DNA Barcoding of Neotropical Sand Flies (Diptera,Psychodidae, Phlebotominae): Species Identification and Discovery within Brazil

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.     

Hunting pressure modulates the composition and size structure of terrestrial and arboreal vertebrates in Amazonian forests

Overhunting is a leading contemporary driver of tropical forest wildlife loss. The absence or extremely low densities of large-bodied vertebrates disrupts plant-animal mutualisms and consequently degrades key ecosystem services. Understanding patterns of defaunation is therefore crucial given that most tropical forests worldwide are now “half-empty”. Here we investigate changes in vertebrate community composition and size structure along a gradient of marked anthropogenic hunting pressure in the Médio Juruá region of western Brazilian Amazonia. Using a novel camera trapping grid design deployed both in the understorey and the forest canopy, we estimated the aggregate biomass of several functional groups of terrestrial and arboreal species at 28 sites along the hunting gradient. Generalized linear models (GLMs) identified hunting pressure as the most important driver of aggregate biomass for game, terrestrial, and arboreal species, as well as nocturnal rodents, frugivores, and granivores. Local hunting pressure affected vertebrate community structure as shown by both GLM and ordination analyses. The size structure of vertebrate fauna changed in heavily hunted areas due to population declines in large-bodied species and apparent compensatory increases in nocturnal rodents. Our study shows markedly altered vertebrate community structure even in remote but heavily settled areas of continuous primary forest. Depletion of frugivore and granivore populations, and concomitant density-compensation by seed predators, likely affect forest regeneration in persistently overhunted tropical forests. These findings contribute to a better understanding of how cascading effects induced by historical defaunation operate, informing wildlife management policy in tropical peri-urban, rural and wilderness areas.

     

Effects of Soy Lecithin Extender on Dog Sperm Cryopreservation

Semen cryopreservation is an essential biotechnology in canine reproduction and during the cryopreservation process commonly egg yolk are used. The discrepancy in the egg yolk composition and the potential risk of disease dissemination are obstacles for semen exportation and use. Therefore, studies aiming to substitute egg yolk are extremely important. In this context, soy lecithin contains a low-density lipoprotein fraction, is an interesting alternative. Thus, the objective of this study was to compare extenders based on soy lecithin (several concentrations and forms) with egg yolk during the cryopreservation process of dog sperm. For this purpose, we used twelve dogs. Semen was evaluated at different time points (after refrigeration, glycerolization, and thawing), by motility analysis (CASA) and functional tests (e.g., membrane integrity—eosin/nigrosin, acrosome integrity—fast green/Bengal rose, mitochondrial activity—3’3 diaminobenzidine, Chromatin susceptibility to acid-induced denaturation—SCSA, and susceptibility to oxidative stress—thiobarbituric acid reactive substances). The results indicated that egg yolk and lower concentrations of lecithin had similar effects on mitochondrial activity and motility. Thus, soy lecithin is a potentially viable alternative to egg yolk for the cryopreservation of dog semen.     

Vitamin C modulates glutamate transport and NMDA receptor function in the retina

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS ). In the retina, a high‐affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N ‐methyl‐d ‐aspartate (NMDA ) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate‐stimulated [³H] MK 801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element‐binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase‐dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.

     

Hyperpalatable Diet and Physical Exercise Modulate the Expression of the Glial Monocarboxylate Transporters MCT1 and 4

Hyperpalatable diets (HP) impair brain metabolism, and regular physical exercise has an apparent opposite effect. Here, we combined a prior long-term exposure to HP diet followed by physical exercise and evaluated the impact on some neuroenergetic components and on cognitive performance. We assessed the extracellular lactate concentration, expression of monocarboxylate transporters (MCTs), pyruvate dehydrogenase (PDH), and mitochondrial function in the hippocampus. Male C57BL/6J mice were fed 4 months with HP or a control diet. Subsequently, they were divided in the following groups: control diet sedentary (CDS), control diet exercise (CDE), HP diet sedentary (HPS), and HP diet exercise (HPE) (n = 15 per group) and were engaged for an additional 30-day period of voluntary exercise and HP diet. Relative to the control situation, exercise increased MCT1, MCT4, and PDH protein levels, while the HP diet increased MCT1 and MCT4 protein levels. The production of hydrogen peroxide (H₂O₂) and the mitochondrial membrane potential (??_m) stimulated by succinate in hippocampal homogenates were not significantly different between groups. ADP phosphorylation and the maximal respiratory rate induced by FCCP showed similar responses between groups, implying a normal mitochondrial function. Also, extracellular brain lactate levels were increased in the HPE group compared to other groups soon after performing the Y-maze task. However, such enhanced lactate levels were not associated with improved memory performance. In summary, hippocampal protein expression levels of MCT1 and 4 were increased by physical exercise and HP diet, whereas PDH was only increased by exercise. These observations indicate that a hippocampal metabolic reprogramming takes place in response to these environmental factors.     

