Cytotoxicity of isolated compounds from Picrasma crenata (Vell.) Engl. in animal tumor cell (HTC).

The study aim was to evaluate the cytotoxic activity, using the MTT test [3-(4,5-Dimethilthiazol-2-yl)-2,5-diphenil tetrazolium bromide], from the crude extract of Picrasma crenata (Pau Tenente) and its isolated compounds, quassin and parain, in culture of rat liver tumor cells (HTC). The test was carried out exposing the cells for 24, 48 and 72 hours to concentrations of 5, 10, 50, 100, 200, 300, 400, 500 and 1000 µg of crude extract of Pau Tenente/mL of culture medium and 1, 5, 10, 15, 20, 40, 60, 80 and 100 µg of quassin or parain compounds/mL of culture medium. The absorbances averages results obtained showed that the crude extract did not present cytotoxicity for the HTC cells in all the concentrations and evaluated times. For quassin, the concentrations of 80 and 100 µg/mL were cytotoxic, after 72 hours of treatment. For parain, the concentrations of 1, 5, 20, 40, 60, 80 and 100 µg/mL, in 72 hours, were cytotoxic, revealing a new activity for this compound. Thus, the results demonstrate a first indication of the cytotoxic activity of compounds quassin and parain, adding an important social and economic value to them, and may have application in future research and in pharmaceutical industry.

Neurotoxicity in zebrafish exposed to carbon nanotubes: Effects on neurotransmitters levels and antioxidant system.

Given the increasing use of carbon nanotubes (CNT) in several industries and technological applications, it is essential to perform in vivo toxicological studies with these nanomaterials to evaluate their potential ecotoxicity. Dopamine (DA) and serotonin (5HT) are key neurotransmitters for brain functions and behavioral responses. Determination of DA and 5HT were performed in brain samples from zebrafish Danio rerio exposed i.p. to single-walled CNT (SWCNT), besides analyzing acetylcholinesterase (AChE) and ectonucleotidases activity, lipid peroxidation and total antioxidant capacity. Results showed that treatment with SWCNT increased between 3 and 6-fold the concentration of DA and 5HT (p < 0.05). Similarly, a significant reduction (p < 0.05) in AChE activity was observed in the brains of SWCNT exposed zebrafish when compared to the control groups. Cholinergic, serotonergic, and dopaminergic systems, through AChE activity and serotonin and dopamine levels, respectively were affected by SWCNT in the zebrafish brain. Alterations in these neurotransmitters can potentially affect several physiological and behavioral that they control.

Oxidative status of the myocardium in response to different intensities of physical training.

The intensity of exercise determines the metabolic pathway and the energetic substrate that is spent. Our study sought to identify the effects of different intensities of swimming on myocardial oxidative status and the blood lipid profile. Eighty Wistar rats (male and female) submitted to different intensities of a swimming regimen (low, LS; moderate, MS; or high, HS) for 16 weeks. Samples of blood and myocardium from the left ventricle were collected to determine lipid profiles and oxidative status. Reactive oxygen species (ROS) and antioxidant capacity against peroxyl radicals (ACAP), lipid profiles and lipid peroxidation was analyzed. ROS levels and ACAP were higher in male rats than in female rats overall (p<0.05). However, ACAP in the myocardium was significantly elevated in LS female rats compared to the MS and HS female rats, which had a significantly lower ACAP compared to all other groups. LS and MS training in both sexes and HS training (in females) led to significant decreases in the heart's lipid peroxidation. Amelioration of the lipid profile and reduction in oxidative damage contributed to a physiological state that benefits cardiovascular function in exercised animals. The results show that low and moderate intensity exercise promotes beneficial adaptations.

Analysis of power transfer on multicore fibers with long-period gratings.

The use of long-period gratings (LPGs) to distribute optical power from one core to all the cores in a multicore fiber (MCF) is theoretically analyzed. Simple analytical expressions that describe the mode power's evolution along the LPGs are derived from the coupled mode equations. This study demonstrated the power transfer between cores in a MCF promoted by identical LPGs. Moreover, with this technique, the pump signal can be distributed to all the cores without interfering with the transmission signals.

