The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human ...patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.
The search for non-invasive signs of oocyte meiotic competence is very important for the development of in vitro follicle culture (IVFC) systems. The aims of the present study were: (1) to ...investigate the effect of in vitro maturation (IVM) of in vivo grown goat COCs, in group or individually, on oocyte chromatin configuration (Experiment 1), and (2) the influence of IVFC period (12 vs. 18 days) on the ability of the oocyte to resume meiosis immediately after IVFC (before in vitro maturation; IVM), or after IVM (Experiment 2). In experiment 1, in vivo grown cumulus-oocyte complexes (COCs) were submitted to IVM in groups (10 COCs/100 μL-drop) or individually (1 COC/10 μL-drop), and chromatin configuration was assessed. In experiment 2, isolated follicles were individually cultured for 12 or 18 days, and submitted to individual IVM afterwards. The following end points were evaluated: follicular growth and morphology, oocyte diameter, viability and chromatin configuration, as well as individual follicular estradiol production. Similar maturation rates were obtained between in vivo grown COCs matured individually and in groups (66.7% vs. 63.6%, respectively) (Experiment 1). Only after 18 days of IVFC, oocytes were able to grow during IVM, reaching a mean oocyte diameter of 119 μm. Also, this treatment produced the highest rate of metaphase II oocytes (46.2% out of the total number of cultured follicles). Finally, it was observed that follicles with a daily growth rate >7.1 μm/day (fast-growing) and that reached at least 600 μm in diameter, were more likely (P < 0.05) to produce oocytes capable of attaining MII. In conclusion, caprine oocytes can be individually matured in vitro, as efficiently as in groups. This result was essential to pair in vitro follicle development and in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to establish non-invasive signs for the efficiency of IVFC based on follicle daily growth rate and diameter, and oocyte diameter: follicle daily growth >7 μm, follicle diameter of at least 600 μm, and oocyte diameter ≥120 μm. In addition, 18 days seems to be the most suitable culture time for caprine early antral follicles.
•Goat oocytes can be matured individually in vitro as efficiently as in groups.•Follicular dynamics in vitro: non-invasive signs of oocyte meiotic competence.•18 days is the most suitable culture time for caprine early antral follicles.
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•We report the effect of microwaves and conventional heating on the crude oil.•Microwaves allowed us to remove water and acidic compounds from the oil phase.•Temperature and pH can be ...managed to alter the profile of acids recovered.
In this work, the application of microwave technology is investigated for breaking water-in-heavy crude oil emulsions focusing upon the identification of acidic species existing in the aqueous phase recovered by the microwave demulsification process. To accomplish this, a methodology for characterization of acidic species based on GC/qMS (gas chromatography coupled to mass spectrometry quadrupole type) has been developed. An experimental study was conducted to assess the effect of operating conditions, including the heating mode (microwaves/conventional heating), process time (30, 60min), pH of the aqueous phase (pH=2, 6, 10) and temperature (90, 120, 150°C) over the migration of acidic species from the crude oil to the aqueous phase during the demulsification of a heavy crude oil. The most influential variable was the temperature, which favors the partitioning of a larger number of acidic species, including cyclic compounds of low molecular weight that were not extracted at low temperature. The microwave heating allowed a wider distribution profile of monocyclic and bicyclic acids than the conventional heating scheme. Changing the aqueous phase pH from acid to the alkaline range modifies the profile of acidic compounds from predominantly acyclic to monocyclic and bicyclic species.
Dengue is an important arboviral infection, causing a broad range symptom that varies from life-threatening mild illness to severe clinical manifestations. Recent studies reported the impairment of ...the central nervous system (CNS) after dengue infection, a characteristic previously considered as atypical and underreported. However, little is known about the neuropathology associated to dengue. Since animal models are important tools for helping to understand the dengue pathogenesis, including neurological damages, the aim of this work was to investigate the effects of intracerebral inoculation of a neuroadapted dengue serotype 2 virus (DENV2) in immunocompetent BALB/c mice, mimicking some aspects of the viral encephalitis. Mice presented neurological morbidity after the 7
day post infection. At the same time, histopathological analysis revealed that DENV2 led to damages in the CNS, such as hemorrhage, reactive gliosis, hyperplastic and hypertrophied microglia, astrocyte proliferation, Purkinje neurons retraction and cellular infiltration around vessels in the pia mater and in neuropil. Viral tropism and replication were detected in resident cells of the brain and cerebellum, such as neurons, astrocyte, microglia and oligodendrocytes. Results suggest that this classical mice model might be useful for analyzing the neurotropic effect of DENV with similarities to what occurs in human.
The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development ...of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300-400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus-oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.
This study investigated: 1) the kinetics of oocyte chromatin configuration during in vitro maturation (IVM) of caprine and bovine oocytes; and 2) the effect of in vitro pre-maturation (IVPM) with ...cilostamide with or without association of the follicular wall (FW) on the same parameters. In experiment I, cumulus-oocyte complexes (COCs) were cultured in vitro in a standard maturation medium for 6, 12, 18 or 30 h. For experiment II, the COCs were cultured for 30 h, either in a standard IVM medium or in IVPM containing cilostamide (10 or 20 μM) and FW alone or in combination, for 6 or 12 h before the onset of maturation. The MII rate was similar (P > .05) between 18 and 30 h of maturation, both of which were higher (P < .05) than 6 and 12 h IVM in both species (Experiment I). Contrary to caprine, all IVPM treatments presented a higher (P < .05) percentage of bovine oocytes arrested at the GV stage than the control treatment after 6 h of culture. The percentage of MII oocytes after 30 h (IVPM+IVM) of culture in bovine oocytes treated with 10 μM cilostamide associated with FW and FW alone cultured for 6 h presented MII percentages similar to the control. However, in caprine, these treatments significantly reduced the percentages of MII in relation to the control treatment (Experiment II). In conclusion, the combination of concentration-exposure time to cilostamide during IVPM delayed meiotic progression in bovine after 6 and 12 h of culture. However, overall the culture period (IVPM+IVM) influenced the oocyte chromatin configuration and kinetics in both species.
