Long-term sertraline treatment increases expression and decreases phosphorylation of glycogen synthase kinase-3B in platelets of patients with late-life major depression

Background: Abnormal regulation of glycogen synthase kinase 3-beta (GSK3B) activity has been implicated in the pathophysiology of mood disorders. Many pharmacological agents, including antidepressants, can modulate GSK3B. The aim of the present study was to investigate the effect of short-and long-term sertraline treatment on the expression and phosphorylation of GSK3B in platelets of patients with late-life major depression. Methods: Thirty-nine unmedicated elderly adults with major depressive disorder (MOD) were initially included in this study. The comparison group comprised 18 age-matched, healthy individuals. The expression of total and Ser-9 phosphorylated GSK3B (pGSK3B) was determined by Enzyme Immunometric Assay (EIA) in platelets of patients and controls at baseline, and after 3 and 12 months of sertraline treatments for patients only. During this period, patients were continuously treated with therapeutic doses of sertraline. GSK3B activity was indirectly estimated by calculating the proportion of inactive (phosphorylated) forms (pGSK3B) in relation to the total expression of the enzyme (i.e.. GSK3B ratio). Results: Depressed patients had significantly higher levels of pGSK3B as compared to controls (p < 0.001). Within the MDD group, after 3 months of sertraline treatment no significant changes were observed in GSK3B expression and phosphorylation state, as compared to baseline levels. However, after 12 months of treatment we found a significant increase in the expression of total GSK3B (p = 0.05), in the absence of any significant changes in pGSK3B (p = 0.12), leading to a significant reduction in GSK3B ratio (p = 0.001). Conclusions: Our findings indicate that GSK3B expression was upregulated by the continuous treatment with sertraline, along with an increment in the proportion of active forms of the enzyme. This is compatible with an increase in overall GSK3B activity, which may have been induced by the long-term treatment of late-life depression with sertraline. (C) 2012 Elsevier Ltd. All rights reserved.

Exploration of the Antiplatelet Activity Profile of Betulinic Acid on Human Platelets

Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including antiretroviral, antibacterial, antimalarial, and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated, and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-14, and arachidonic acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR-derived structure of betulinic acid and prostacyclin agonists (PGI2), and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that deserves further investigation.

A radioenzymatic assay to identify three groups of phospholipase A(2) in platelets

Phospholipases A(2) (PLA(2)) are key enzymes in membrane metabolism. The release of fatty acids and lysophospholipids by PLA(2) activates several intra-cellular second messenger cascades that regulate a wide variety of physiological responses. The aim of the present study is to describe a radioenzymatic assay to determine the activity of three main PLA(2) subtypes in platelets, namely extracellular calcium-dependent PLA(2) (sPLA(2)) and intracellular calcium-dependent (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)). The differentiation of these distinct PLA(2) subtypes was based on the enzyme substrate preference (arachdonic acid or palmitoyl acid) and calcium concentration. Our results indicate that this new assay is feasible, precise and specific to measure the activity of the aforementioned subtypes of PLA(2). Therefore, this protocol can be used to investigate modifications of PLA(2) homeostasis in distinct biological models addressing the pathophysiology of many medical and neuropsychiatric disorders such as schizophrenia and Alzheimer's disease. (C) 2012 Elsevier Ltd. All rights reserved.

Redescription of Parotocinclus bahiensis (Miranda-Ribeiro, 1918) (Pisces, Ostariophysi, Loricariidae)

Microlepidogaster bahiensis Miranda-Ribeiro (1918), assigned to Parotocinclus Eigenmann & Eigenmann, 1889, by Garavello (1977), was described in a short and uninformative way which does not allow for its distinction from other species of Parotocinclus; besides, the two syntypes of the species are regarded as lost. For these reasons a neotype is herein designated and a detailed description of the species is presented based on topotypes. The species is characterized by the combination of the following features: scapular bridge almost completely exposed ventrally, arrector fossae, when present, small or very reduced; abdomen covered by 5-7 wide lateral plates on each side and very small platelets in between, leaving abundant naked areas surrounding them; a small group of larger plates in front of anus; caudal peduncle ellipsoid in cross section; total plates in median series 21-23; longitudinal series with 23-26 plates; 18-29 teeth on premaxillary and 12-24 on dentary; it can also be distinguished by its characteristic caudal-fin color pattern. Features that allow us to assign the species to Parotocinclus and a hypothesis about its relationships with other species of the genus are also presented.

