Sequence analysis of the internal transcribed spacer 2 (ITS2) region of rDNA for identifying Trichogramma species and evaluating genetic diversity / Análise de sequência do rDNA da região espaçadora do transcrito interno 2 (ITS2) para identificar espécies de Trichogramma e avaliar a diversidade genética

Abstract Species of Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammtidae) are frequently used as biological control agents against Lepidoptera, but practical application of these egg endoparasitoids are complicated because of their complex taxonomy. This study aimed to compare sequences of internal transcribed spacer regions of ribosomal DNA (ITS2-rDNA) of Trichogramma accessions with those deposited in GenBank in order to access the reliability of the ITS2 as a barcode for discriminating species and evaluating the genetic diversity. ITS2-rDNA sequences obtained from seventeen specimens of Trichogramma confirmed previous identifications based on morphological characteristics. Multiple sequence alignment revealed the existence of highly conserved regions in ITS2 sequences while the neighbour-joining dendrogram indicated that the specimens formed three clusters comprising T. manicobai and T. marandobai (group I), T. galloi (group II) and T. pretiosum (group III). The ITS2 marker was shown to be a powerful DNA barcode for discriminating Trichogramma species and could be used to complement the morphological approach.

Resumo Espécies de Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammatidae) são freqüentemente usadas como agentes de controle biológico contra Lepidoptera, esses endoparasitóides de ovos apresentam taxonomia complexa, o que dificulta sua aplicação prática. Este estudo teve como objetivo comparar seqüências de regiões espaçadoras internas transcritas de DNA ribossômico (ITS2-rDNA) de acessos de Trichogramma com aquelas depositadas no GenBank, a fim de avaliar a confiabilidade do ITS2 barcode para discriminar espécies e avaliar a diversidade genética. As seqüências de ITS2-rDNA obtidas de dezessete espécimes de Trichogramma confirmaram identidades anteriores com base em características morfológicas. O alinhamento de múltiplas sequências revelou a existência de regiões altamente conservadas nas sequências ITS2, enquanto o dendrograma indicou que os espécimes formavam três grupos compreendendo T. manicobai e T. marandobai (grupo I), T. galloi (grupo II) e T. pretiosum (grupo III). O marcador ITS2 mostrou ser um poderoso DNA barcode para discriminar espécies de Trichogramma podendo ser usado como complemento da abordagem morfológica.

Strain of Bacillus thuringiensis from Restinga, toxic to Aedes (Stegomyia) aegypti (Linnaeus) (Diptera, Culicidae) / Estirpe de Bacillus thuringiensis da restinga, tóxico ao Aedes (Stegomyia) aegypti (Linnaeus) (Diptera, Culicidae)

Abstract Bacillus thuringiensis is the most commonly used entomopathogen in the control of Aedes aegypti, which is a vector for different etiological agents that cause serious infections in humans. Several studies aim to isolate strains of this bacterium from different environments, with the perspective of selecting isolates with larvicidal activity for mosquitoes. Aiming at the insecticidal action of B. thuringiensis, the present study aimed to prospect B. thuringiensis of restinga and mangrove soils from the state of Maranhão, Brazil, with toxic potential for use in the biological control of Ae. aegypti. Bioassays were performed to determine the entomopathogenic activity of the bacilli against Ae. aegypti and lethal concentrations (LC50 and CL90) were estimated after the tests. Polymerase Chain Reaction and SDS-PAGE techniques were performed to verify the gene and protein content of the isolates, respectively. The soil of the mangrove and restinga ecosystems showed potential for obtaining B. thuringiensis. This isolate, in addition to having proteins with molecular mass similar to the toxins Cry and Cyt, also presented several diptera-specific genes cry and cyt, demonstrating that it has high potential to be used in the biological control of Ae. aegypti.

