N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

Viability of ovine spermatozoa collected from epididymides stored at 18°-25°C for 48 hours post mortem / Viabilidade dos espermatozoides ovinos coletados de epidídimos armazenados a 18°-25°C até 48 horas após a morte

The objectives of this study were to verify the time during which viable ovine spermatozoa could be recovered from the cauda epididymis kept at ambient temperature (18-25°C). Sperm collected in an artificial vagina (AV) were used as control. Spermatozoa samples were collected with an AV and from epididymis at 0 (G0), 6 (G6), 12 (G12), 24 (G24), and 48 (G48) hours post mortem. Total motility (TM), progressive motility (PM), hypo-osmotic membrane integrity test (HOST) and morphological changes were assessed. TM decreased (P<0.05) from 24 hours post mortem (70.0±1.9%) compared to AV (86.4±1.0%). PM decreased (P<0.05) from 12 hours after death (31.3±4.0%) compared to AV group (73.2±1.4%). The percentage of viable cells in HOST decreased (P<0.05) in the G48 (60.0±8.9%). Spermatozoa recovery was lower (P<0.05) 48 hours after death (2064.2±230.7 x 106 spermatozoa) compared to G0(2623.6±288.4 x 106 spermatozoa). In conclusion, under the conditions of this study, it would be possible to use epididymal spermatozoa recovered up to 24 hours after death for artificial insemination or in vitro fertilization; however, fertility trials are necessary to prove this hypothesis.(AU)

Os objetivos deste estudo foram avaliar o período pelo qual era possível recuperar espermatozoides ovinos viáveis da cauda de epidídimos mantidos em temperatura ambiente (18-25°C). O sêmen coletado em vagina artificial (AV) foi utilizado como controle. Os espermatozoides foram coletados dos epidídimos à zero hora (G0), às seis (G6), 12 (G12), 24 (G24) e 48 (G48) horas post mortem. A motilidade total (TM), a motilidade progressiva (PM), a integridade de membrana plasmática em solução hiposmótica (HOST) e a morfologia espermática foram avaliadas. A TM diminuiu (P<0,05) a partir de 24 horas após a morte (70,0±1,9%) comparado ao sêmen coletado em AV (86,4±1,0%). A PM diminuiu (P<0,05) a partir de 12 horas após a morte (31,3±4,0%) comparado ao grupo AV (73,2±1,4%). A porcentagem de espermatozoides viáveis no HOST diminuiu (P<0,05) no G48 (60,0±8,9%). A recuperação espermática foi menor (P<0,05) 48 horas após a morte (2064,2±498,1 x 106 espermatozoides) comparado ao G0 (2298,4±288,4 x 106 espermatozoides). Em conclusão, nas condições deste estudo, é possível utilizar espermatozoides epididimários recuperados até 24 horas após a morte para inseminação artificial ou fertilização in vitro, porém testes de fertilidade são necessários para comprovar essa hipótese.(AU)

Effect of waterfalls and the flood pulse on the structure of fish assemblages of the middle Xingu River in the eastern Amazon basin / Efeito das cachoeiras e do pulso de inundação na estrutura das assembleias de peixes do Médio Rio Xingu, Amazônia oriental

The structure of fish assemblages in Neotropical rivers is influenced by a series of environmental, spatial and/or temporal factors, given that different species will occupy the habitats that present the most favourable conditions to their survival. The present study aims to identify the principal factors responsible for the structuring of the fish assemblages found in the middle Xingu River, examining the influence of environmental, spatial, and temporal factors, in addition to the presence of natural barriers (waterfalls). For this, data were collected every three months between July 2012 and April 2013, using gillnets of different sizes and meshes. In addition to biotic data, 17 environmental variables were measured. A total of 8,485 fish specimens were collected during the study, representing 188 species. Total dissolved solids, conductivity, total suspended matter, and dissolved oxygen concentrations were the variables that had the greatest influence on the characteristics of the fish fauna of the middle Xingu. Only the barriers and hydrological periods played a significant deterministic role, resulting in both longitudinal and lateral gradients. This emphasizes the role of the connectivity of the different habitats found within the study area in the structuring of its fish assemblages.

A estrutura da ictiofauna em rios neotropicais é constantemente influenciada por fatores ambientais, espaciais e/ou temporais, uma vez que as espécies tendem a ocupar ambientes com condições favoráveis à sua sobrevivência. Dessa forma, esta pesquisa tem como objetivo responder qual o principal fator responsável pela estruturação das assembleias de peixes no Médio Rio Xingu, testando a influência dos fatores ambientais, espaciais e temporais, além da presença de barreiras naturais (cachoeiras). Os dados foram coletados, trimestralmente, entre os meses de julho de 2012 e abril de 2013, utilizando redes de emalhe de tamanhos de malha variados. Foram mensuradas 17 variáveis ambientais. Foram coletados 8.485 indivíduos distribuídos em 188 espécies. Observou-se que sólidos dissolvidos totais, condutividade, material em suspensão total e oxigênio dissolvido foram as variáveis que mais influenciaram a ictiofauna do médio Rio Xingu. Observou-se que apenas as barreiras naturais e os períodos hidrológicos foram determinantes, ocorrendo tanto variação longitudinal quanto lateral, ficando claro que a conectividade entre os diferentes trechos do médio rio Xingu é de suma importância na estruturação das assembleias de peixes.

