Hematological analysis of Micropogonias Furnieri, Desmarest, 1823, Scianidae, from two estuaries of Baixada Santista, São paulo Brazil

Hematological alterations in fish are considered a useful tool to evaluate pathological processes resulting from the exposure to environmental pollutants. The whitemouth croaker Micropogonias furnieri is a common species in estuarine areas and potentially exposed to many contaminants. In the present study, the hematological characteristics of fish collected at two sites in Baixada Santista (Santos Estuarine System - SES, a polluted site; and the Estuary of Itanhaém River - EIR, unpolluted site) del was analysed. The following blood descriptors were analyzed: number of Erythrocytes (Er), Hematocrit (Ht), Hemoglobin (Hb), Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin concentration (MCHC). Fish from SES exhibited significant lower levels of Ht and increase on MCHC and Hb. Such differences are likely related to the different contamination levels found in these estuaries.

Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae)

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Cardiac arrhythmia emergency room visits and environmental air pollution in Sao Paulo, Brazil

Objectives: Air-pollution exposure has been associated with increased cardiovascular hospital admissions and mortality in time-series studies. We evaluated the relation between air pollutants and emergency room (ER) visits because of cardiac arrhythmia in a cardiology hospital. Methods: In a time-series study, we evaluated the association between the emergency room visits as a result of cardiac arrhythmia and daily variations in SO2, CO, NO2, O-3 and PM10, from January 1998 to August 1999. The cases of arrhythmia were modelled using generalised linear Poisson regression models, controlling for seasonality (short-term and long-term trend), and weather. Results: Interquartile range increases in CO (1.5 ppm), NO2 (49,5 mu g/m(3)) and PM10 (22.2 mu g/m(3)) on the concurrent day were associated with increases of 12.3% (95% CI: 7.6% to 17.2%), 10.4% (95% CI: 5.2% to 15.9%) and 6.7% (95% CI: 1.2% to 12.4%) in arrhythmia ER visits, respectively. PM10, CO and NO2 effects were dose-dependent and gaseous pollutants had thresholds. Only CO effect resisted estimates in models with more than one pollutant. Conclusions: Our results showed that air pollutant effects on arrhythmia are predominantly acute starting at concentrations below air quality standards, and the association with CO and NO2 suggests a relevant role for pollution caused by cars.

Cavity Preparation and Influence of Restorative Materials on the Prevention of Secondary Caries

Objective: This in vitro study evaluated the influence of cavity preparation using the Er:YAG laser and restorative materials containing fluoride on preventing caries lesions. Background: It has been suggested that cavity preparation using the Er:YAG laser has a potential for improving resistance to secondary caries on enamel. Methods: Forty unerupted human third molars teeth were sectioned into 72 blocks of dental enamel and distributed into two groups to prepare cavities measuring (1.6 mm diameter) with diamond burs (DB) or Er:YAG laser (LA; 6 Hz, 300 mJ, 47 J/cm(2)). After that, each group was divided into three subgroups and restored with a glass-ionomer cement (GI), a resin-modified glass-ionomer (RM), or a composite resin (CR). Blocks were thermal cycled and submitted to a pH challenge to develop artificial caries-like lesions. Lesions were evaluated by Knoop microhardness test. An average of four indentations was used. Statistical analyses were performed by ANOVA followed by Tukey's test. Results: The results (in Knoop hardness number) for DB cavity preparation were GI, 235.5 (+/- 75.5); RM, 137.1 (+/- 64.1); and CR, 39.3 (+/- 26.5). For LA cavity preparation, the results were GI, 410.0 (+/- 129.7); RM, 310.3 (+/- 119.5); and CR, 96.4 (+/- 57.4). Conclusions: There was less development of caries lesion around LA-prepared cavities than around the DB-prepared cavities; however, no synergistic cariostatic effect was observed between the Er:YAG laser and glass ionomer cement.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Background: Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods: Twenty-four adult Wistar rats, 60 days old (+/-250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [ 100 mu g/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a. m. Results: Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions: We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