Resumo Bacillus thuringiensis é o entomopatógeno mais utilizado no controle do Aedes aegypti, vetor de diferentes agentes etiológicos que causam infecções graves em humanos. Diversos estudos têm como objetivo isolar cepas dessa bactéria de diferentes ambientes, com a perspectiva de selecionar isolados com atividade larvicida para mosquitos. Visando a ação inseticida de B. thuringiensis, o presente estudo teve como objetivo prospectar B. thuringiensis de solos de restinga e mangue do estado do Maranhão, Brasil, com potencial tóxico para uso no controle biológico de Ae. aegypti. Bioensaios foram realizados para determinar a atividade entomopatogênica do bacilo contra Ae. aegypti e as concentrações letais (CL50 e CL90) foram estimadas após os testes. As técnicas de Reação em Cadeia da Polimerase e SDS-PAGE foram realizadas para verificar o conteúdo de genes e proteínas dos isolados, respectivamente. Os solos dos ecossistemas de mangue e restinga apresentaram potencial para obtenção de B. thuringiensis. O isolado BtMA-750, obtido a partir da amostra de solo da restinga, foi interessantemente distinguido por sua alta toxicidade para Ae. aegypti. Este isolado, além de apresentar proteínas com massa molecular semelhante às toxinas Cry e Cyt, apresentou também diversos genes díptero-específicos cry e cyt, demonstrando que tem alto potencial para ser usado no controle biológico de Ae. aegypti.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae) / Isolados de Bacillus thuringiensis de biomas maranhenses com potencial ação inseticida contra larvas de Aedes aegypti (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Early resynchronization of non-pregnant beef cows based in corpus luteum blood flow evaluation 21 days after Timed-AI.

The study aimed to verify whether a hormone protocol started at Day 13 (D13) after Timed Artificial Insemination (TAI) influences the conception rate. Nelore cows (primiparous and multiparous) from two commercial beef farms (n = 1,431) were first TAI (D0). Timed AI was performed in lots (TAI Lots) ranging from 187 to 346 cows. On D13, regarding the TAI lot, cows were assigned for either receiving (Resynch group, n = 1,002) or not (Control group, a subset of approximately 30%, n = 429) another hormone protocol for resynchronization. The same hormone protocol was used for the first TAI and for the resynchronization, except for 1 mg instead of 2 mg of estradiol benzoate (EB) at the begging of the protocol. Eight days later (D21), the Resynch group was checked for corpus luteum blood flow by color Doppler ultrasonography, and in those detected as non-pregnant, the protocol was completed and a 2nd TAI was performed at D23. Pregnancy diagnosis was later (D30) performed by B-mode ultrasonography in the control group and confirmed in the presumptive pregnant cows from the 1st TAI of the Resynch group. The remaining cows were checked for pregnancy 30 days after the 2nd TAI (experimental Day 53). The statistical model to explain conception rate considered the effects of Group (Control or Resynch), Farm, Parity (primiparous or multiparous), Sire, Technician (who perform AI), TAI Lot and pertinent interactions (Group*Parity, Group*Farm and Group*TAI Lot). The statistical analyses of the model were performed using the Proc Glimmix (SAS virtual University Edition). The conception rate for the 1st TAI was similar (P > 0.4) between Control (50.3%, 216/429) and Resynch group (52.6%, 527/1002). The positive predictive diagnostic on D21 showed high relation with PD30 (90.7%, 527/581). In Resynch group, non-pregnant cows (n = 421, 1002 minus 581) were re-inseminated. The conception rate of the 2nd TAI (42.8%, 180/421) was affected (P < 0.002) by side effects of the Farm (48.5 vs. 33.1%) and Parity (51.2 vs. 40.3%, for multiparous vs. primiparous, p < 0.001). Nevertheless, after the 2 TAIs of the Resynch group, the cumulative conception rate was 70.5% (707/1002). In conclusion, the early resynchronization of cows with a low (1 mg) EB dose and progesterone device at D13 after TAI can be used as a strategy to reduce conception interval in beef cattle, and thus to increase the number of pregnant cows from artificial insemination after the breeding season.

