Peptide fingerprinting of the neurotoxic fractions isolated from the secretions of sea anemones Stichodactyla helianthus and Bunodosoma granulifera. New members of the APETx-like family identified by a 454 pyrosequencing approach

Sea anemones are known to contain a wide diversity of biologically active peptides, mostly unexplored according to recent peptidomic and transcriptomic studies. In the present work, the neurotoxic fractions from the exudates of Stichodactyla helianthus and Bunodosoma granulifera were analyzed by reversed-phase chromatography and mass spectrometry. The first peptide fingerprints of these sea anemones were assessed, revealing the largest number of peptide components (156) so far found in sea anemone species, as well as the richer peptide diversity of B. granulifera in relation to S. helianthus. The transcriptomic analysis of B. granulifera, performed by massive cDNA sequencing with 454 pyrosequencing approach allowed the discovery of five new APETx-like peptides (U-AITX-Bg1a-e - including the full sequences of their precursors for four of them), which together with type 1 sea anemone sodium channel toxins constitute a very distinguishable feature of studied sea anemone species belonging to genus Bunodosoma. The molecular modeling of these new APETx-like peptides showed a distribution of positively charged and aromatic residues in putative contact surfaces as observed in other animal toxins. On the other hand, they also showed variable electrostatic potentials, thus suggesting a docking onto their targeted channels in different spatial orientations. Moreover several crab paralyzing toxins (other than U-AITX-Bg1a-e), which induce a variety of symptoms in crabs, were isolated. Some of them presumably belong to new classes of crab-paralyzing peptide toxins, especially those with molecular masses below 2 kDa, which represent the smallest peptide toxins found in sea anemones. (C) 2011 Elsevier Inc. All rights reserved.

Characterization of a Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) - derived chitinase and its potential for pest control

The larval endoparasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) has a toolbox of biological weapons to secure for host colonization and the successful parasitization of its host Heliothis virescens (F.) (Lepidoptera: Noctuidae). The cDNA of a putative chitinase has been previously isolated and initially characterized from teratocytes of this parasitoid among the plethora of molecules available in the venom and calyx fluids injected by females, oral and/or anal secretions released by the parasitoid larvae and/or produced by the expression of genes of the symbiotic associated polydnavirus. This putative chitinase has been initially associated with the host cuticle digestion to allow for parasitoid egression and with the asepsis of the host environment, acting as an antimicrobial. As chitinases are commonly expressed in plants against plant pathogens, the chitinase derived from the teratocytes of T. nigriceps is a potential tool for the development of insect pest control methods based on the disruption of the perithrophic membrane of herbivores. Therefore, we aimed to characterize the activity of the putative chitinase from teratocytes of T. nigriceps (Tnchi) produced using the Escherichia coli expression system and its potential to control H. virescens larvae when expressed into transgenic tobacco plants. The purified E. coli-produced Tnchi protein showed no chitinolitic activity, but was active in binding with colloidal and crystalline chitins in water and with colloidal chitin in buffered solution (pH = 6.74). Transgenic tobacco plants showed no enhanced chitinolitic activity relative to control plants, but survival of three-day old larvae of H. virescens was severely affected when directly fed on transgenic tobacco leaves expressing the recombinant Tnchi protein. Some properties of the Tnchi protein and the potential use of Tnchi-transgenic plants to control plant pests are discussed. (c) 2012 Elsevier Inc. All rights reserved.

Up-regulation of chemokine C-C ligand 2 (CCL2) and C-X-C chemokine 8 (CXCL8) expression by monocytes in chronic idiopathic urticaria

The disturbed cytokinechemokine network could play an important role in the onset of diseases with inflammatory processes such as chronic idiopathic urticaria (CIU). Our main objectives were to evaluate the relation between proinflammatory chemokine serum levels from CIU patients and their response to autologous skin test (ASST) and basophil histamine release (BHR). We also aimed to assess the chemokine secretion by peripheral blood mononuclear cells (PBMC) upon polyclonal stimulus and to evaluate chemokine CC ligand 2/C-X-C chemokine 8 (CCL2/CXCL8) and Toll-like receptor-4 (TLR-4) expression in monocytes. We observed significantly higher serum levels of the CXCL8, CXCL9, CXCL10 and CCL2 in CIU patients compared to the healthy group, regardless of the BHR or ASST response. The basal secretion of CCL2 by PBMC or induced by Staphylococcus aureus enterotoxin A (SEA) was higher in CIU patients than in the control group, as well as for CXCL8 and CCL5 secretions upon phytohaemagglutinin stimulation. Also, up-regulation of CCL2 and CXCL8 mRNA expression was found in monocytes of patients upon SEA stimulation. The findings showed a high responsiveness of monocytes through CCL2/CXCL8 expression, contributing to the creation of a proinflammatory environment in CIU.