Preparation and characterization of poly(vinyl butyral)-polyaniline-montmorillonite nanocomposites

Poly(vinyl butyral)-polyaniline-sodium montmorillonite nanocomposites were prepared via polymerization of aniline between clay mineral platelets at two different pH levels (2.0 and 5.0), followed by dispersion of the polyaniline-sodium montmorillonite nanocomposite in a poly(vinyl butyral) solution. A comparison was made of the effect of the pH levels and the polyaniline-sodium montmorillonite nanocomposite precursor on the final structures of the poly(vinyl butyral) nanocomposites and their electrical conductivities. X-ray diffraction patterns revealed the formation of nanocomposites at both pH levels. UV-Vis spectra indicated that the polyaniline formed at both pH levels was conductive, with the UV-Vis spectra presenting a band at 420 nm corresponding to the polaronic form and the beginning of a new band at 600 nm indicating the presence of polaronic segments. FTIR spectra revealed the peaks of the groups present in polyaniline and poly(vinyl butyral) nanocomposites. The electrical conductivities of the polyaniline and poly(vinyl butyral) nanocomposites prepared at pH 2.0 were lower than those of the same nanocomposites prepared at pH 5.0, probably due to the lower formation of polyaniline chains in a more acidic dispersion and to the final configuration of polyaniline in the nanocomposites.

Blood transfusion utilization and recipient survival at Hospital das Clinicas in Sao Paulo, Brazil

BACKGROUND: The characteristics of blood recipients including diagnoses associated with transfusion and posttransfusion survival are unreported in Brazil. The goals of this analysis were: 1) to describe blood utilization according to clinical diagnoses and patient characteristics and 2) to determine the factors associated with survival of blood recipients. STUDY DESIGN AND METHODS: A retrospective cross-sectional analysis was conducted on all inpatients in 2004. Data came from three sources: The first two files consist of data about patient characteristics, clinical diagnosis, and transfusion. Analyses comparing transfused and nontransfused patients were conducted. The third file was used to determine survival recipients up to 3 years after transfusion. Logistic regression was conducted among transfused patients to examine characteristics associated with survival. RESULTS: In 2004, a total of 30,779 patients were admitted, with 3835 (12.4%) transfused. These patients had 10,479 transfusions episodes, consisting of 39,561 transfused components: 16,748 (42%) red blood cells, 15,828 (40%) platelets (PLTs), and 6190 (16%) plasma. The median number of components transfused was three (range, 1-656) per patient admission. Mortality during hospitalization was different for patients whose admissions included transfusion or not (24% vs. 4%). After 1 year, 56% of transfusion recipients were alive. The multivariable model of factors associated with mortality after transfusion showed that the most significant factors in descending order were hospital ward, increasing age, increasing number of components transfused, and type of components received. CONCLUSION: Ward and transfusion are markers of underlying medical conditions and are associated with the probability of survival. PLT transfusions are common and likely reflect the types of patients treated. This comprehensive blood utilization study, the first of its kind in Brazil, can help in developing transfusion policy analyses in South America.

Reduced platelet amyloid precursor protein ratio (APP ratio) predicts conversion from mild cognitive impairment to Alzheimer's disease

Studies have shown that platelet APP ratio (representing the percentage of 120-130 kDa to 110 kDa isoforms of the amyloid precursor protein) is reduced in patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). In the present study, we sought to determine if baseline APP ratio predicts the conversion from MCI to AD dementia after 4 years of longitudinal assessment. Fifty-five older adults with varying degrees of cognitive impairment (34 with MCI and 21 with AD) were assessed at baseline and after 4 years. MCI patients were re-classified according to the conversion status upon follow-up: 25 individuals retained the diagnostic status of MCI and were considered as stable cases (MCI-MCI); conversely, in nine cases the diagnosis of dementia due to AD was ascertained. The APP ratio (APPr) was determined by the Western blot method in samples of platelets collected at baseline. We found a significant reduction of APPr in MCI patients who converted to dementia upon follow-up. These individuals had baseline APPr values similar to those of demented AD patients. The overall accuracy of APPr to identify subjects with MCI who will progress to AD was 0.74 +/- A 0.10, p = 0.05. The cut-off of 1.12 yielded a sensitivity of 75 % and a specificity of 75 %. Platelet APPr may be a surrogate marker of the disease process in AD, with potential implications for the assessment of abnormalities in the APP metabolism in patients with and at risk for dementia. However, diagnostic accuracy was relatively low. Therefore, studies in larger samples are needed to determine whether APPr may warrant its use as a biomarker to support the early diagnosis of AD.