Efficacy and limitations of different approaches to anticipate the diagnosis of pregnancy in cattle / Eficácia e limitações de diferentes abordagens para antecipar o diagnóstico de gestação em bovinos

The study evaluated sonographic and serologic exams performed for early (20 to 30d) diagnosis of pregnancy. One hundred-twenty (n= 120) bovine recipients were synchronized (estrous=D0) and timed embryo transferred (TET, D7) with fresh in vitro produced embryos. In the first trial (n= 46), diagnosis of pregnancy was performed on day 20 (D20) by detecting CL blood flow (BF) and by Pregnancy-Associated Glycoproteins (PAGs) serology. In the second trial (n= 30), pregnancy diagnosis was performed on D25 by ultrasound visualization of uterine contents and by PAGs serology. In the last trial, PAG's serology was performed on D30. Ultrasonographic detection of the uterine contents and embryo viability performed on D30 (DG30) was considered the gold standard. The PROC FREQ procedure was used to test the agreement between diagnostic methods. On D20, the Doppler ultrasonography of the CL had showed high sensitivity (100%), but only moderate specificity (53.3%). On the same day, serologic diagnostic had no agreement (k= -0.08, P< 0.46) with the gold standard, with very low sensitivity (6.3%). However, the sensitivity of the serologic exam increased dramatically (from 6.3 to 100%) from D20 to D25, and it contributed to detect false negatives from the ultrasound diagnosis, improving the overall accuracy from 90% to 96.7%.(AU)

O estudo foi planejado para correlacionar exames ultrassonográficos e sorológicos realizados para o diagnóstico precoce (20 a 30d) de gestação. Cento e vinte (n= 120) receptoras bovinas foram sincronizadas (estro=D0), e embriões frescos produzidos in vitro foram transferidos em tempo fixo (TETF, D7). No experimento 1 (n= 46), o diagnóstico de gestação foi realizado no D20, pela detecção do fluxo sanguíneo do CL e pela sorologia de glicoproteínas associadas à gestação (PAGs). No experimento 2 (n= 30), a detecção da gestação foi realizada por meio da visualização do conteúdo do útero e também pela sorologia para PAGs. No experimento 3, a sorologia para PAGs foi realizada no D30. Em todos os experimentos, a visualização ultrassonográfica da vesícula e da viabilidade embrionária, realizada no D30, foi considerada padrão-ouro. O procedimento PROC FREQ testou o nível de concordância dos métodos diagnósticos. No D20, o diagnóstico baseado na vascularização do CL mostrou alta sensibilidade (100%) e apenas moderada especificidade (53,3%). Nesse mesmo dia, o diagnóstico sorológico não apresentou concordância (k=-0,08, P<0,46) com o padrão-ouro, além de baixa sensibilidade (6,3%). No entanto, a sensibilidade do exame sorológico aumentou drasticamente (6,3 para 100%) do D20 para o D25, contribuindo para detectar falsos negativos diagnosticados pela ultrassonografia, melhorando a acurácia (90 para 96,7%).(AU)

To be or not to be hospitalised with tuberculosis in Portugal.

SETTING: In Portugal, as in other countries, tuberculosis (TB) is considered a disease that should be managed on an ambulatory basis. However, hospitalisation remains important to manage some at-risk groups and complications.OBJECTIVE: To identify the possible risk factors associated with hospitalisations in TB patients in Portugal.DESIGN: Data extraction through two national databases (one for registration of TB cases and the other with hospitalisation information in public health facilities) between 2007 and 2013. Univariate and multivariate analysis of demographic and clinical variables was performed.RESULTS: We identified 4421 hospitalisations. Chronic diseases, cancer, substance abuse, a higher social/economic risk, extra-pulmonary TB, lung cavitary disease and previous uncompleted treatment were more frequent among hospitalised patients. Human immunodeficiency virus coinfection, cancer, alcohol abuse, extra-pulmonary TB and uncompleted previous TB treatment were the most important predictors of hospitalisation with TB. The hospitalisation rate among TB patients in Portugal was lower when compared with other countries with lower and higher incidences.CONCLUSION: Immune dysfunctions and progression of chronic diseases are associated with more severe forms of TB and frequent adverse effects which can be sufficiently severe to necessitate hospital admission. Despite having an intermediate TB incidence, the hospitalisation rate in Portugal is not higher than that of other countries.

Sex-specific transcription and DNA methylation profiles of reproductive and epigenetic associated genes in the gonads and livers of breeding zebrafish.