Immunohistochemical assessment of PTEN in vulvar cancer: best practices for tissue staining, evaluation, and clinical association.

BACKGROUND: Pten encodes a well-characterized protein that is important in several cancers due to its tumor suppressor function. Yet, the detection and evaluation of PTEN by immunohistochemistry (IHC) for clinical practice have not been standardized. Thus, in this study, we performed a literature review of protocols for PTEN assessment by IHC and the possible differences in evaluation, based on our experience with vulvar carcinomas. Also, we report some of our most recent findings regarding the clinical impact of PTEN in this type of tumor. METHODS: In total, 150 FFPE vulvar carcinoma samples in a tissue microarray were examined by IHC with regard to PTEN, PI3K, AKT, and mTOR. All evaluations were performed by slide digitalization and quantification using APERIO ImageScope software. All measurements were converted into HScore values for the statistical analysis. RESULTS: Sharp and specific PTEN expression was observed in the nuclei and cytoplasmic compartments. Its HScore values ranged from 3.5 to 226, with a median of 92.5. mTOR expression was robust in all cases (mean HScore=248.1). AKT and PI3K had median HScore values of 200.5 and 156.5, respectively. In addition, PTEN expression was associated with higher rates of patient survival. CONCLUSION: The preanalytical step is the first issue in the immunohistochemical evaluation of PTEN. With regard to the analytical procedure, the antigen retrieval step yielded better stains for protocols with high-pH buffers, and antibody clone 6H2.1 effected the most reliable results. PTEN is a good prognostic marker for vulvar cancer, correlating with higher rates of patient survival. Our data underscore the importance of technical standardization to ensure more reliable and reproducible evaluation of PTEN in clinical practice.

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.

Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. By contrast, sperm treated with methyl-ß-cyclodextrin showed a significant reduction in cholesterol levels, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in all three stallions. These results show that presence of serum albumin is not associated with a reduction in membrane cholesterol levels in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in a standard capacitating medium. These data indicate an atypical relationship among cholesterol efflux, ROS production, and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine IVF under standard conditions.

Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen.

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap-freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm(2) of membrane was assessed by conventional microscopy (CM) and computer-assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r(2) = 0.91 and CASA: r(2) = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm-egg-binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.

Semen analysis of Golden Retriever healthy dogs and those affected by muscular dystrophy.

This study aimed to characterise canine flow cytometry semen analysis, as well as seminal reactive oxygen species dosage using the Golden Retriever breed as model of study. Moreover, we searched for the influence of muscular dystrophy in Golden Retriever dogs on semen parameters. Thirty-seven semen samples were obtained from healthy Golden Retrievers (n = 15) and from muscular dystrophy affected dogs (n = 22). Sperm-rich fractions were analysed by standardised breeding soundness examination in addition to the assay of fluorescence assisted cell sorting for acrosome integrity, mitochondrial activity and DNA fragmentation. Volume of ejaculate, per cent of motile spermatozoa and vigour were similar between groups; there were no differences in the per cent of minor and major defects. Integrity of acrosomal membrane, mitochondrial potential and sperm DNA fragmentation had no significant differences between groups either. Animals from control group had higher concentration of spontaneous seminal oxidative species in comparison with affected animals. Dogs affected by dystrophy had seminal parameters similar to those observed in healthy dogs except for the lower concentration of oxidative species. Future studies aiming to establish reference values for canine seminal parameters should be considered preferably with distinction of breeds.

