Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Background: Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2 , clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 kDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.Fil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Cajade, Rodrigo. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología. Laboratorio de Herpetología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Piñeiro, Jose Miguel. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología. Laboratorio de Herpetología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres, Ana M.. Universidad Nacional del Nordeste; ArgentinaFil: da Silva, Igor R. F.. Universidade Estadual de Campinas; BrasilFil: Hyslop, Stephen. Universidade Estadual de Campinas; BrasilFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Pimenta, Daniel C.. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Bustillo, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentin

Neuromuscular activity of bothrops fonsecai snake venom in vertebrate preparations

CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOThe neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160μg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160μg/ml, respectively) and attenuated the contractures to 110μM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200μg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200μg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160mg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160mg/ml, respectively) and attenuated the contractures to 110mM ACh (78–100% inhibition) and 40mM KCl (45–90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200mg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200mg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K+ were abolished by preincubating venom with commercial antivenom.These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom5615CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãosem informaçã

Genetic comparison of sickle cell anaemia cohorts from Brazil and the United States reveals high levels of divergence

Genetic analysis of admixed populations raises special concerns with regard to study design and data processing, particularly to avoid population stratification biases. The point mutation responsible for sickle cell anaemia codes for a variant hemoglobin, sickle hemoglobin or HbS, whose presence drives the pathophysiology of disease. Here we propose to explore ancestry and population structure in a genome-wide study with particular emphasis on chromosome 11 in two SCA admixed cohorts obtained from urban populations of Brazil (Pernambuco and Sao Paulo) and the United States (Pennsylvania). Ancestry inference showed different proportions of European, African and American backgrounds in the composition of our samples. Brazilians were more admixed, had a lower African background (43% vs. 78% on the genomic level and 44% vs. 76% on chromosome 11) and presented a signature of positive selection and Iberian introgression in the HbS region, driving a high differentiation of this locus between the two cohorts. The genetic structures of the SCA cohorts from Brazil and US differ considerably on the genome-wide, chromosome 11 and HbS mutation locus levels9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP8367/2011-1; 150398/2013-1; 304455/2012-1; 310938/2014-7; 305218/2017-48367/2011-1; 150398/2013-1; 304455/2012-1; 310938/2014-7; 305218/2017-42008/57441-0; 2014/00984-3; 2012/06438-5; 2015/13152-9; 2008/10596-

Nanopartículas de poli-'épsilon'-caprolactona carregadas com hidrocortisona: preparação usando planejamento fatorial e sua avaliação

Polymeric-nanoparticle systems such as nanocapsules and nanospheres have a great potential in applications for nanoencapsulation of corticosteroids which show low solubility in water. The physicochemical characteristics of nanoparticle suspensions are important pre-requisites for the successful development of new dosage form. In this study, hydrocortisone-loaded poly-ε-caprolactone nanoparticles have been prepared by the interfacial deposition method. A 3-factor 2-level factorial design was used to study and optimize nanoparticles formulation. This factorial design was used to study the contrasts and effects of independent variables on particle size distribution, morphology, surface charge, drug content, entrapment efficiency and in vitro drug release profiles. The screened independent variables were: the concentration of hydrocortisone, poly-ε-caprolactone and isodecyl oleate. A High Performance Liquid Chromatography method was developed and validated for hydrocortisone quantification. Special attention was given to both absolute recovery and entrapment efficiency. The results of optimized formulations showed a narrow size distribution with a polydispersity index near to 0.200. The particle sizes were on average 109.2 and 236.5nm to nanospheres and nanocapsules, respectively. In the best formulations the zeta potential was higher than 30 mV (in module) and the absolute recovery and entrapment efficiency were higher 82% and nearly 60%, respectively. The main variables were the quantity of the polymer and of the oil. Nanoparticles observed by the Scanning Electron Microscope depicted extremely spherical shape. In vitro release studies were performed through dialysis with continuous stream. Nanocapsules and nanospheres showed a similar pure diffusion release mechanism according to Korsmeyer-Peppas’s model.CNPqCAPESFAPES

Análise das demandas judiciais para aquisição de medicamentos no estado da Paraíba

