Recovery and partitioning of fibrinolytic protease from Bacillus sp. UFPEDA 485 by aqueous two-phase systems

Fibrinolytic proteases produced by Bacillus sp. has attracted interest in the pharmaceutical industry as a promising alternative in thrombolytic therapy due to their effectiveness in degrading fibrin, its production requiring the development of an efficient recovery process. Aqueous two-phase systems (ATPS) have been recognized as an efficient and economical process for recovering enzymes. To optimize the recovery of fibrinolytic protease from the fermentation broth of Bacillus sp. UFPEDA 485, a 23 full factorial design was used to evaluate the influence of the three independent variables PEG molar mass (MPEG), PEG concentration (CPEG) and sodium sulfate concentration (CNa2SO4) on the partition coefficient (K), purification factor (PF) and yield recovery (Y) of fibrinolytic protease in PEG/Na2SO4 aqueous two-phase system. For all ATPS studied, enzymes partitioned to the top phase and the highest extraction was obtained for MPEG 6000 g.mol-1, CPEG 24 % (w/w) and CNa2SO4 11.6 % (w/w) with K = 5.03; PF = 3.30; Y = 91.40% and Fibrinolytic activity in the top phase 821 U.mL-1. Findings reported here show that ATPS composed of PEG/Na2SO4 is a valuable strategy for the extraction of fibrinolytic protease and can be considered a promising method for the extraction of enzymes in industrial scale

CONSTITUCIONAIS PLENAS: UMA ANÁLISE DA CONCRETUDE DOS DIREITOS DA PESSOA COM DEFICIÊNCIA SOB A ÓTICA DA LEGISLAÇÃO ADMINISTRATIVA, TRIBUTÁRIA E CONSUMERISTA

O presente estudo tem por escopo demonstrar que algumas disposições constitucionais passaram a demandar uma série de medidas do legislador infraconstitucional para que se pudesse conferir-lhes aplicabilidade plena, especificamente, as disposições relativas à tutela da igualdade material relativa às pessoas com deficiência. Nesse contexto, o presente estudo tem por objeto investigar a produção e atualização legislativa referente à legislação: a) consumerista; b) administrativista; e c) tributária, com o objetivo de averiguar se essas foram suficientes a conferir efetividade ao texto constitucional. Parte-se da hipótese confirmável que, em que pese as disposições legislativas, a relutância na atualização do limite da tabela de isenção tributária do ICMS na aquisição de veículos automotores e a expressiva regressividade em boa parte dos impostos sobre o consumo, aliada a inaplicabilidade empírica da legislação administrativa, sobretudo relativa ao Estatuto das Cidades, acabam por desamparar, exatamente, esta parcela da população que já era bastante onerada pela inerente adaptação dos seus bens de consumo a suas respectivas realidades. A metodologia funda-se em pesquisa documental hipotético-indutiva com o objetivo de investigar como tem sido feita a atualização da legislação administrativista, consumerista e tributária, no que tange à efetiva tutela da pessoa com deficiência. Os achados demonstram defasagem na aplicação do Estatuto das Cidades, na atualização do teto de isenção e alta regressividade na tributação sobre o consumo, o que, aliado aos gastos de adaptações necessários às demandas desta parcela da população, ao longo do tempo, vêm tornando o mandamento constitucional da igualdade material mais programático do que, efetivamente, plen

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 angstrom) and complexed with bovine trypsin (at 2 angstrom). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. the structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1' pockets, respectively. the overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15 degrees is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.United States Department of Energy, Office of Science, Office of Basic Energy SciencesCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)National Institutes of Health, National Cancer Institute, Center for Cancer ResearchNCI, Ctr Canc Res, Macromol Crystallog Lab, Frederick, MD 21701 USAUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilInst Butantan, Lab Bioquim & Biofis, Unidade Sequenciamento Prot & Peptideos, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUnited States Department of Energy, Office of Science, Office of Basic Energy Sciences: W-31-109-Eng-38FAPESP: 09/53766-5Web of Scienc

Optimization of clavulanic acid production by Streptomyces daufpe 3060 by response surface methodology

Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.Brazilian Research Funding InstitutionsCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Fluoranthene Biodegradation by Serratia sp. AC-11 Immobilized into chitosan beads.

