In silico enzyme engineering - successful stories and future outlook

Part of the goals of the multi-partner INDOX European project (indoxproject.eu) was to identify, select and tailor oxidoreductases to obtain ad hoc industrial biocatalysts with optimized properties. Thanks to the combined effort of experimental and computational researchers we have successfully improved a variety of enzymatic processes including the industrial synthesis of: 1-naphthol1, 25-hydroxyvitamin D32, and conductive polyaniline3. The computational methodologies employed, in some design efforts, will be presented and the importance of fast and accurate computer-guided protein engineering solutions4,5 discussed. Please click Additional Files below to see the full abstract

Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

New Monte Carlo Based Technique To Study DNA–Ligand Interactions

We present a new all-atom Monte Carlo technique capable of performing quick and accurate DNA–ligand conformational sampling. In particular, and using the PELE software as a frame, we have introduced an additional force field, an implicit solvent, and an anisotropic network model to effectively map the DNA energy landscape. With these additions, we successfully generated DNA conformations for a test set composed of six DNA fragments of A-DNA and B-DNA. Moreover, trajectories generated for cisplatin and its hydrolysis products identified the best interacting compound and binding site, producing analogous results to microsecond molecular dynamics simulations. Furthermore, a combination of the Monte Carlo trajectories with Markov State Models produced noncovalent binding free energies in good agreement with the published molecular dynamics results, at a significantly lower computational cost. Overall our approach will allow a quick but accurate sampling of DNA–ligand interactions.The authors thank the Barcelona Supercomputing Center for computational resources. This work was supported by grants from the European Research Council—2009-Adg25027-PELE European project and the Spanish Ministry of Economy and Competitiveness CTQ2013-48287 and “Juan de la Cierva” to F.L.Peer ReviewedPostprint (author's final draft

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high-redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a lignin-related phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction were affected. Thus, quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets

Low Back Pain: A Pain That May Not Be Harmless

Abdominal aortic aneurysms are defined as vascular dilatations greater than 50% of the normal proximal segment or those that have a maximum diameter above 3 cm. Risk factors include male gender, age over 75 years, history of vascular pathology, hypertension and arteriosclerosis. The Authors describe a case of a 74-year-old man, evaluated in an emergency setting for right lumbar pain lasting 4 days. The pain did not respond to analgesia and became progressively worse. Due to the severity of symptoms, CT angiography was performed, which showed an active rupture of a partially contained aneurysm associated with aortic dissection. Early diagnosis and timely management of aortic aneurysms are essential in preventing complications, namely rupture (50-83% patients die after rupture and before receiving medical care). Acute aortic dissection is a surgical emergency and the risk of rupture is proportional to the size of the aneurysm and its rate of growth

RELATO DE EXPERIÊNCIA: ATIVIDADE DE EXTENSÃO UNOESC NA ESCOLA – ORIENTAÇÃO PROFISSIONAL

Pelo segundo ano consecutivo o Curso de Psicologia da Unoesc Chapecó desenvolve palestras de Orientação Profissional inseridas no projeto de extensão Unoesc na Escola. A orientação profissional é uma das atividades que podem ser desenvolvidas pelo profissional Psicólogo que visa à promoção do autoconhecimento bem como do conhecimento acerca do mercado e do mundo das profissões para os alunos concluintes do Ensino Médio. Nesse ano, a atividade contou com a participação de 19 acadêmicos contemplados com bolsa de estudo. Estes foram capacitados para realizar as palestras nas escolas do Município de Chapecó, SC e região. Foram atendidas seis escolas com um total de  aproximadamente 115 alunos contemplados. O objetivo da atividade foi sensibilizar e orientar os alunos que cursam o terceiro ano do Ensino Médio por meio de palestras que abordaram aspectos os quais envolvem o momento da escolha profissional. Em contrapartida, os acadêmicos bolsistas desenvolveram competências relacionadas ao perfil do egresso do Curso de Psicologia, bem como competências e habilidades específicas. Essa atividade vai ao encontro da missão do Curso de Psicologia que é proporcionar uma formação sólida e ampla com adequada fundamentação teórico-prática para a atuação nas áreas de processos, clínicas e/ou promoção e prevenção da saúde. Percebeu-se que as palestras atenderam aos objetivos e que há demanda para novas atividades relacionadas à orientação profissional na Unoesc Chapecó.Palavras-chave: Orientação Profissional. Palestra. Capacitação de acadêmicos bolsistas

EXPERIÊNCIA PEDAGÓGICA INOVADORA NO CONTEXTO DA EDUCAÇÃO BÁSICA NO BRASIL: IDENTIFICAÇÃO E ANÁLISE A PARTIR DO PECEB

This article presents the problematizations arising from a research that sought to identify and analyze innovative pedagogical experiences in the context of basic education in Brazil. To this end, it identified among the actions of the federal government, specifically the Ministry of Education, the Program to Stimulate Creativity and Innovation in Basic Education (PECEB). This program certified 178 schools and educational institutions with the title of innovative and creative. The analysis focuses on the concept of innovation, having as a methodological resource the exploratory research in projects classified as innovative in three schools in the state of Santa Catarina.Este artigo apresenta as problematizações decorrentes de uma pesquisa que buscou identificar e analisar experiências pedagógicas inovadoras no contexto da educação básica no Brasil. Para tanto, identificou entre as ações do governo federal, especificamente do Ministério da Educação, o Programa de Estímulo a Criatividade e Inovação na Educação Básica (PECEB). Este programa certificou 178 escolas e instituições de educação conferindo o título de inovadoras e criativas. Centra-se a análise no conceito de inovação tendo como recurso metodológico a pesquisa exploratória em projetos classificados como inovadores em três escolas do estado de Santa Catarina