Aerial organ anatomy of Smilax syphilitica (Smilacaceae)

Smilax L. in Brazil is represented by 32 taxa and it is a taxonomically difficult genus because the plants are dioecious and show wide phenotypic variation. The analysis and use of leaf anatomy characters is recognized as a frequently successful taxonomic method to distinguish between individual taxon, when floral material is absent or minute differences in flowers and foliage exist such as in Smilax. The aim of this study was to characterize the anatomical features of the aerial organs in Smilax syphilitica collected from the Atlantic Rainforest, in Santa Teresa-ES and the Smilax aff syphilitica from the Amazon Rainforest, in Manaus, Brazil. For this, a total of three samples of Smilax were collected per site. Sample leaves and stems were fixed with FAA 50, embedded in historesin, sectioned on a rotary microtome, stained and mounted in synthetic resin. Additionally, histochemical tests were performed and cuticle ornamentation was analyzed with standard scanning electron microscopy. S. syphilitica and S. aff syphilitica differed in cuticle ornamentation, epidermal cell arrangement and wall thickness, stomata type and orientation, calcium oxalate crystal type, and position of stem thorns. Leaf blades of S. syphilitica from the Amazon Rainforest have a network of rounded ridges on both sides, while in S. aff syphilitica, these ridges are parallel and the spaces between them are filled with numerous membranous platelets. Viewed from the front, the epidermal cells of S. syphilitica have sinuous walls (even more pronounced in samples from the Amazon); while in S. aff syphilitica, these cells are also sinuous but elongated in the cross-section of the blade and arranged in parallel. Stomata of S. syphilitica are paracytic, whereas in S. aff syphilitica, are both paracytic and anisocytic, and their polar axes are directed towards the mid-vein. Calcium oxalate crystals in S. syphilitica are prisms, whereas in S. aff syphilitica, crystal sand. Thorns occur in nodes and internodes in S. syphilitica but only in internodes in S. aff syphilitica. These features have proven to be of diagnostic value and may support a separation into two species, but future studies are needed to confirm that S. aff syphilitica is indeed a new taxon. Rev. Biol. Trop. 60(3): 1137-1148. Epub 2012 September 01.

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LTG33D), originally produced by some enterotoxigenic Escherichia coil (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LIG33D. Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LTG33D elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LTG33D. Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LTG33D. Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine. (C) 2011 Elsevier Ltd. All rights reserved.

Thrombocytopenia and platelet activity in dogs experimentally infected with Rangelia vitalii

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia. (C) 2011 Elsevier B.V. All rights reserved.

Influence of the relative amounts of crystalline and amorphous phases on the mechanical properties of polyamide-6 nanocomposites

The relative amounts of amorphous and crystalline ?- and a-phases in polyamide-6 nanocomposites, estimated from the deconvolution of X-ray diffraction peaks using Gaussian functions, correlates with their mechanical, thermomechanical, and barrier properties. The incorporation of organoclay platelets (Cloisite 15A and 30B) induced the crystallization of the polymer in the ? form at expense of the amorphous phase, such that 12 wt % of Cloisite is enough to enhance the mechanical and the thermomechanical properties. However, higher nanofiller loads were necessary to achieve good barrier effects, because this property is mainly dependent on the tortuous path permeation mechanism of the gas molecules through the nanocomposite films. (C) 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Cuticle of 'Gala' and 'Galaxy' Apples Cultivars under Different Environmental Conditions

This study aimed to analyze the cuticle thickness and pattern of epicuticular wax deposition in 'Gala' and 'Galaxy' apple cultivars (Malus domestica Borkh,) from three Brazilian producing areas: Vacaria (RS), Fraiburgo (SC) and Sao Joaquim (SC) with altitudes of 971, 1,048 and 1,353m, respectively. Harvested fruit were kept under two storage conditions: regular atmosphere (RA) (0 degrees C and 90% RH) and controlled atmosphere (CA) (1.5% O-2, 2.5% CO2, 0 degrees C and 90% RH). Cuticle thickness measurements were made using LM and the deposition pattern of epicuticular wax observed with a SEM. Altitude among the apple producing areas was not a factor in deposition pattern of waxes between the cultivars but at higher altitudes, the cuticle was thicker in both the cultivars. In the freshly-harvested fruits, waxes deposition in the form of platelets and the mechanism of "tear and repair" were observed. Severity of microcracks in the cuticle was more evident on the fruits from CA.

