras, desenvolveram-se em quatro instares. A duracão média do estágio ninfal foi de 11,4 dias, com mortalidade de 36,5por cento. O período médio de pré-larviposicão foi de 1,8 dias; longevidade média das fêmeas de 25,2 dias; fecundidade média de 18,7 ninfas/fêmea, variando de 2 a 41 ninfas/fêmea.

Consumption of Cinara spp. (Hemiptera, Aphididae) by Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae)

Adults and larvae of coccinellids were observed feeding on populations of the giant conifer aphids Cinara spp. on Pinus spp., in Southern Brazil. The objective of this research is to evaluate the consumption capacity of Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae) on these aphid species, in order to obtain information for biological control programs. Ten larvae of each predator species were fed with aphids of small size (nymphs of 1st and 2nd instars), and 10 with aphids of medium size (nymphs of 3rd and 4th instars), maintained under 15°C, 20°C and 25°C, 12 h photophase and 70 ± 10 percent relative humidity. The aphids were counted every 24 hours, replacing those that were consumed. The total consumption of Cinara by the larvae of C. sanguinea was not statistically different at the three temperatures: 325.5; 322.2 and 324.8 of small aphids and 121.3; 140.4 and 109.9 of medium ones, respectively at 15°C, 20°C and 25°C. The consumption by H. convergens was higher than by C. sanguinea and increased noticeably with temperature: 444 aphids at 15°C; 491.3 at 20°C and 513.3 at 25°C, considering the small aphids, and 187.1; 205.1 and 216.6 of medium aphids at the three temperatures. The small aphids weigh about half as much as medium ones and were preferred by all larval instars probably because they are easier to manipulate than the large ones. Both predators, especially the 4th instar larvae, showed high consumption capacity on the Cinara nymphs at all temperatures and can be regarded as promising biological control agents.

Comparative biology of Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae) focusing on the control of Cinara spp. (Hemiptera, Aphididae)

The giant conifer aphids Cinara pinivora (Wilson, 1919) and Cinara atlantica (Wilson, 1919) (Hemiptera, Aphididae) have been observed attacking Pinus spp. in Southern and Southeastern Brazil. The coccinellids, on the other hand, were found feeding on these aphids in the field, which can be regarded as potential biological control agents. The biological cycle and mortality rate of larvae of Cycloneda sanguinea (Linnaeus, 1763) and Hippodamia convergens Guérin-Méneville, 1842 (Coleoptera, Coccinellidae) were evaluated using twenty larvae of each predator species fed with nymphs of Cinara. The vials with the insects were kept under 15 ºC, 20 ºC and 25 ºC, with 12h photophase and 70 ± 10 percent relative humidity. The consumption was evaluated every 24 hours and the nymphs replaced. For C. sanguinea, the egg incubation time was 10.5, 5.0 and 4.0 days; the average larval development period was 33.3, 15.8 and 8.6 days and the larval mortality rate 20 percent,0 percent and 15 percent, respectively at 15 ºC, 20 ºC and 25 ºC. For H. convergens, the larval development time was 41.9, 19.3 and 10.9 days at 15 ºC, 20 ºC and 25 ºC, respectively. The larval mortality rate was 35 percent, 15 percent and 0 percent under the three temperatures. Both species developed adequately when fed nymphs of Cinara, however, C. sanguinea performed better than H. convergens, even at 15 ºC, at which temperature the biological cycles of the coccinellids are prolonged, but the temperature is favorable for the development of Cinara populations in the field.

A new species of Impatientinum Mordvilko (Hemiptera: Aphididae) from Brazil / Uma nova espécie de Impatientinum Mordvilko (Hemiptera: Aphididae) no Brasil

Impatientinum paranaense sp. nov. é descrita ocorrendo em Cuphea calophylla (Lythraceae) no Brasil. Fêmeas ápteras e aladas vivíparas são descritas e ilustradas. A chave de Remaudière (1981) para o gênero Impatientinum foi modificada para incluir a espécie nova.

Impatientinum paranaense sp. nov. on Cuphea calophylla (Lythraceae) from Brazil is described. Descriptions and illustrations of apterous and alate viviparous females are given. The new taxon is included into the Remaudière (1981)'s key to Impatientinum species.