Optimization of the Extraction of Polyphenols and Antioxidant Capacity from Byrsonima crassifolia (L.) Kunth Fruit by Response Surface Methodology

The purpose of this research was to optimize the extraction conditions of polyphenols from murici (Byrsonima crassifolia (L.) Kunth) using the response surface methodology. Temperature (from 10 to 70°C), acetone concentration (from 10 to 100%), extraction time (from 0 to 160 min), and solid-liquid ratio (from 20 to 140 mg/mL) were investigated as independent variables in order to obtain the optimal conditions for extraction and to maximize the total phenolic content (TPC) and antioxidant activity (DPPH) of obtained extracts. Experimental results were fitted to the second-order polynomial model where multiple regression and analysis of variance were used to determine the fitness of the model and optimal condition for investigated responses. The solid-liquid did not interfere in the two responses. The results showed that for TPC extraction, the optimal conditions were obtained with an acetone concentration of 44%, a temperature of 29°C, and an extraction time of 51 min. For DPPH, the optimal conditions were the following: an acetone concentration of 45%, a temperature of 40°C, and an extraction time of 53 min. The use of such conditions allowed the maximum extraction of antioxidant murici at a lower cost of production, which may contribute to large-scale industrial applications and future pharmacological research

Differential transcript profile of inhibitors with potential anti-venom role in the liver of juvenile and adult Bothrops jararaca snake

Background. Snakes belonging to the Bothrops genus are vastly distributed in Central and South America and are responsible for most cases of reported snake bites in Latin America. The clinical manifestations of the envenomation caused by this genus are due to three major activities-proteolytic, hemorrhagic and coagulant-mediated by metalloproteinases, serine proteinases, phospholipases A(2) and other toxic compounds present in snake venom. Interestingly, it was observed that snakes are resistant to the toxic effects of its own and other snake's venoms. This natural immunity may occur due the absence of toxin target or the presence of molecules in the snake plasma able to neutralize such toxins. Methods. In order to identify anti-venom molecules, we construct a cDNA library from the liver of B. jararaca snakes. Moreover, we analyzed the expression profile of four molecules-the already known anti-hemorrhagic factor Bj46a, one gamma-phospholipase A(2) inhibitor, one inter-alpha inhibitor and one C1 plasma protease inhibitor-in the liver of juvenile and adult snakes by qPCR. Results. The results revealed a 30-fold increase of gamma-phospholipase A(2) inhibitor and a minor increase of the inter-alpha inhibitor (5-fold) and of the C1 inhibitor (3-fold) in adults. However, the Bj46a factor seems to be equally transcribed in adults and juveniles. Discussion. The results suggest the up-regulation of different inhibitors observed in the adult snakes might be a physiological adaptation to the recurrent contact with their own and even other snake's venoms throughout its lifespan. This is the first comparative analysis of ontogenetic variation of expression profiles of plasmatic proteins with potential anti-venom activities of the venomous snake B. jararaca. Furthermore, the present data contributes to the understanding of the natural resistance described in these snakes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Tecnologico (CNPq)INCT - Entomologia MolecularInst Butantan, Lab Herpetol, Sao Paulo, BrazilUniv Sao Paulo, Interunidades Biotecnol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilUniv Fed Rio de Janeiro, Dept Bioquim, Rio De Janeiro, BrazilInst Nacl Ciencia & Tecnol Entomol Mol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilFAPESP: 2010/10266-5FAPESP: 2012/03657-8FAPESP: 2013/05357-4FAPESP: 2014/11108-0CNPq: 308780/2013-2Web of Scienc

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Proteases digestivas do hepatopâncreas dos camarões marinhos Farfantepenaeus subtilis e Farfantepenaeus paulensis