Reproduction is an essential process for life and is regulated by complex hormone networks and environmental factors. To date, little is known about the contribution of epigenetic mechanisms to the regulation of reproduction, particularly in lower vertebrates. We used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. We found evidence for associations between DNA promotor methylation and transcription for esr1 (gonads and female livers), amh (gonads) and dnmt1 (livers). In the liver, esr1 was shown to be significantly over-expressed in females compared to males, and its promoter was significantly hypo-methylated in females compared to males. In the gonads, genes involved in epigenetic processes including dnmt1, dnmt3 and hdac1 were over-expressed in the ovary compared to the testis. In addition, dnmt1 and dnmt3 transcription in the testis was found to be strongly correlated with global DNA methylation. These data provide evidence of the sex-specific epigenetic regulation and transcription of genes involved in reproduction and epigenetic signalling in a commonly used vertebrate model.

Freezing goat embryos at different developmental stages and quality using ethylene glycol and a slow cooling rate / Congelação de embriões caprinos em diferentes estádios de desenvolvimento e qualidade utilizando etileno glicol e taxa de resfriamento lenta

The efficiency of an alternative freezing protocol for goat embryos of different morphology and quality was tested. Fifty-eight embryos on Day 6-7 stage were transferred as fresh or after freeze-thawing (n=29/group). For freezing, embryos were placed into 1.5M ethylene-glycol solution for 10min. During this time, they were loaded in the central part of 0.25mL straw, separated by air bubble from columns containing PBS/BSA 0.4% plus 20% BFS. Straws were then frozen using a freezing machine from 20ºC to -6ºC at a cooling rate of 3ºC/min, stabilization for 15min (seeding after 5min), from -6 C to -32ºC at 0.6 C/min,and plunged into liquid nitrogen. Frozen embryos were thawed for 30s at 37ºC in a water bath. Embryos subjected to fresh transfer were maintained in holding medium (37ºC). Fresh and frozen-thawed embryos were transferred at day 7 post-estrus to 30 recipients. Kidding and kid born rates were similar (P> 0.05), respectively, for recipients receiving fresh (66.7% or 10/15; 55.2% or 16/29) or frozen-thawed (60% or 9/15; 51.7% or 15/29) embryos. The cryopreservation of goat embryos using slow-freezing protocol and 1.5MEG resulted in similar efficiency rates of fresh embryos.(AU)

Este estudo testou a eficiência de protocolo alternativo de criopreservação de embriões caprinos de diferentes qualidades morfológicas. Foram utilizados 58 embriões, coletados entre o sexto e o sétimo dia do ciclo estral (n=29/grupo). Embriões congelados passaram por solução 1,5M etilenoglicol por 10min e foram aspirados durante esse tempo para parte central de palheta 0,25mL, separada por bolhas de ar de colunas contendo PBS 0,4% BSA e 20% SFB. As palhetas foram congeladas em máquina de congelação de 20ºC a -6ºC, com taxa de resfriamento de 3ºC/min, estabilização por 15min (seeding após 5min), -6ºC a -32ºC a 0,6ºC/min, e imersas em nitrogênio líquido. Os embriões foram descongelados por 30s a 37ºC, em água. Embriões frescos foram mantidos em solução de manutenção (37ºC). Embriões frescos e congelados/descongelados foram transferidos para 30 receptoras no sétimo dia do ciclo estral. A taxa de partos e a de crias nascidas (respectivamente) foram similares (P>0,05) para receptoras recebendo embriões frescos (66,7% ou 10/15; 55,2% ou 16/29) ou congelados/descongelados (60,0% ou 9/15; 51,7% ou 15/29). O protocolo de criopreservação de embriões utilizado no presente estudo resultou em índices de eficiência semelhantes aos de embriões frescos.(AU)

Intrafollicular oestradiol production, expression of the LH receptor (LHR) gene and its isoforms, and early follicular deviation in Bos indicus.