Adhesion of Endodontic Sealers to Human Root Dentine Submitted to Different Surface Treatments

Objective: To evaluate the adhesion of the endodontic sealers Epiphany, Apexit Plus, and AH Plus to root canal dentin submitted to different surface treatments, by using the push-out test. Methods: One hundred twenty-eight root cylinders obtained from maxillary canines were embedded in acrylic resin, had the canals prepared, and were randomly assigned to four groups (n = 32), according to root dentin treatment: (I) distilled water (control), (II) 17% EDTAC, (III) 1% NaOCl and (IV) Er:YAG laser with 16-Hz, 400-mJ input (240-mJ output) and 0.32-J/cm(2) energy density. Each group was divided into four subgroups (n = 8) filled with Epiphany (either dispensed from the automix syringe supplied by the manufacturer or prepared by hand mixing), Apexit Plus, or AH Plus. Data (MPa) were analyzed by ANOVA and Tukey's test. Results: A statistically significant difference (p < 0.01) was found among the root-canal sealers, except for the Epiphany subgroups, which had statistically similar results to each other (p > 0.01): AH Plus (4.77 +/- 0.85), Epiphany/hand mixed (3.06 +/- 1.34), Epiphany/automix syringe (2.68 +/- 1.35), and Apexit Plus (1.22 +/- 0.33). A significant difference (p < 0.01) was found among the dentin surface treatments. The highest adhesion values were obtained with AH Plus when root dentin was treated with Er: YAG laser and 17% EDTAC. Epiphany sealer presented the lowest adhesion values to root dentin treated with 17% EDTAC. Conclusions: The resin-based sealers had different adhesive behaviors, depending on the treatment of root canal walls. The mode of preparation of Epiphany (automix syringe or hand mixing) did not influence sealer adhesion to root dentin.

Ultrastructural analysis of root canal dentine irradiated with 980-nm diode laser energy at different parameters

Objective: The purpose of this in vitro study was to investigate using the scanning electron microscope (SEM) the ultrastructural morphological changes of the radicular dentine surface after irradiation with 980-nm diode laser energy at different parameters and angles of incidence. Background Data: There have been limited reports on the effects of diode laser irradiation at 980 nm on radicular dentin morphology. Materials and Methods: Seventy-two maxillary canines were sectioned and roots were biomechanically prepared using K3 rotary instruments. The teeth were irrigated with 2 mL of distilled water between files and final irrigation was performed with 10 mL of distilled water. The teeth were then randomly divided into five groups (n = 8 each) according to their diode laser parameters: Group 1: no irradiation (control); group 2: 1.5 W/continuous wave (CW) emission (the manufacturer's parameters); group 3: 1.5 W/100 Hz; group 4: 3 W/CW; and group 5: 3 W/100 Hz. Laser energy was applied with helicoid movements (parallel to the canal walls) for 20 sec. Eight additional teeth for each group were endodontically prepared and split longitudinally and irradiation was applied perpendicularly to the root surface. Results: Statistical analysis showed no difference between the root canal thirds irradiated with the 980-nm diode laser, and similar results between the parameters 1.5 W/CW and 3 W/100 Hz (p > 0.05). Conclusion: When considering different output powers and delivery modes our results showed that changes varied from smear layer removal to dentine fusion.

Effectiveness of 980-mm Diode and 1064-nm Extra-Long-Pulse Neodymium-Doped Yttrium Aluminum Garnet Lasers in Implant Disinfection