A comparative study of ectonucleotidase and P2 receptor mRNA profiles in C6 cell line cultures and C6 <Emphasis Type="Italic">ex vivo</Emphasis> glioma model

Glioblastoma multiforme is the most common type of primary brain tumour and has the worst clinical outcome. Nucleotides represent an important class of extracellular molecules involved in cell proliferation, differentiation and apoptosis. Alterations in purinergic signalling have been implicated in pathological processes, such as cancer, and glioma cell lines are widely employed as a model to study the biology of brain tumours. Increasing evidence, however, suggests that glioma cell lines may not present all the phenotypic and genetic characteristics of the primary tumours. We have compared the biological characteristics of C6 rat glioma cells in culture and the same cells after their implantation in the rat brain and growth in culture (denominated as the C6 ex vivo culture model). Parameters evaluated included cell morphology, differentiation, angiogenic markers, purinergic receptors and ecto-nucleotidase mRNA profile/enzymatic activity. Analysis of the C6 glioma cell line and C6 ex vivo glioma cultures revealed distinct cell morphologies, although cell differentiation and angiogenic marker expressions were similar. Both glioma models co-expressed multiple P2X and P2Y receptor subtypes with some differences. In addition, the C6 glioma cell line and C6 ex vivo glioma cultures exhibited similar extracellular ATP metabolism and cell proliferation behaviour when exposed to cytotoxic ATP concentrations. Thus, the disruption of purinergic signalling is a feature shown not only by glioma cell lineages, but also by primary glioma cultures. Our results therefore suggest the participation of the purinergic system in glioma malignancy. This study was supported by grants from the Brazilian agencies CNPq, FAPERGS and Fundo de Incentivo à Pesquisa e Eventos (HCPA). E. Braganhol and D. Huppes were recipients of Brazilian CNPq fellowships; A. Bernardi was the recipient of a CAPES fellowship.     

In Vitro Antifungal Activity of Dihydroxyacetone Against Causative Agents of Dermatomycosis

Dihydroxyacetone (DHA), a three-carbon sugar, is the browning ingredient in commercial sunless tanning formulations. DHA preparations have been used for more than 50 years and are currently highly popular for producing temporary pigmentation resembling an ultraviolet-induced tan. In this work, the in vitro antifungal activity of dihydroxyacetone was tested against causative agents of dermatomycosis, more specifically against dermatophytes and Candida spp. The antifungal activity was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines for yeasts and filamentous fungi. The data obtained show that the fungicidal activity varied from 1.6 to 50 mg ml⁻¹. DHA seems to be a promising substance for the treatment of dermatomycosis because it has antifungal properties at the same concentration used in artificial suntan lotions. Therefore, it is a potential low-toxicity antifungal agent that may be used topically because of its penetration into the corneal layers of the skin.     

Detection of the Influenza A(H1N1)pdm09 Virus Carrying the K-15E,P83S and Q293H Mutations in Patients Who Have Undergone Bone Marrow Transplant

The 2009 pandemic influenza A(H1N1)pdm09 virus emerged and caused considerable morbidity and mortality in the third world, especially in Brazil. Although circulating strains of A(H1N1)pdm09 are A/California/04/2009-like (CA-04-like) viruses, various studies have suggested that some mutations in the viral hemagglutinin (HA) may be associated with enhanced severity and fatality. This phenomenon is particularly challenging for immunocompromised individuals, such as those who have undergone bone marrow transplant (BMT), because they are more likely to display worse clinical outcomes to influenza infection than non-immunocompromised individuals. We studied the clinical and viral aspects of post-BMT patients with confirmed A(H1N1)pdm09 diagnosis in the largest cancer hospital in Brazil. We found a viral strain with K-15E, P83S and Q293H polymorphisms in the HA, which is presumably more virulent, in these individuals. Despite that, these patients showed only mild symptoms of infection. Our findings complement the discovery of mild cases of infection with the A(H1N1)pdm09 virus with the K-15E, P83S and Q293H mutations in Brazil and oppose other studies that have linked these changes with increased disease severity. These results could be important for a better comprehension of the impact of the pandemic influenza in the context of BMT.     

LITAF Mutations Associated with Charcot-Marie-Tooth Disease 1C Show Mislocalization from the Late Endosome/Lysosome to the Mitochondria

Charcot-Marie-Tooth (CMT) disease is one of the most common heritable neuromuscular disorders, affecting 1 in every 2500 people. Mutations in LITAF have been shown to be causative for CMT type 1C disease. In this paper we explore the subcellular localization of wild type LITAF and mutant forms of LITAF known to cause CMT1C (T49M, A111G, G112S, T115N, W116G, L122V and P135T). The results show that LITAF mutants A111G, G112S, W116G, and T115N mislocalize from the late endosome/lysosome to the mitochondria while the mutants T49M, L122V, and P135T show partial mislocalization with a portion of the total protein present in the late endosome/lysosome and the remainder of the protein localized to the mitochondria. This suggests that different mutants of LITAF will produce differing severity of disease. We also explored the effect of the presence of mutant LITAF on wild-type LITAF localization. We showed that in cells heterozygous for LITAF, CMT1C mutants T49M and G112S are dominant since wild-type LITAF localized to the mitochondria when co-transfected with a LITAF mutant. Finally, we demonstrated how LITAF transits to the endosome and mitochondria compartments of the cell. Using Brefeldin A to block ER to Golgi transport we demonstrated that wild type LITAF traffics through the secretory pathway to the late endosome/lysosome while the LITAF mutants transit to the mitochondria independent of the secretory pathway. In addition, we demonstrated that the C-terminus of LITAF is necessary and sufficient for targeting of wild-type LITAF to the late endosome/lysosome and the mutants to the mitochondria. Together these data provide insight into how mutations in LITAF cause CMT1C disease.