CPAP Has No Effect on Clearance, Sputum Properties, or Expectorated Volume in Cystic Fibrosis

BACKGROUND: Positive expiratory pressure (PEP) is used for airway clearance in cystic fibrosis (CF) patients. Hypertonic saline (HTS) aerosol increases sputum expectoration volume and may improve respiratory secretion properties. CPAP may also be used to maintain airway patency and mobilize secretions. To evaluate if CPAP would increase the beneficial clearance effect of HTS in subjects with CF, we investigated the effects of CPAP alone and CPAP followed by HTS on sputum physical properties and expectoration volume in CF subjects. METHODS: In this crossover study, 15 CF subjects (mean age 19 y old) were randomized to interventions, 48 hours apart: directed coughs (control), CPAP at 10 cm H2O, HTS 7%, and both CPAP and HTS (CPAP+HTS). Sputum collection was performed at baseline and after interventions. Expectorated volume was determined and in vitro sputum properties were analyzed for contact angle and cough clearability. RESULTS: There were no significant differences between any treatment in arterial blood pressure, heart rate, or pulse oximetry, between the 2 time points. HTS and CPAP+HTS improved cough clearability by 50% (P = .001) and expectorated volume secretion by 530% (P = .001). However, there were no differences between control and CPAP on sputum contact angle, cough clearability, or volume of expectorated secretion. CONCLUSIONS: CPAP alone had no effect on mucus clearance, sputum properties, or expectorated volume, and did not potentiate the effect of HTS alone in CF subjects.

High Rates of Detection of Respiratory Viruses in Tonsillar Tissues from Children with Chronic Adenotonsillar Disease

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.

Correlation between mandibular gland secretion and cuticular hydrocarbons in the stingless bee Melipona quadrifasciata

We investigated whether Melipona quadrifasciata worker mandibular gland secretions contribute directly to their cuticular hydrocarbon profile. The mandibular gland secretion composition and cuticular surface compounds of newly emerged worker bees, nurse bees, and foragers were determined by gas chromatography and mass spectrometry and compared. Both the mandibular gland secretions and the cuticular surface compounds of all worker stages were found to be composed almost exclusively of hydrocarbons. Although the relative proportion of hydrocarbons from the cuticular surface and gland secretion was statistically different, there was a high similarity in the qualitative composition between these structures in all groups of bees.

Prey digestion in the midgut of the predatory bug Podisus nigrispinus (Hemiptera: Pentatomidae)

Pre-oral digestion is described as the liquefaction of the solid tissues of the prey by secretions of the predator. It is uncertain if pre-oral digestion means pre-oral dispersion of food or true digestion in the sense of the stepwise bond breaking of food polymers to release monomers to be absorbed. Collagenase is the only salivary proteinase, which activity is significant (10%) in relation to Podisus nigrispinus midgut activities. This suggests that pre-oral digestion in P. nigrispinus consists in prey tissue dispersion. This was confirmed by the finding of prey muscles fibers inside P. nigrispinus midguts. Soluble midgut hydrolases from P. nigrispinus were partially purified by ion-exchange chromatography, followed by gel filtration. Two cathepsin L-like proteinases (CAL1 and CAL2) were isolated with the properties: CAL1 (14.7 kDa, pH optimum (pHo) 5.5, km with carbobenzoxy-Phe-Arg-methylcoumarin, Z-FR-MCA, 32 mu M); CAL2 (17 kDa, pHo 5.5, km 11 mu M Z-FR-MCA). Only a single molecular species was found for the other enzymes with the following properties are: amylase (43 kDa, pHo 5.5, km 0.1% starch), aminopeptidase (125 kDa, pHo 5.5, km 0.11 mM L-Leucine-p-nitroanilide), alpha-glucosidase (90 kDa, pHo 5.0, km 5 mM with p-nitrophenyl alpha-D-glucoside). CAL molecular masses are probably underestimated due to interaction with the column. Taking into account the distribution of hydrolases along P. nigrispinus midguts, carbohydrate digestion takes place mainly at the anterior midgut, whereas protein digestion occurs mostly in middle and posterior midgut, as previously described in seed- sucker and blood-feeder hemipterans. (C) 2012 Elsevier Ltd. All rights reserved.