Efeito da suplementação com vitamina E sobre os metabolismos oxidativo e cardíaco em equinos submetidos a exercício de alta intensidade / Effect of vitamin E on oxidative and cardiac metabolism in horses submitted to high intensity exercise

A suplementação antioxidante visa prevenir os danos oxidativos induzidos pelo exercício físico em diversos tecidos, como o miocárdio. Nesse contexto, este estudo objetivou avaliar os marcadores cardíacos e a lipoperoxidação em equinos no teste de exercício de rápida aceleração e curta duração (TRA), em esteira de alta velocidade, antes e após a suplementação com vitamina E. Para tanto, foram utilizados 10 equinos sem treinamento, que realizaram o primeiro TRA (TRA1) com carga de trabalho fundamentada no consumo máximo de oxigênio individual (VO2max) e que induziu a concentração de lactato maior que 4mmol/L, sendo considerado predominantemente anaeróbico. Em seguida, os equinos receberam vitamina E (dl-alfa-tocoferol) na dose de 1.000UI/dia, por via oral, durante 52 dias, e, posteriormente, realizaram um segundo TRA (TRA2) com o mesmo protocolo de TRA1. As amostras de sangue foram colhidas nos momentos antes do exercício, imediatamente após o término do teste e em 1h, 3h, 6h, 12h e 24h subsequentes. Determinou-se o malondialdeído (MDA) plasmático como índice de lipoperoxidação, e as concentrações séricas de troponina I cardíaca (cTnI), isoenzima MB da creatinoquinase (CK-MB) e mioglobina, como marcadores cardíacos. Como efeito do exercício, observou-se aumento discreto de MDA, de cTnI e de CK-MB, sendo significativo apenas para CK-MB. A suplementação foi capaz de amenizar a produção das espécies reativas de oxigênio, evidenciada pela menor concentração de MDA em TRA2, em 24h, além de causar um efeito protetor no miocárdio, devido ao menor valor de cTnI em 6h no TRA2 em relação ao TRA1. Não houve grandes alterações na concentração de mioglobina. Concluiu-se que o exercício de alta intensidade promoveu estresse no miocárdio nos equinos avaliados, bem como houve efeito benéfico da vitamina E na proteção miocárdica e sobre a lipoperoxidação.

Antioxidant supplementation aims to prevent oxidative damage induced by physical exercise in several tissues, including myocardium. This study aimed to assess cardiac and lipid peroxidation markers of horses submitted to a high intensity and short duration exercise test (TRA) on a high speed treadmill, before and after vitamin E supplementation. Ten untrained horses performed the first TRA test (TRA1), with workload based on maximal oxygen uptake (VO2max), which induced a blood lactate higher than 4mmol/L, i.e. predominantly anaerobic exercise test. After TRA1, the horses were supplemented with oral vitamin E (dl-alpha-tocopherol) at a daily dose of 1,000IU for 52 days. Then they performed the second test (TRA2) with the same protocol of TRA1. Blood samples were collected before exercise, immediately after and at 1h, 3h, 6h, 12h and 24h after the end of test. Plasma malondialdehyde (MDA), as lipid peroxidation index, and serum cardiac troponin I (cTnI), creatine kinase isoenzyme MB (CK-MB) and myoglobin, as cardiac markers, were determined. There was a slight increase in MDA, cTnI and CK-MB levels caused by the exercise, but statistically significant only for CK-MB levels. Vitamin E was effective on scavenging reactive oxygen species, as evidenced by lower MDA level after supplementation (TRA2) at 24h. Also, vitamin E caused myocardial protection showed by lower cTnI concentration at 6h in TRA2 when compared to TRA1. There were no changes on myoglobin levels. In conclusion, high intensity exercise induced myocardial stress in horses and we also verified the beneficial effect of vitamin E on myocardial protection and lipid peroxidation.