The aim of the present study was to characterise the roles of intrafollicular oestradiol production and granulosa cell (GC) expression of the LH receptor (LHR) gene and its isoforms during follicular deviation in Bos indicus. Follicular wave emergence was synchronised in heifers from a Bos taurus dairy (Holstein; n=10) and a B. indicus dairy breed (Gir; n=10). Follicles were aspirated individually at sizes corresponding to the periods of predeviation, deviation and postdeviation. Intrafollicular oestradiol (IF-E2) and progesterone (IF-P4) concentrations were determined in the follicular fluid (FF) by radioimmunoassay, and relative expression of P450 aromatase (CYP19A1) and LHR forms was evaluated in GC using real-time quantitative-polymerase chain reaction. Despite differences in the size of the dominant follicle at deviation, changes in CYP19A1 expression and IF-E2 concentrations were similar in follicles of the same diameter in both breeds. A peak in total LHR expression occurred after follicular deviation in association with low expression of LHR isoforms. The results suggest that regulation of LHR function by sequential changes in the expression pattern of LHR isoforms may play a role in the early deviation of the dominant follicle, as observed in B. indicus breeds.

Strain of Bacillus thuringiensis from Restinga, toxic to Aedes (Stegomyia) aegypti (Linnaeus) (Diptera, Culicidae)

Abstract Bacillus thuringiensis is the most commonly used entomopathogen in the control of Aedes aegypti, which is a vector for different etiological agents that cause serious infections in humans. Several studies aim to isolate strains of this bacterium from different environments, with the perspective of selecting isolates with larvicidal activity for mosquitoes. Aiming at the insecticidal action of B. thuringiensis, the present study aimed to prospect B. thuringiensis of restinga and mangrove soils from the state of Maranhão, Brazil, with toxic potential for use in the biological control of Ae. aegypti. Bioassays were performed to determine the entomopathogenic activity of the bacilli against Ae. aegypti and lethal concentrations (LC50 and CL90) were estimated after the tests. Polymerase Chain Reaction and SDS-PAGE techniques were performed to verify the gene and protein content of the isolates, respectively. The soil of the mangrove and restinga ecosystems showed potential for obtaining B. thuringiensis. This isolate, in addition to having proteins with molecular mass similar to the toxins Cry and Cyt, also presented several diptera-specific genes cry and cyt, demonstrating that it has high potential to be used in the biological control of Ae. aegypti.

Resumo Bacillus thuringiensis é o entomopatógeno mais utilizado no controle do Aedes aegypti, vetor de diferentes agentes etiológicos que causam infecções graves em humanos. Diversos estudos têm como objetivo isolar cepas dessa bactéria de diferentes ambientes, com a perspectiva de selecionar isolados com atividade larvicida para mosquitos. Visando a ação inseticida de B. thuringiensis, o presente estudo teve como objetivo prospectar B. thuringiensis de solos de restinga e mangue do estado do Maranhão, Brasil, com potencial tóxico para uso no controle biológico de Ae. aegypti. Bioensaios foram realizados para determinar a atividade entomopatogênica do bacilo contra Ae. aegypti e as concentrações letais (CL50 e CL90) foram estimadas após os testes. As técnicas de Reação em Cadeia da Polimerase e SDS-PAGE foram realizadas para verificar o conteúdo de genes e proteínas dos isolados, respectivamente. Os solos dos ecossistemas de mangue e restinga apresentaram potencial para obtenção de B. thuringiensis. O isolado BtMA-750, obtido a partir da amostra de solo da restinga, foi interessantemente distinguido por sua alta toxicidade para Ae. aegypti. Este isolado, além de apresentar proteínas com massa molecular semelhante às toxinas Cry e Cyt, apresentou também diversos genes díptero-específicos cry e cyt, demonstrando que tem alto potencial para ser usado no controle biológico de Ae. aegypti.