Os camarões Farfantepenaeus subtilis e Farfantepenaeus paulensis são espécies nativas e aparecem como uma alternativa ao cultivo do Litopenaeus vannamei, que hoje corresponde a mais de 90% do cultivo de camarões marinhos da região nordeste. Proteases do hepatopâncreas de F. subtilis jovens e adultos e F. paulensis jovens foram estudadas quanto aos seguintes aspectos: inibição enzimática, pH e temperatura ótima, estabilidade térmica, eletroforese e zimogramas. No extrato bruto de F. subtilis, não houve diferenças significativas nas atividades proteolíticas em ambas as fases de vida utilizando azocaseína, leucina pnitroanilida (Leu-p-Nan) e substratos b-naftilamida (p³0,05). No entanto, as atividades tríptica (Benzoil arginina p-nitroanilida-BApNA) e quimotríptica (Succinil alanina alanina prolina fenilalanina p-nitroanilida-SAPNA) foram mais elevadas em jovens do que em adultos (p<0,05). Atividades de tripsina e quimotripsina também foram detectadas em F. paulensis. Tosil lisina clorometil cetona (TLCK) e benzamidina inibiram fortemente as atividades proteolíticas nos extratos de F. subtilis e F. paulensis. Tosil fenilalanina clorometil cetona (TPCK) foi capaz de inibir 59,34% da atividade de quimotripsina em F. paulensis utilizando SAPNA como substrato. A temperatura ótima para tripsina-símile e quimotripsina-símile em ambas as fases de vida foi 55°C, enquanto que para aminopeptidases houve uma diferença entre jovens (55°C) e adultos (45°C). Tripsina-símile reteve aproximadamente 15% de sua atividade inicial quando incubada a 55°C por 30 minutos, enquanto quimotripsina-símile e leucina aminopeptidase-símile retiveram 60% e 45% de atividade respectivamente. No extrato bruto de F. paulensis a tripsina-símile apresentou atividade máxima em pH 8,0 e temperatura ótima de 40°C. A atividade mais elevada de quimotripsina-símile foi detectada numa faixa de pH alcalino (7,2-9,0) e na temperatura de 55°C. O zimograma de estabilidade térmica apresentou um padrão similar de bandas com atividade proteolítica em F. subtilis jovens e adultos, exceto que o extrato de camarões juvenis apresentou uma banda termoestável a 65°C. PMSF inibiu todas as bandas proteolíticas. Duas isoformas de quimotripsina (31,6 e 36,4 kDa) foram estáveis a 85°C em F. paulensis. A caracterização de enzimas digestivas de camarões nativos pode gerar informações importantes para o entendimento da fisiologia e da capacidade digestiva destes organismos, principalmente pelo fato dessas enzimas estarem diretamente ligadas ao aproveitamento de aminoácidos dietários, sendo estes importantes fontes de monômeros para a síntese de proteínas funcionais que serão responsáveis por uma série de eventos fisiológicos como: crescimento, reprodução, defesa imunológica, entre outro

Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 degrees C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2-9.0) and 55 degrees C. CaCl(2) did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 degrees C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P < 0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.FINEP/RECARCINEFinanciadora de Estudos e Projetos (FINEP)SEAP/PRSEAP/PRConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPqFACEPEFACEPEPETROBRASPETROBRASFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP[05/50578-2]FAPESP[07/07051-9]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq/SEAP[504031/2003-1]CNPq/SEAPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq fellowship[308444/2006-0]National Research System (Brazil)National Research System (Brazil

A new antimicrobial protein from the anterior midgut of Triatoma infestans mediates Trypanosoma cruzi establishment by controlling the microbiota

The Reduviid Triatoma infestans is a vector for the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. The parasite must address the defense molecules and microbiota that colonize the anterior midgut of T. infestans. To obtain insight into T cruzi - microbiota interactions in triatomine insects, we characterized a new antimicrobial product from the anterior midgut of T infestans (TiAP) that may be involved in these relationships. The TiAP DNA fragment was cloned and expressed in a bacterial system, and the effect of the protein on bacteria and T. cruzi was evaluated by RNAi, qPCR and antimicrobial experiments. The number of T. cruzi in T infestans anterior midguts was significantly lower in TiAP knockdown insects than in unsilenced groups. We also verified that the amount of bacteria in silenced T. infestans is approximately 600-fold higher than in unsilenced insects by qPCR. The 327-bp cDNA fragment that encodes mature TiAP was cloned into the pET-14b vector and expressed fused to a His-tag in Escherichia coli C43. The recombinant protein (rTiAP) was purified using an Ni-NTA column, followed by a HiTrap SP column. According to a trypanocidal assay, rTiAP did not interfere with the viability of T cruzi trypomastigotes. Moreover, in antimicrobial experiments using E. coli and Micrococcus luteus, the protein was only bacteriostatic for Gram-negative bacteria. The data indicate that infection by T. cruzi increases the expression of TiAP to modulate the microbiota. The inhibition of microbiota growth by TiAP is important for parasite establishment in the T. infestans anterior midgut. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conseiho Nacional de Desenvolvimento Tecnologico (CNPq)INCT-Entomologia MolecularUniv Fed Sao Paulo, Escola Paulista Med, Dept Biochem, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Parasitol, BR-31270901 Belo Horizonte, MG, BrazilFiocruz MS, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Biochem, BR-04044020 Sao Paulo, SP, BrazilFAPESP: 2012/03657-8FAPESP: 2012/00723-0CNPq: 308780/2013-2Web of Scienc

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K-i = 3.29 nM) and human cathepsin L (K-i= 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan. (C) 2011 Elsevier Inc. All rights reserved