Objective: To evaluate the potential of 980-nm gallium aluminum arsenide (GaAlAs) and 1064-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) lasers to reduce bacteria after irradiation of implant surfaces contaminated with Enterococcus faecalis and Porphyromonas gingivalis and on irradiated implant surface morphology. Background: Despite the frequency of implant success, some implant loss is related to peri-implantitis because of difficulty in eliminating the biofilm. Methods: Implants (3.75 x 13 mm) with machined surfaces, surfaces sand blasted with titanium oxide (TiO(2)), and sand-blasted and acid-etched surfaces were exposed to P. gingivalis and E. faecalis cultures and irradiated with 980-nm GaAlAs or 1064-nm Nd: YAG lasers. After laser treatments, the number of remaining colony-forming units and implant surface morphology were analyzed using scanning electron microscopy (SEM). Results: The Nd: YAG laser was able to promote a total contamination reduction on all implants irradiated. The results with the GaAlAs laser showed 100% bacteria reduction on the implants irradiated with 3 W. Irradiation with 2.5 W and 3 W achieved 100% of bacteria reduction on P. gingivalis-contaminated implants. Decontamination was not complete for the sand-blasted TiO(2) (78.6%) and acid-etched surfaces (49.4%) contaminated with E. faecalis and irradiated with 2.5 W. SEM showed no implant surface changes. Conclusion: The wavelengths used in this research provided bacteria reduction without damaging implant surfaces. New clinical research should be encouraged for the use of this technology in the treatment of peri-implantitis.

Gene Expression Complex Networks: Synthesis, Identification, and Analysis

Thanks to recent advances in molecular biology, allied to an ever increasing amount of experimental data, the functional state of thousands of genes can now be extracted simultaneously by using methods such as cDNA microarrays and RNA-Seq. Particularly important related investigations are the modeling and identification of gene regulatory networks from expression data sets. Such a knowledge is fundamental for many applications, such as disease treatment, therapeutic intervention strategies and drugs design, as well as for planning high-throughput new experiments. Methods have been developed for gene networks modeling and identification from expression profiles. However, an important open problem regards how to validate such approaches and its results. This work presents an objective approach for validation of gene network modeling and identification which comprises the following three main aspects: (1) Artificial Gene Networks (AGNs) model generation through theoretical models of complex networks, which is used to simulate temporal expression data; (2) a computational method for gene network identification from the simulated data, which is founded on a feature selection approach where a target gene is fixed and the expression profile is observed for all other genes in order to identify a relevant subset of predictors; and (3) validation of the identified AGN-based network through comparison with the original network. The proposed framework allows several types of AGNs to be generated and used in order to simulate temporal expression data. The results of the network identification method can then be compared to the original network in order to estimate its properties and accuracy. Some of the most important theoretical models of complex networks have been assessed: the uniformly-random Erdos-Renyi (ER), the small-world Watts-Strogatz (WS), the scale-free Barabasi-Albert (BA), and geographical networks (GG). The experimental results indicate that the inference method was sensitive to average degree k variation, decreasing its network recovery rate with the increase of k. The signal size was important for the inference method to get better accuracy in the network identification rate, presenting very good results with small expression profiles. However, the adopted inference method was not sensible to recognize distinct structures of interaction among genes, presenting a similar behavior when applied to different network topologies. In summary, the proposed framework, though simple, was adequate for the validation of the inferred networks by identifying some properties of the evaluated method, which can be extended to other inference methods.

Endurance exercise training ameliorates insulin resistance and reticulum stress in adipose and hepatic tissue in obese rats

Obesity-induced endoplasmatic reticulum (ER) stress has been demonstrated to underlie the induction of obesity-induced JNK and NF-kappa B activation inflammatory responses, and generation of peripheral insulin resistance. On the other hand, exercise has been used as a crucial tool in obese and diabetic patients, and may reduce inflammatory pathway stimulation. However, the ability of exercise training to reverse endoplasmatic reticulum stress in adipose and hepatic tissue in obesity has not been investigated in the literature. Here, we demonstrate that exercise training ameliorates ER stress and insulin resistance in DIO-induced rats. Rats were fed with standard rodent chow (3,948 kcal kg(-1)) or high-fat diet (5,358 kcal kg(-1)) for 2 months. After that rats were submitted to swimming training (1 h per day, 5 days for week with 5% overload of the body weight for 8 weeks). Samples from epididymal fat and liver were obtained and western blot analysis was performed. Our results showed that swimming protocol reduces pro-inflammatory molecules (JNK, I kappa B and NF-kappa B) in adipose and hepatic tissues. In addition, exercise leads to reduction in ER stress, by reducing PERK and eIF2 alpha phosphorylation in these tissues. In parallel, an increase in insulin pathway signaling was observed, as confirmed by increases in IR, IRSs and Akt phosphorylation following exercise training in DIO rats. Thus, results suggest that exercise can reduce ER stress, improving insulin resistance in adipose and hepatic tissue.