Epithelial-mesenchymal transition-like events in vulvar cancer and its relation with HPV.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) still remains an obscure event in vulvar squamous cell carcinoma (VSCC). METHODS: Immunohistochemistry (IHC) expression of E-cadherin, ß-catenin, Snail, Slug, Twist and Vimentin was analysed in 87 VSCC, controlled for human papillomavirus (HPV) positivity, considering tumour front and central tumour as different morphological categories from the same tumour. RESULTS: Lower ß-catenin and higher Vimentin expression was associated with invasive front when compared with the central tumour (P=0.013 and P≤0.001, respectively). Higher expression of E-cadherin in central tumour was significantly related to absence of vascular and perineural invasion, lower invasion depth and ≥2 lymph node involvement. Loss of ß-catenin and high Slug, Snail and Twist expression was associated with HPV-negative tumours. Moreover, ß-catenin lower expression associated with gain in Slug expression predicts a subgroup with worst outcome (P=0.001). Lower expression of ß-catenin in both central tumour and invasive front correlated with lower overall survival (P=0.021 and P=0.011, respectively). Also, multivariate analysis showed that lower ß-catenin expression was independently associated with poorer outcome (P=0.044). CONCLUSION: Human papillomavirus-related tumours show better prognosis and outcome; besides, they do not progress through EMT phenomenon. Immunohistochemical analysis of ß-catenin in invasive tumour front is a key issue for establishing prognosis of vulva cancer.

Halting the fuse discharge propagation using optical fiber microwires.

We report and analyze the halting of the fuse effect propagation in optical fiber microwires. The increase of the mode field diameter in the tapered region decreases the optical intensity resulting in the extinction of the fuse effect. This fiber element presents a low insertion loss and can be introduced in the optical network in order to protect the active equipment from the damage caused by the fuse effect.

The GHEP-EMPOP collaboration on mtDNA population data--A new resource for forensic casework.

Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org).

The use of dried blood spots for assessing antibody response to hepatitis A virus after natural infection and vaccination.

During recent years, vaccination against hepatitis A has been implemented in several countries. It is expected that the increase in mass vaccination against hepatitis A will eventually result in a decreased prevalence of anti-HAV antibodies in the general population. For this reason, a suitable clinical sample for diagnosis of hepatitis A must be sufficiently sensitive to enable detection of lower antibodies titers. In this study, the feasibility of using dried blood spots (DBS) was assessed for the detection of anti-HAV antibodies after a natural infection and vaccination. Seventy-four DBS and paired plasma samples were obtained from a group of college students for a cross-sectional hepatitis A seroepidemiological study. Forty-six students seronegative for anti-HAV were selected randomly and immunized with an inactivated hepatitis A vaccine using an 0-6 month schedule. Seroconversion was monitored in paired plasma and DBS samples 6 months after the first dose followed by a period of 8 and 24 months after the second dose. A strong correlation between OD/CO rates of paired plasma and DBS samples for the detection of anti-HAV was observed. The sensitivity and specificity of the DBS compared with plasma for the detection of anti-HAV antibodies after natural infection was 100%. The sensitivity of DBS in samples collected 24 months after the second dose of hepatitis A vaccine was 95.4%. The results showed that DBS samples can be used for the detection of anti-HAV antibodies both after natural infection or vaccination.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation.

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 x 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.

Seasonal and organ variations in antioxidant capacity, detoxifying competence and oxidative damage in freshwater and estuarine fishes from Southern Brazil.

This study analyzed biochemical biomarkers of freshwater and estuarine fish species from Southern Brazil. It analyzed three organs (muscle, liver and gills), in four fish species (Micropogonias furnieri, Pimelodus pintado, Loricariichthys anus and Parapimelodus nigribarbis) in order to perform an environmental diagnosis. Obtained results showed that liver of L. anus and gills of M. furnieri presented higher total antioxidant capacity against peroxyl radicals during fall, whereas a clear seasonality was found for gill reduced glutathione (GSH) levels of all studied species, with higher concentration during spring. In terms of oxidative damage (TBARS), liver of M. furnieri and gills of P. nigribarbis showed higher TBARS levels during fall, whereas P. pintado showed the lowest TBARS value. Finally, a conspicuous seasonal effect was observed for purified and non-purified glutathione-S-transferase (GST), where minimum values were registered during fall, pointing to this season as one where fish species are less competent to perform detoxifying reactions.