Sequence analysis of the internal transcribed spacer 2 (ITS2) region of rDNA for identifying Trichogramma species and evaluating genetic diversity

Abstract Species of Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammtidae) are frequently used as biological control agents against Lepidoptera, but practical application of these egg endoparasitoids are complicated because of their complex taxonomy. This study aimed to compare sequences of internal transcribed spacer regions of ribosomal DNA (ITS2-rDNA) of Trichogramma accessions with those deposited in GenBank in order to access the reliability of the ITS2 as a barcode for discriminating species and evaluating the genetic diversity. ITS2-rDNA sequences obtained from seventeen specimens of Trichogramma confirmed previous identifications based on morphological characteristics. Multiple sequence alignment revealed the existence of highly conserved regions in ITS2 sequences while the neighbour-joining dendrogram indicated that the specimens formed three clusters comprising T. manicobai and T. marandobai (group I), T. galloi (group II) and T. pretiosum (group III). The ITS2 marker was shown to be a powerful DNA barcode for discriminating Trichogramma species and could be used to complement the morphological approach.

Resumo Espécies de Trichogramma Westwood, 1833 (Hymenoptera: Trichogrammatidae) são freqüentemente usadas como agentes de controle biológico contra Lepidoptera, esses endoparasitóides de ovos apresentam taxonomia complexa, o que dificulta sua aplicação prática. Este estudo teve como objetivo comparar seqüências de regiões espaçadoras internas transcritas de DNA ribossômico (ITS2-rDNA) de acessos de Trichogramma com aquelas depositadas no GenBank, a fim de avaliar a confiabilidade do ITS2 barcode para discriminar espécies e avaliar a diversidade genética. As seqüências de ITS2-rDNA obtidas de dezessete espécimes de Trichogramma confirmaram identidades anteriores com base em características morfológicas. O alinhamento de múltiplas sequências revelou a existência de regiões altamente conservadas nas sequências ITS2, enquanto o dendrograma indicou que os espécimes formavam três grupos compreendendo T. manicobai e T. marandobai (grupo I), T. galloi (grupo II) e T. pretiosum (grupo III). O marcador ITS2 mostrou ser um poderoso DNA barcode para discriminar espécies de Trichogramma podendo ser usado como complemento da abordagem morfológica.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia.

This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.

[Central nervous system in IgG4-related disease: case report and literature review]. / Afeccion del sistema nervioso central en la enfermedad relacionada con IgG4: descripcion de un caso y revision de la bibliografia.

INTRODUCTION: IgG4-related disease is a recently described multisystemic clinical entity that can occur with different clinical manifestations. The most often affected organs are the pancreas, bile duct and salivary glands, with unusual central nervous system affection. CASE REPORT: A 33 year old woman who presented with cognitive impairment, hallucinations, headache, convulsive syndrome, maxillary sinus inflammation with bone involvement and evidence of pachymeningitis and panhypopytuirarism with meningeal biopsy that confirmed IgG4-related disease, after ruling out secondary causes. Treatment was started with steroids and azathioprine without relapses after 12 months follow-up. CONCLUSIONS: IgG4-related disease should be considered in cases of hypertrophic pachymeningitis and hypophysitis especially when no other cause has been found, even if they are not accompanied by other systemic disease manifestations, having ruled out other common causes. The treatment of choice is glucocorticoids and it could be needed to add another immuno-suppressant agent as steroid sparing and to prevent relapses. Prospective studies are needed to evaluate the different clinical and paraclinical manifestations and to establish the results of long-term treatment.

TITLE: Afeccion del sistema nervioso central en la enfermedad relacionada con IgG4: descripcion de un caso y revision de la bibliografia.Introduccion. La enfermedad relacionada con IgG4 es una entidad clinica multisistemica recientemente descrita y que se presenta con diferentes manifestaciones clinicas. Los organos que estan afectados con mayor frecuencia son el pancreas, la via biliar y las glandulas salivales, y es menos frecuente la afeccion del sistema nervioso central. Caso clinico. Mujer de 33 años con alteraciones cognitivas, alucinaciones, cefalea, sindrome convulsivo, sinusitis maxilar con afeccion osea y evidencia de paquimeningitis y panhipopituitarismo, con biopsia meningea que confirmo una enfermedad relacionada con IgG4, tras haberse descartado causas secundarias. Se inicio tratamiento con glucocorticoides y azatioprina, sin recaidas despues de 12 meses de seguimiento. Conclusiones. Se debe considerar el diagnostico de enfermedad relacionada con IgG4 en casos de paquimeningitis hipertrofica e hipofisitis, incluso sin que se acompañen de otras manifestaciones sistemicas, siempre que se hayan descartado otras causas mas frecuentes. El tratamiento de eleccion son los glucocorticoides, y puede ser necesario añadir otro inmunosupresor como ahorrador de esteroides y para evitar las recaidas. Se necesitan estudios prospectivos para evaluar las diferentes manifestaciones clinicas y paraclinicas y establecer los resultados del tratamiento a largo plazo.