Carbon and Nitrogen Dynamics in an Oxisol as Affected by Liming and Crop Residues under No-Till

The short-term effects of surface lime application and black oat (Avena strigosa Schreb.) residues, with or without N fertilization, were evaluated in a long-term no-till (NT) system on a sandy clay loam, a kaolinitic, thermic Typic Hapludox from the state of Parana, Brazil. The main plot treatments were: control and dolomitic lime applied on soil surface at 8 Mg ha(-1). Three treatments with crop residues were evaluated on the subplots: (i) fallow, (ii) black oat residues, and (iii) black oat residues aft er N fertilization at 180 kg ha(-1). Black oat dry biomass was not affected by the treatments during 3 yr. Surface liming increased soil pH, microbial biomass, microbial activity, and bacterial/fungal ratio at the soil surface (0-5 cm), resulting in increased amino acid turnover, water-soluble humic substances formation, and N mineralization and nitrification. While the application of black oat did increase the soil pH, overall it had much less effect on soil biological processes and C and N pools than did lime. We concluded that black oat cannot replace the need for lime to optimize crop production in these tropical NT systems. In the long term, however, black oat should aid in the amelioration of acidity and replenishment of soil organic C pools and should help reduce erosion. Overall, this study suggests that overapplication of inorganic fertilizer N may occur in some tropical NT systems. Further experiments are required in NT systems to investigate the use of slow-release N fertilizers in combination with lime and black oat as a mechanism to reduce acidification and promote sustainability.

Production and characteristics comparison of crude beta-glucosidases produced by microorganisms Thermoascus aurantiacus e Aureobasidium pullulans in agricultural wastes

The purified beta-glucosidase of Aureobasidium pullulans ER-16 is one of more thermostable enzyme reported to date. Considering the unfeasibility of using purified enzyme for industrial application, it was interesting to analyze this property for the crude enzyme. Thermophilic fungus Thermoascus aurantiacus CBMAI-756 and mesophilic A. pullulans ER-16 were cultivated in different hemicellulosic materials on solid-state cultivation for beta-glucosidase production. Wheat bran was most appropriate for beta-glucosidase production by both microorganisms. T. aurantiacus exhibited maximum enzyme production (7.0 U/ml or 70 U/g) at 48-72 h and A. pullulans a maximum (1.3 U/ml or 13 U/g) at 120 h. Maximum activities were at 75 degrees C with optimum pH at 4.5 and 4.0, for T aurantiacus and A. pullulans, respectively. A. pullulans`s beta-glucosidase was more pH stable (4.5-10.0 against 4.5-8.0) and more thermostable (90% after 1 h at 75 degrees C against 85% after 1 h at 70 degrees C) than the enzyme from the thermophilic T. aurantiacus. The t((1/2)) at 80 degrees C were 50 and 12.5 min for A. pullulans and T. aurantiascus, respectively. These data confirm the high thermostability of crude beta-glucosidase from A. pullulans. Both beta-glucosidases were strongly inhibited by glucose, but ethanol significantly increased the activity of the enzyme from T. aurantiacus. (C) 2008 Elsevier Inc. All rights reserved.

Erbium-activated silica-zirconia planar waveguides prepared by sol-gel route

Er(3+) doped (100-x)SiO(2)-xZrO(2) planar waveguides were prepared by the sol-gel route, with x ranging from 10 up to 30 mol%. Multilayer films doped with 0.3 mol% Er(3+) ions were deposited on fused quartz substrates by the dip-coating technique. The thickness and refractive index were measured by m-line spectroscopy at different wavelengths. The fabrication protocol was optimized in order to confine one propagating mode at 1.5 mu m. Photoluminescence in the near and visible region indicated a crystalline local environment for the Er(3+) ion. (c) 2007 Elsevier B.V. All rights reserved.