Sources of organic trace minerals in diets for weaned piglets

One hundred and twenty six piglets from a commercial genetic strain weaned at 21 days of age (6.11 +/- 0.42 kg) were used to evaluate the effects of supplementation levels of organic sources of trace minerals in the diets of weaned piglets on performance, occurrence of diarrhea, excretion of copper and zinc in the feces, and hematological parameters. A completely randomized block design was adopted, composed of six treatments (diets containing 100% of inorganic trace minerals premix at 3.00 kg/T; diets containing 0, 25, 50, 75 or 100% of organic trace minerals premix, equivalent to 0; 0.75; 1.50; 2.25 or 3.00 kg/T, respectively), seven replicates and three animals per plot. During the experimental period (from 21 to 63 days of age), the increasing levels of organic trace minerals premix in the diets determined a quadratic effect on daily weight gain (DWG), feed conversion (FC), percentage of hematocrit (Ht), hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), platelets (Pt), and a linear increase of Cu and Zn concentrations in feces. The comparison of means between the treatment with inorganic trace minerals premix and the other treatments showed that piglets fed diets without trace minerals premix had lower values of DWG, Ht, Hb, MCH, MCV, Pt and the worst FC value of piglets fed diets containing 25% of organic trace minerals premix presented lower values of Ht and Hb, in comparison with those fed diets with 100% of inorganic trace minerals premix. Inorganic trace minerals premix can be substituted by organic trace minerals premix at a lower level of inclusion in diets for weaned piglets.

Vorapaxar in the Secondary Prevention of Atherothrombotic Events

BACKGROUND Thrombin potently activates platelets through the protease-activated receptor PAR-1. Vorapaxar is a novel antiplatelet agent that selectively inhibits the cellular actions of thrombin through antagonism of PAR-1. METHODS We randomly assigned 26,449 patients who had a history of myocardial infarction, ischemic stroke, or peripheral arterial disease to receive vorapaxar (2.5 mg daily) or matching placebo and followed them for a median of 30 months. The primary efficacy end point was the composite of death from cardiovascular causes, myocardial infarction, or stroke. After 2 years, the data and safety monitoring board recommended discontinuation of the study treatment in patients with a history of stroke owing to the risk of intracranial hemorrhage. RESULTS At 3 years, the primary end point had occurred in 1028 patients (9.3%) in the vorapaxar group and in 1176 patients (10.5%) in the placebo group (hazard ratio for the vorapaxar group, 0.87; 95% confidence interval [CI], 0.80 to 0.94; P<0.001). Cardiovascular death, myocardial infarction, stroke, or recurrent ischemia leading to revascularization occurred in 1259 patients (11.2%) in the vorapaxar group and 1417 patients (12.4%) in the placebo group (hazard ratio, 0.88; 95% CI, 0.82 to 0.95; P=0.001). Moderate or severe bleeding occurred in 4.2% of patients who received vorapaxar and 2.5% of those who received placebo (hazard ratio, 1.66; 95% CI, 1.43 to 1.93; P<0.001). There was an increase in the rate of intracranial hemorrhage in the vorapaxar group (1.0%, vs. 0.5% in the placebo group; P<0.001). CONCLUSIONS Inhibition of PAR-1 with vorapaxar reduced the risk of cardiovascular death or ischemic events in patients with stable atherosclerosis who were receiving standard therapy. However, it increased the risk of moderate or severe bleeding, including intracranial hemorrhage. (Funded by Merck; TRA 2P-TIMI 50 ClinicalTrials.gov number, NCT00526474.)

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the B beta chain and BpirSP41 on both A alpha and B beta chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of similar to 3.5 mu g versus 20 mu g for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu2+ ion and specific serine protease inhibitors. In addition. BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure-function relationship of SVSPs. (C) 2012 Elsevier Masson SAS. All rights reserved.