Maternal Melatonin Programs the Daily Pattern of Energy Metabolism in Adult Offspring

Background: Shift work was recently described as a factor that increases the risk of Type 2 diabetes mellitus. In addition, rats born to mothers subjected to a phase shift throughout pregnancy are glucose intolerant. However, the mechanism by which a phase shift transmits metabolic information to the offspring has not been determined. Among several endocrine secretions, phase shifts in the light/dark cycle were described as altering the circadian profile of melatonin production by the pineal gland. The present study addresses the importance of maternal melatonin for the metabolic programming of the offspring. Methodology/Principal Findings: Female Wistar rats were submitted to SHAM surgery or pinealectomy (PINX). The PINX rats were divided into two groups and received either melatonin (PM) or vehicle. The SHAM, the PINX vehicle and the PM females were housed with male Wistar rats. Rats were allowed to mate and after weaning, the male and female offspring were subjected to a glucose tolerance test (GTT), a pyruvate tolerance test (PTT) and an insulin tolerance test (ITT). Pancreatic islets were isolated for insulin secretion, and insulin signaling was assessed in the liver and in the skeletal muscle by western blots. We found that male and female rats born to PINX mothers display glucose intolerance at the end of the light phase of the light/dark cycle, but not at the beginning. We further demonstrate that impaired glucose-stimulated insulin secretion and hepatic insulin resistance are mechanisms that may contribute to glucose intolerance in the offspring of PINX mothers. The metabolic programming described here occurs due to an absence of maternal melatonin because the offspring born to PINX mothers treated with melatonin were not glucose intolerant. Conclusions/Significance: The present results support the novel concept that maternal melatonin is responsible for the programming of the daily pattern of energy metabolism in their offspring.

Effects of neonatal castration and androgenization on sexual dimorphism in bone, leptin and corticosterone secretion

This study investigated the role of neonatal sex steroids in rats on sexual dimorphism in bone, as well as on leptin and corticosterone concentrations throughout the lifespan. Castration of males and androgenization of females were used as models to investigate the role of sex steroids shortly after birth. Newborn Wistar rats were divided into four groups, two male groups and two female groups. Male pups were cryoanesthetized and submitted to castration or sham-operation procedures within 24 h after birth. Female pups received a subcutaneous dose of testosterone propionate (100 mu g) or vehicle. Rats were euthanized at 20, 40, or 120 postnatal days. Body weight was also measured at 20, 40, and 120 days of age, and blood samples and femurs were collected. The length and thickness of the femurs were measured and the areal bone mineral density (areal BMD) was determined by dual-energy X-ray absorptiometry (DEXA). Biomechanical three-point bending testing was used to evaluate bone breaking strength, energy to fracture, and extrinsic stiffness. Blood samples were submitted to a biochemical assay to estimate calcium, phosphorus, alkaline phosphatase, leptin, and corticosterone levels. Weight gain, areal BMD and bone biomechanical properties increased rapidly with respect to age in all groups. In control animals, skeletal sexual dimorphism, leptin concentration, and dimorphic corticosterone concentration patterns were evident after puberty. However, androgen treatment induced changes in growth, areal BMD, and bone mass properties in neonatal animals. In addition, neonatally-castrated males had bone development and mechanical properties similar to those of control females. These results suggest that the exposure to neonatal androgens may represent at least one covariate that mediates dimorphic variation in leptin and corticosterone secretions. The study indicates that manipulation of the androgen environment during the critical period of sexual differentiation of the brain causes long-lasting changes in bone development, as well as serum leptin and corticosterone concentrations. In addition, this study provides useful models for the investigation of bone disorders induced by hypothalamic hypogonadism. (C) 2011 Elsevier Inc. All rights reserved.

ANTIFUNGAL ACTIVITY OF LECTINS AGAINST YEAST OF VAGINAL SECRETION

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256 mu g/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

Correlates of human immunodeficiency virus cervicovaginal shedding among postmenopausal and fertile-aged women

Objective: The aims of this study were to compare the intensity of human immunodeficiency virus (HIV)-RNA genital shedding among postmenopausal (PM) and fertile-aged (F) women and to investigate the association between viral shedding and gynecological features, HIV plasma viral loads, and other markers of HIV disease progression. Methods: We interviewed 146 HIV-infected women (73 PM/73 F) in search of gynecological complaints and signs and symptoms of HIV disease and obtained additional information concerning HIV infection by medical chart review. Cervicovaginal lavages (CVLs) were collected for assessment of HIV shedding. Laboratory analyses included CD4(+) cell counts, HIV-RNA quantitation in plasma and CVL, and screening for concurrent genital infections. Results: HIV-RNA genital shedding was detected in 16.4% of PM and 21.9% of F women (P = 0.400), and the intensity of HIV shedding did not differ between both groups (means-PM: 1.4log/mL; F: 1.4log/mL; P = 0.587). Three women (2 PM/1 F) exhibited viral shedding in the absence of detectable viremia. HIV plasma viral loads correlated with HIV shedding in both groups. In multivariable analysis, HIV plasma viral loads were independently associated with HIV shedding in both groups. Moreover, the intensity of shedding was independently associated with vaginal pH, tumor necrosis factor a concentrations in CVL, and HIV plasma viral loads. Conclusions: Despite significant changes that occur in the vaginal mucosa of PM women, HIV cervicovaginal shedding was not significantly influenced by this state in our cohort. In contrast, increased vaginal pH and genital inflammation, evidenced by increased tumor necrosis factor alpha concentrations in CVL and HIV plasma viral loads, were independently associated with the intensity of HIV shedding in PM and F women.

Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils

Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 mu M; 2 h) and subsequently incubated with LPS (5 mu g/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or beta(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor kappa B (NF-kappa B) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure.

Oral Transmission of Chagas Disease

Chagas disease is now an active disease in the urban centers of countries of nonendemicity and endemicity because of congenital and blood and/or organ transplantation transmissions and the reactivation of the chronic disease in smaller scale than vectorial transmission, reported as controlled in countries of endemicity. Oral transmission of Chagas disease has emerged in unpredictable situations in the Amazon region and, more rarely, in areas of nonendemicity where the domiciliary triatomine cycle was under control because of exposition of the food to infected triatomine and contaminated secretions of reservoir hosts. Oral transmission of Chagas disease is considered when >1 acute case of febrile disease without other causes is linked to a suspected food and should be confirmed by the presence of the parasite after direct microscopic examination of the blood or other biological fluid sample from the patient.

Behavioral analysis of the interaction between the spitting spider Scytodes globula (Araneae: Scytodidae) and the harvestman Discocyrtus invalidus (Opiliones: Gonyleptidae)

Spitting spiders (Scytodidae) have a distinct predatory strategy in which they eject a sticky secretion from their cheliceral fangs to immobilize prey. This behavior could potentially allow the spider not only to avoid defensive secretions but also to bite specific vulnerable spots of a potential prey such as a harvestman. We used an ethogram, a fluxogram and an experiment to analyze the interaction between the harvestman Discocyrtus invalidus Piza 1938 (Arachnida: Opiliones) and the syntopic spider Scytodes globula (Nicolet 1849) (Arachnida: Araneae). These spiders, while readily taking crickets as prey, seldom spat at and never bit the harvestmen, which apparently did not exude repugnatorial secretions. We therefore tested, by clogging the glands and using appropriate controls, whether non-visible amounts of secretions could cause the rejection, but the harvestmen were still refused. This is the first detailed and quantified description of an interaction between a spitting spider and a harvestman. The general conclusions are that S. globula avoids preying on D. invalidus, S. globula behaves differently when attacking harvestmen and crickets and the scent gland secretions of D. invalidus do not play a direct role in this predator-prey interaction.

PEEP-ZEEP technique: cardiorespiratory repercussions in mechanically ventilated patients submitted to a coronary artery bypass graft surgery

Abstract Background The PEEP-ZEEP technique is previously described as a lung inflation through a positive pressure enhancement at the end of expiration (PEEP), followed by rapid lung deflation with an abrupt reduction in the PEEP to 0 cmH2O (ZEEP), associated to a manual bilateral thoracic compression. Aim To analyze PEEP-ZEEP technique's repercussions on the cardio-respiratory system in immediate postoperative artery graft bypass patients. Methods 15 patients submitted to a coronary artery bypass graft surgery (CABG) were enrolled prospectively, before, 10 minutes and 30 minutes after the technique. Patients were curarized, intubated, and mechanically ventilated. To perform PEEP-ZEEP technique, saline solution was instilled into their orotracheal tube than the patient was reconnected to the ventilator. Afterwards, the PEEP was increased to 15 cmH2O throughout 5 ventilatory cycles and than the PEEP was rapidly reduced to 0 cmH2O along with manual bilateral thoracic compression. At the end of the procedure, tracheal suction was accomplished. Results The inspiratory peak and plateau pressures increased during the procedure (p < 0.001) compared with other pressures during the assessment periods; however, they were within lung safe limits. The expiratory flow before the procedure were 33 ± 7.87 L/min, increasing significantly during the procedure to 60 ± 6.54 L/min (p < 0.001), diminishing to 35 ± 8.17 L/min at 10 minutes and to 36 ± 8.48 L/min at 30 minutes. Hemodynamic and oxygenation variables were not altered. Conclusion The PEEP-ZEEP technique seems to be safe, without alterations on hemodynamic variables, produces elevated expiratory flow and seems to be an alternative technique for the removal of bronchial secretions in patients submitted to a CABG.