Challenges in the research and development of new human vaccines

The field of vaccinology was born from the observations by the fathers of vaccination, Edward Jenner and Louis Pasteur, that a permanent, positive change in the way our bodies respond to life-threatening infectious diseases can be obtained by specific challenge with the inactivated infectious agent performed in a controlled manner, avoiding the development of clinical disease upon exposure to the virulent pathogen. Many of the vaccines still in use today were developed on an empirical basis, essentially following the paradigm established by Pasteur, "isolate, inactivate, and inject" the disease-causing microorganism, and are capable of eliciting uniform, long-term immune memory responses that constitute the key to their proven efficacy. However, vaccines for pathogens considered as priority targets of public health concern are still lacking. The literature tends to focus more often on vaccine research problems associated with specific pathogens, but it is increasingly clear that there are common bottlenecks in vaccine research, which need to be solved in order to advance the development of the field as a whole. As part of a group of articles, the objective of the present report is to pinpoint these bottlenecks, exploring the literature for common problems and solutions in vaccine research applied to different situations. Our goal is to stimulate brainstorming among specialists of different fields related to vaccine research and development. Here, we briefly summarize the topics we intend to deal with in this discussion.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Coping with genetic diversity: the contribution of pathogen and human genomics to modern vaccinology

Vaccine development faces major difficulties partly because of genetic variation in both infectious organisms and humans. This causes antigenic variation in infectious agents and a high interindividual variability in the human response to the vaccine. The exponential growth of genome sequence information has induced a shift from conventional culture-based to genome-based vaccinology, and allows the tackling of challenges in vaccine development due to pathogen genetic variability. Additionally, recent advances in immunogenetics and genomics should help in the understanding of the influence of genetic factors on the interindividual and interpopulation variations in immune responses to vaccines, and could be useful for developing new vaccine strategies. Accumulating results provide evidence for the existence of a number of genes involved in protective immune responses that are induced either by natural infections or vaccines. Variation in immune responses could be viewed as the result of a perturbation of gene networks; this should help in understanding how a particular polymorphism or a combination thereof could affect protective immune responses. Here we will present: i) the first genome-based vaccines that served as proof of concept, and that provided new critical insights into vaccine development strategies; ii) an overview of genetic predisposition in infectious diseases and genetic control in responses to vaccines; iii) population genetic differences that are a rationale behind group-targeted vaccines; iv) an outlook for genetic control in infectious diseases, with special emphasis on the concept of molecular networks that will provide a structure to the huge amount of genomic data.

Efeito inibidor do soro urêmico sobre o metabolismo oxidativo dos neutrófilos de cães / Inhibitory effect of uremic serum on neutrophils oxidative metabolism from dogs

Foi testada a hipótese de que, à semelhança do que ocorre em humanos, os componentes do soro urêmico inibem o metabolismo oxidativo dos neutrófilos de cães. Para isto, o sangue total de 10 cães foi incubado com soro homólogo urêmico e não urêmico e posteriormente comparado quanto à produção neutrofílica de superóxido estimada pelo método citoquímico de redução do tetrazólio nitroazul (NBT). A produção de superóxido gerada pelo metabolismo oxidativo dos neutrófilos tratados com soro urêmico apresentou significante redução (P<0,05) em relação aos tratados com plasma autólogo e homólogo com níveis normais de ureia. Concluiu-se que os componentes presentes no soro urêmico inibem ex vivo o metabolismo oxidativo dos neutrófilos de cães portadores de insuficiência renal e que, portanto, têm sua imunidade inata comprometida.

The hypothesis that the uremic compounds decrease the neutrophil oxidative metabolism in dogs, as occurs in humans was tested. Whole blood from 10 dogs was incubated with uremic and non-uremic homologous sera and the superoxide production by activated neutrophils was quantified by the nitroblue tetrazolium reduction test (NBT). The superoxide production generated by neutrophils in the presence of uremic serum was significantly decreased (P<0.05) in comparison with autologous and homologous sera with normal urea levels. It was concluded that the presence of components of uremic serum inhibits the ex vivo neutrophil oxidative metabolism in dogs, suggesting that, similarly to humans, dogs with renal failure may have the innate immunity response compromised.

Drug resistance in Chromobacterium violaceum

Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world. This bacterium is able to produce a large variety of products of biotechnological and pharmacological use. Although C. violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported. Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes. A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism. Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous. In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found. It is postulated that these genes contribute to the ability of C. violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium

Flora bacteriana heterotrófica em ostras (Crassostrea rhizophorae) e águas da Baía Norte, Ilha de Santa Catarina, Brasil / Heterotrophic bacteria in oysters and waters of North Bay, Santa Catarina, Brazil

A população bacteriana das águas da Baía Norte da Ilha de Santa Catarina foi estudada utilizando-se a ostra Crassostrea rhizophorae como biondicador. Foram realizadas avaliações quali-quantitativas de bactérias heterotróphicas originárias da água do mar e de ostras de três locais da Baía. Nas ostras, analisou-se a massa digestiva e o corpo inteiro do animal. Os resultados mostram altas concentrações de bactérias na água e nos bivalves. A maior acumulação de microrganismos foi detectada no trato digestivo das ostras. Enterobactérias predominam na água enquanto as Pseudomonadaceae são mais frequentes nos bivalves.