Resumo O objetivo deste estudo foi descrever os aspectos médico-científicos e sanitários dos mandados judiciais para fornecimento de medicamentos impetrados ao Tribunal de Justiça da Paraíba (TJ-PB) contra agentes públicos no estado da Paraíba. Teve caráter descritivo, transversal, e foi realizado no Tribunal de Justiça da Paraíba. Foram analisados processos de medicamentos julgados na segunda instância entre os anos de 2009 e 2010 com decisão definitiva. Quanto às evidências científicas da eficácia e segurança, os medicamentos foram avaliados por meio de revisões sistemáticas na base de dados do Centro Cochrane do Brasil. Os grupos de medicamentos solicitados com maior frequência foram os agentes neoplásicos, seguidos de insulinas e antagonistas da angiotensina II. Os custos com medicamentos antineoplásicos representaram 76% do gasto total com demandas judiciais, representando um custo aproximado de R$ 343 mil por mês. Desses, um item não tinha registro em orgão sanitário brasileiro. Os resultados apontam para um elevado número de demandas judiciais com prevalência de solicitações de medicamentos padronizados pelo Sistema Único de Saúde (SUS), dos quais os antineoplásicos obtêm destaque e reforçam a necessidade de revisão da Política Nacional de Assistência Farmacêutica tanto das estratégias de implantação como da premência em ampliar a relação de medicamentos disponibilizados pelo SUS

Infrastructure for Detector Research and Development towards the International Linear Collider

The EUDET-project was launched to create an infrastructure for developing and testing new and advanced detector technologies to be used at a future linear collider. The aim was to make possible experimentation and analysis of data for institutes, which otherwise could not be realized due to lack of resources. The infrastructure comprised an analysis and software network, and instrumentation infrastructures for tracking detectors as well as for calorimetry.Comment: 54 pages, 48 picture

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Evaluating the effects of SARS-CoV-2 Spike mutation D614G on transmissibility and pathogenicity

SummaryGlobal dispersal and increasing frequency of the SARS-CoV-2 Spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of Spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large data set, well represented by both Spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the Spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.</jats:p

Intraperitoneal but Not Intravenous Cryopreserved Mesenchymal Stromal Cells Home to the Inflamed Colon and Ameliorate Experimental Colitis

BACKGROUND AND AIMS: Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. RESULTS: Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1β decreased, while VEGF and TGF-β did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. CONCLUSIONS: Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis

Inhibitors of trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease.

Chagas disease remains one of the most neglected diseases in the world despite being the most important parasitic disease in Latin America. The characteristic chronic manifestation of chagasic cardiomyopathy is the region's leading cause of heart-related illness, causing significant mortality and morbidity. Due to the limited available therapeutic options, new drugs are urgently needed to control the disease. Sirtuins, also called Silent information regulator 2 (Sir2) proteins have long been suggested as interesting targets to treat different diseases, including parasitic infections. Recent studies on Trypanosoma cruzi sirtuins have hinted at the possibility to exploit these enzymes as a possible drug targets. In the present work, the T. cruzi Sir2 related protein 1 (TcSir2rp1) is genetically validated as a drug target and biochemically characterized for its NAD+-dependent deacetylase activity and its inhibition by the classic sirtuin inhibitor nicotinamide, as well as by bisnaphthalimidopropyl (BNIP) derivatives, a class of parasite sirtuin inhibitors. BNIPs ability to inhibit TcSir2rp1, and anti-parasitic activity against T. cruzi amastigotes in vitro were investigated. The compound BNIP Spermidine (BNIPSpd) (9), was found to be the most potent inhibitor of TcSir2rp1. Moreover, this compound showed altered trypanocidal activity against TcSir2rp1 overexpressing epimastigotes and anti-parasitic activity similar to the reference drug benznidazole against the medically important amastigotes, while having the highest selectivity index amongst the compounds tested. Unfortunately, BNIPSpd failed to treat a mouse model of Chagas disease, possibly due to its pharmacokinetic profile. Medicinal chemistry modifications of the compound, as well as alternative formulations may improve activity and pharmacokinetics in the future. Additionally, an initial TcSIR2rp1 model in complex with p53 peptide substrate was obtained from low resolution X-ray data (3.5 Å) to gain insight into the potential specificity of the interaction with the BNIP compounds. In conclusion, the search for TcSir2rp1 specific inhibitors may represent a valuable strategy for drug discovery against T. cruzi