Efficacy of induction of luteolysis in superovulated cows is dependent on time of prostaglandin F2alpha analog treatment: effects on plasma progesterone and luteinizing hormone profiles.

The objectives were to (1) evaluate the effectiveness of induction of luteolysis in superovulated (SOV) cows at two distinct time points after embryo flushing; and (2) compare the pattern of LH release after treatment with PGF in cows with single vs. multiple ovulations. In the first experiment, Holstein cows were SOV with 400 IU of FSH following standard procedures. Uterine flushing for embryo recovery was performed 7 days after artificial insemination (Day 0), and cows were randomly allocated into two groups to receive PGF (0.5-mg sodium cloprostenol, intramascular) either immediately after flushing (Day 7 group, N = 19) or 4 days later (Day 11 group, N = 20). Time of luteolysis was determined on the basis of plasma progesterone (P4) concentrations. There was no difference (P > 0.05) in plasma P4 before treatment between Day 7 and Day 11 groups. A decline in plasma P4 was observed 48 hours after PGF treatment in both the groups (P < 0.0001). In Day 11 cows, P4 continued to decrease thereafter, whereas Day 7 animals had no further reduction in plasma P4. Luteolysis (P4 < 1 ng/mL) occurred in all Day 11 cows. In the Day 7 group, however, luteolysis failure was observed for 11 of 19 cows (57.9%). In cows without luteolysis, plasma P4 increased after the initial PGF-induced decline. The second experiment compared luteolysis in (SOV, N = 6) vs. non-SOV (control, N = 8) cows. Both groups received a single PGF treatment on Day 11 after estrus, and luteolysis was monitored daily by ovarian ultrasonography and plasma P4 measurements. In addition, plasma LH was measured in blood samples taken every 20 minutes for 1 hour during five consecutive days after treatment. A similar percentage of reduction in P4 was observed in both groups 24 hours after treatment; however, SOV cows only reached plasma P4 values similar (P > 0.05) to controls 96 hours after treatment. There was no difference in initial LH values between SOV and controls (P > 0.05). The slower decrease in plasma P4 in the SOV group prevented an increase in LH for up to 96 hours after luteolysis induction, whereas LH values increased (P < 0.05) in controls 24 hours after treatment. In conclusion, (1) luteolysis may fail or be incomplete when PGF treatment is given on the day of uterine flushing (Day 7) in SOV cows; (2) induction of luteolysis 4 days later (Day 11) is effective, but the initial high-plasma P4 concentrations result in a slower slope of P4 decline to basal levels, and consequently, delayed increase in LH pulses.

ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia

This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.

Corpus luteum blood flow evaluation on Day 21 to improve the management of embryo recipient herds.

The aim of the present study was to use blood flow evaluation of the CL at 14 days after embryo transfer to detect nonpregnant animals and optimize the management of bovine recipients. The estrous cycle was synchronized in 165 recipients, and the day of expected ovulation was considered to be Day 0. Embryo transfer was performed 7 days later, on Day 7. On Day 21, pregnancy was diagnosed on the basis of blood flow evaluation of the CL (DG21-predictive diagnostic). To validate this methodology, visual scores for blood flow were compared to objective data extracted from CL ultrasound images recorded in the Doppler mode. The size was also evaluated using recorded images of the CL in the B mode. Blood samples were also collected for further analysis of the progesterone (P4) concentration. The diagnosis of pregnancy was confirmed at 35 days after estrus (DG35-definitive diagnostic). The DG21 showed that 55.2% (90 of 163) of the animals were presumptively pregnant, and this value was higher (P < 0.04) than that obtained at DG35 (43.6%, 71 of 163). The predictive diagnostic achieved moderate specificity (79.3%) for the detection of pregnancy, but most importantly, high sensitivity (100%) for the detection of nonpregnant recipients. The overall accuracy of the diagnosis was 88.3%. The P4 concentrations were different (P < 0.02) and correlated with each visual score assigned for the CL size. Visual scores for CL blood flow were also efficient (P < 0.0001) to distinguish animals with different levels of P4; however, P4 concentrations were higher for scores 1 and 2 (high and regular blood flow, respectively) than those for score 3 (low blood flow). This technique showed high sensitivity and facilitated the early detection of nonpregnant animals. The DG21 would allow about 79.3% of nonpregnant animals to be resynchronized 9 to 14 days earlier, when compared to conventional management based on pregnancy diagnosis at Days 30 to 35.