•The combination (concentration-exposure time) to cilostamide during IVPM influenced oocyte chromatin configuration after IVM.•Cilostamide impacts differently the kinetics of oocyte in vitro maturation in caprine and bovine.•Oocyte exposure to cilostamide + FW in bovine caused a temporary blockage and further yielded MII rates similar to control.
Abstract We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a ...(ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.
Trypanosoma cruzi depends on the effectiveness of redox metabolism to survive and ensure infection in the host. Homeostasis of redox metabolism in T. cruzi is achieved by the actions of several ...proteins that differ in many aspects from host proteins. Although extensive research has been performed examining hydroperoxide cytosolic antioxidant defense centered on trypanothione, the mechanisms of mitochondrial antioxidant defense are not yet known. The aim of this study was to elucidate the partners of TcMPx antioxidant pathway and to determine the influence of the cellular context (physiological versus oxidative stress). Through co-precipitation coupled with a mass spectrometry approach, a variety of proteins were detected under physiological and oxidative stress conditions. Interestingly, functional category analysis of the proteins identified under physiological conditions showed that they were involved in the stress response, oxidoreduction, thiol transfer, and metabolic processes; this profile is distinct under oxidative stress conditions likely due to structural alterations. Our findings help to elucidate the reactions involving TcMPx and most importantly also reveal that this protein is present throughout the cell and that its interaction partners change following oxidative stress exposure. The involvement and significance of the proteins found to interact with TcMPx and other possible functions for this protein are discussed widening our knowledge regarding T. cruzi mitochondrial antioxidant defenses.
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•TcMPx is distributed throughout the cell.•Possible other functions for TcMPx are provided.•TcMPx interaction partners change following oxidative stress exposure.
ABSTRACT Coffee growers who produce the robusta species (Conilon) have sought to increase productivity and drink quality by improving production techniques. Artificial vision systems can assist in ...increasing the efficiency of operations associated with crop management. This study aimed to obtain colorimetric characteristics of robusta coffee fruits at different stages of maturity and use them for classifying fruits from digital images. A digital camera with spectral resolution in the visible was used to acquire images from 60 samples of coffee fruits at the green, cherry, and over-ripe stages of maturity. Colorimetric variables were extracted from the RGB, HIS, and L*a*b* color models and correlated with the physicochemical attributes of the fruits. The principal component analysis associated with the k-means technique was applied to the colorimetric variables that showed a significant correlation with the physical-chemical attributes. The colorimetric variables were reduced to a principal component, which presented an explanatory percentage of the variance of 82.33%. The clustering obtained by the application of the k-means technique showed the feasibility of using images for the automatic classification of robusta coffee fruits, with an overall accuracy of 100%.
ABSTRACT Results of evaluations using optical evaluation methods may be correlated with tomato quality and maturation. In this context, the objective of this study was to evaluated the correlation ...between tomato colorimetric and physico-chemical variables, clustering them as a function of maturation stages, using multivariate analysis. The experiment was conducted using 150 fruits and three maturation stages (immature, light red and mature). The physico-chemical variables were evaluated through traditional methods. The colorimetric variables were assessed on images in RGB color model taken with a digital camera. The correlation between colorimetric and physico-chemical variables was analyzed using the Pearson’s coefficient. Principal components analysis and k-means clustering method was applied to three data set: RGB isolated variables; colorimetric variables calculated by relation between the RGB bands (colorimetric indexes); and physico-chemical variables. The colorimetric variables present higher explanatory capacity of the maturation variation than physico-chemical variables. The colorimetric indexes presented higher performance in clustering (accuracy of 0.98) tomatoes as a function of maturation.
RESUMO Variáveis obtidas por métodos ópticos podem apresentar correlação com características relacionadas à qualidade e maturação de tomates. Neste contexto, objetivou-se com este estudo analisar a correlação entre as variáveis colorimétricas e físico-químicas de tomates, agrupando-os em função do estádio de maturação, utilizando análise multivariada. O experimento foi realizado utilizando 150 frutos em três estádios de maturação (imaturos, coloridos e maduros). As variáveis físico-químicas foram obtidas por meio de métodos tradicionais. As variáveis colorimétricas foram obtidas a partir de uma câmera digital no espaço de cor RGB. A análise da correlação entre as variáveis colorimétricas e físico-químicas foi realizada por meio do coeficiente de Pearson. A análise de componentes principais associada à técnica de agrupamento k-means foi aplicada em três grupos de dados: variáveis RGB isoladas; variáveis colorimétricas calculadas pela relação entre as bandas RGB (índices colorimétricos); e variáveis físico-químicas. As variáveis colorimétricas apresentam maior capacidade explicativa da variação da maturação quando comparadas às variáveis físico-químicas. O conjunto de índices colorimétricos apresentou o maior desempenho no agrupamento (exatidão global = 0,98) dos tomates em função da maturação.
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