Differences in peripheral blood lymphocyte phenotypes between Helicobacter pylori-positive children and adults with duodenal ulcer.

The immunological mechanisms involved in the development of duodenal ulcer, especially in childhood, are unclear. Helicobacter pylori-positive children and adults, with and without duodenal ulcer, were therefore compared with respect to CD4(+) T-cells, and CD8(+) T-cells, B-cells and B1a-cells, as well as cell activation (CD4(+)/HLA-DR(+) and CD8(+)/HLA-DR(+)) and co-stimulatory (CD4(+)/CD28(+) and CD8(+)/CD28(+)) markers, in peripheral blood. Children with and without duodenal ulcer differed significantly. In particular, there was a phenotypic change in CD8(+) T-cells from children with ulcer that involved a 200% increase in the number of CD8(+)/HLA-DR(+) cells/mm(3) and a decrease of 34.2% in the number of CD8(+)/CD28(+) cells/mm(3). This phenotype of chronically activated memory CD8(+) T-cells, which has also been observed in patients with AIDS and tuberculosis, is associated with disease severity and progression. A lower frequency of B1a-cells was also observed in the group of children with ulcer. Conversely, no difference between infected adults with and without ulcer was observed, but the percentage of CD4(+)/HLA-DR(+) cells was lower in adults with ulcer, suggesting that a down-regulated immune response may play a role in the development of duodenal ulcer in adults. Gastric inflammation correlated positively with CD4(+) and chronically activated CD4(+) T-cells in children and adults without duodenal ulcer, respectively. These results suggest that there are differences in the immunophenotyping profile between H. pylori-positive children and adults with duodenal ulcer, indicating the possibility of distinct immune mechanisms in the development of the disease according to age.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection.

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma(-/-) C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4(-/-) than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4(-/-) than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 +/- 3.07 pg/mL, mean +/- SD) and -negative (11.07 +/- 3.5 pg/mL) WT BALB/c mice, in the group of IL-4(-/-) animals, the serum concentration of IFN-gamma was significantly higher in the infected ones (38.16 +/- 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma-/- C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4-/- than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4-/- than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 ± 3.07 pg/mL, mean ± SD) and -negative (11.07 ± 3.5 pg/mL) WT BALB/c mice, in the group of IL-4-/- animals, the serum concentration of IFN-g was significantly higher in the infected ones (38.16 ± 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

Quantification of fecal estradiol and progesterone metabolites in Syrian hamsters (Mesocricetus auratus).

Alternative methods to the utilization of laboratory animal blood and its by-products are particularly attractive, especially regarding hamsters due to their small size and difficulties in obtaining serial blood samples. Steroid hormone metabolite quantification in feces, widely used in studies of free-ranging or intractable animals, is a non-invasive, non-stressor, economical, and animal saving technique which allows longitudinal studies by permitting frequent sampling of the same individual. The present study was undertaken to determine the suitability of this method for laboratory animals. Estradiol and progesterone metabolites were quantified by radioimmunoassay in feces of intact, sexually mature female Syrian hamsters during the estrous cycle (control) and in feces of superovulated females. Metabolites were extracted by fecal dilution in ethanol and quantified by solid phase radioimmunoassay. Median estrogen and progesterone concentrations were 9.703 and 180.74 ng/g feces in the control group, respectively. Peaks of estrogen (22.44 +/- 4.54 ng/g feces) and progesterone (655.95 +/- 129.93 ng/g feces) mean fecal concentrations respectively occurred 12 h before and immediately after ovulation, which is easily detected in this species by observation of a characteristic vaginal postovulatory discharge. Median estrogen and progesterone concentrations (28.159 and 586.57 ng/g feces, respectively) were significantly higher in superovulated animal feces (P < 0.0001). The present study demonstrated that it is possible to monitor ovarian activity in Syrian hamsters non-invasively by measuring fecal estradiol and progesterone metabolites. This technique appears to be a quite encouraging method for the development of new endocrinologic studies on laboratory animals.