Viable offspring after successful non-surgical embryo transfer in goats / Nascimento de fetos viáveis após transferência de embriões não-cirúrgica em caprinos

O objetivo do presente estudo foi avaliar a viabilidade da técnica de transferência não cirúrgica em cabras. Quatro cabras não-lactantes pluríparas da raça Toggenburg foram utilizadas como receptoras de embriões, sendo que duas receberam um embriões e duas receberam dois embriões coletados não cirurgicamente cabras doadoras. Os corpos lúteos das receptoras foram detectados um dia antes da transferência de embriões por ultrassonografia transretal. Uma seringa de 5mL contendo 2mL de meio holding foi acoplada em um cateter tomcat, no qual os embriões foram aspirados em uma coluna central a duas outras colunas. Um espéculo Colin número 2 foi inserido na vulva e na vagina, e com o uso de uma fonte de luz, a cerviz foi localizada e imobilizada com uma pinça de Allis. Um cateter uretral número seis acoplado a um mandril e lubrificado com meio PBS foi inserido na cérvix, e assim os aneis cervicais foram gradualmente transpostos. Após perder a resistência, o cateter uretral foi movido lateralmente para o corno uterino desejado. O mandril e a pinça de Allis foram retirados e o conjunto seringa e tomcat foi acoplado ao cateter uretral e o conteúdo injetado no corno uterino ipsilateral ao corpo lúteo com posterior retirada do cateter. Cabras que ovularam em apenas um ovário foram usadas para testar a eficiência da deposição do embrião. O tempo gasto entre a inserção do espéculo e a sua remoção foi inferior a três minutos. O tempo para transpor a cérvix foi inferior a um minuto. A ultrassonografia revelou a deposição de líquido no corno desejado. Receptoras que receberam dois embriões tornaram-se gestantes e pariram três crias. Estes primeiros resultados encorajam a técnica e demonstram que a transferência de embriões em caprinos pode ser feita totalmente por procedimentos não cirúrgicos...

Florfenicol associado ou não ao cloprostenol no tratamento de retenção de placenta em vacas leiteiras / Florfenicol and sodium cloprostenol for the treatment of retained placenta in dairy cows

The aim of this study was to compare the reproductive efficiency in dairy cows with retained placenta (RP), treated with Florfenicol associated or not with Cloprostenol. One hundred and eight cows with RP from dairy herds in southern Minas Gerais state and northern São Paulo state, were included for this purpose. The experiment was designed in factorial 2X2 . The four treatments encompassed two levels of each factor : 1 ) Florfenicol - 4 0mg/kg ( IM) divided in two injections or 40mg / kg ( SC) in one injection after calving; 2) Cloprostenol (0.530mg) - two injections associated with florfenicol treatment or without cloprostenol. The analysis of discrete variables used the Kruskal - Wallis (fou r groups) or Wilcoxon (two groups) statistic to compare groups. The Student "T" test was used to compare means of continuous variables. The overall occurrence of RP was 20.7%. The florfenicol groups had no effects on postpartum uterine health or subsequent reproductive efficiency of the cows. The cloprostenol injections of RP hastened the uterine involution and decreased the occurrence of uterine infection, and increased the percentage of animals with corpus luteum between 25 and 40 days after parturition. The intervals from parturition to first artificial insemination (AI) and to conception, as well as the number of AI per conception were reduced in the cloprostenol treatment.