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HFE variants and the expression of iron-related proteins in breast cancer-associated lymphocytes and macrophages

Disponível em: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264664/The association of HFE (High Iron FE) major variants with breast cancer risk and behavior has been a matter of discussion for a long time. However, their impact on the expression of iron-related proteins in the breast cancer tissue has never been addressed. In the present study, hepcidin, ferroportin 1, transferrin receptor 1 (TfR1), and ferritin expressions, as well as tissue iron deposition were evaluated in a collection of samples from breast cancers patients and analyzed according to the patients’ HFE genotype. Within the group of patients with invasive carcinoma, those carrying the p.Cys282Tyr variant in heterozygosity presented a higher expression of hepcidin in lymphocytes and macrophages than wild-type or p.His63Asp carriers. An increased expression of TfR1 was also observed in all the cell types analyzed but only in p.Cys282Tyr/p.His63Asp compound heterozygous patients. A differential impact of the two HFE variants was further noticed with the observation of a significantly higher percentage of p.Cys282Tyr heterozygous patients presenting tissue iron deposition in comparison to p.His63Asp heterozygous. In the present cohort, no significant associations were found between HFE variants and classical clinicopathological markers of breast cancer behavior and prognosis. Although limited by a low sampling size, our results provide a new possible explanation for the previously reported impact of HFE major variants on breast cancer progression, i.e., not by influencing systemic iron homeostasis but rather by differentially modulating the local cellular expression of iron-related proteins and tissue iron deposition.OM is a recipient of the PhD grant SFRH/BD/2011/78184 from Fundação para a Ciência e Tecnologia (FCT). The authors also acknowledge financial support from ICBAS/AI&NSUMIB and by national funds through FCT and Ministério da Educação e Ciência (MEC) and when applicable co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number 4293.info:eu-repo/semantics/publishedVersio

Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method.This work was supported by the National Council of Technological and Scientific Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Sara Silvério also acknowledges her post-doc grant (SFRH/BPD/88584/2012) from FCT (SFRH/BPD/88584/2012) (Portuguese Science and Technology Foundation), Portugal

Influence of fluid ingestion on heart rate, cardiac autonomic modulation and blood pressure in response to physical exercise : a systematic review with meta-analysis and meta-regression

A systematic review was undertaken to investigate the involvement of hydration in heart rate (HR), HR variability (HRV) and diastolic (DBP) and systolic (SBP) blood pressure in response to exercise. Data synthesis: The EMBASE, MEDLINE, Cochrane Library, CINAHL, LILACS and Web of Science databases were searched. In total, 977 studies were recognized, but only 36 were included after final screening (33 studies in meta-analysis). This study includes randomized controlled trials (RCTs) and non-RCTs with subjects > 18 years old. The hydration group consumed water or isotonic drinks, while the control group did not ingest liquids. For the hydration protocol (before, during and after exercise), the HR values during the exercise were lower compared to the controls (−6.20 bpm, 95%CI: −8.69; −3.71). In the subgroup analysis, “water ingested before and during exercise” showed lower increases in HR during exercise (−6.20, 95%CI: 11.70 to −0.71), as did “water was ingested only during exercise” (−6.12, 95%CI: −9.35 to −2.89). Water intake during exercise only revealed a trend of avoiding greater increases in HR during exercise (−4,60, 95%CI: −9.41 to 0.22), although these values were not significantly different (p = 0.06) from those of the control. “Isotonic intake during exercise” showed lower HRs than the control (−7.23 bpm, 95% CI: −11.68 to −2.79). The HRV values following the exercise were higher in the hydration protocol (SMD = 0.48, 95%CI: 0.30 to 0.67). The values of the SBP were higher than those of the controls (2.25 mmHg, 95%CI: 0.08 to 4.42). Conclusions: Hydration-attenuated exercise-induced increases in HR during exercise, improved autonomic recovery via the acceleration of cardiac vagal modulation in response to exercise and caused a modest increase in SBP values, but did not exert effects on DBP following exercise