In Silico enzyme engineering – The importance of fast and accurate algorithms

Computer simulations are already widely used to rationally engineer new enzymes with improved properties. But if we can accurately screen millions of enzyme variants in a computer, then we can move into a new generation of in silico enzyme evolution. At ZYMVOL we are to be able to produce, model and rank protein-substrate interactions (including full protein dynamics) for over 50.000 enzyme variants per day. We have accelerated physics-based simulations, and combined experimental data with quantum approaches to develop a highly predictive computational platform. Our ZYMEVOLVER software can effectively reduce experimental validation to a few hundred variants and enzyme optimization campaigns to less than 6 months. We will illustrate how we are creating custom-made enzymes for industrial applications

The ins and outs of vanillyl alcohol oxidase: Identification of ligand migration paths

Vanillyl alcohol oxidase (VAO) is a homo-octameric flavoenzyme belonging to the VAO/PCMH family. Each VAO subunit consists of two domains, the FAD-binding and the cap domain. VAO catalyses, among other reactions, the two-step conversion of p-creosol (2-methoxy-4-methylphenol) to vanillin (4-hydroxy-3-methoxybenzaldehyde). To elucidate how different ligands enter and exit the secluded active site, Monte Carlo based simulations have been performed. One entry/exit path via the subunit interface and two additional exit paths have been identified for phenolic ligands, all leading to the si side of FAD. We argue that the entry/exit path is the most probable route for these ligands. A fourth path leading to the re side of FAD has been found for the co-ligands dioxygen and hydrogen peroxide. Based on binding energies and on the behaviour of ligands in these four paths, we propose a sequence of events for ligand and co-ligand migration during catalysis. We have also identified two residues, His466 and Tyr503, which could act as concierges of the active site for phenolic ligands, as well as two other residues, Tyr51 and Tyr408, which could act as a gateway to the re side of FAD for dioxygen. Most of the residues in the four paths are also present in VAO’s closest relatives, eugenol oxidase and p-cresol methylhydroxylase. Key path residues show movements in our simulations that correspond well to conformations observed in crystal structures of these enzymes. Preservation of other path residues can be linked to the electron acceptor specificity and oligomerisation state of the three enzymes. This study is the first comprehensive overview of ligand and co-ligand migration in a member of the VAO/PCMH family, and provides a proof of concept for the use of an unbiased method to sample this process.Enzymes are bionanomachines, which speed up chemical reactions in organisms. To understand how they achieve that, we need to study their mechanisms. Computational enzymology can show us what happens in the enzyme’s active site during a reaction. But molecules need first to reach the active site before a reaction can start. The process of substrate entry and product exit to the active site is often neglected when studying enzymes. However, these two events are of fundamental importance to the proper functioning of any enzyme. We are interested in these dynamic processes to complete our understanding of the mode of action of enzymes. In our work, we have studied substrate and product migration in vanillyl alcohol oxidase. This enzyme can produce the flavour vanillin and enantiopure alcohols, but also catalyses other reactions. The named products are of interest to the flavour- and fine-chemical industries.This work was supported by: FP7-KBBE- 2013-7-613549 http://cordis.europa.eu/ programme/rcn/851_en.html (see INDOX project (http://indoxproject.eu), funding received by: GG MFL VG WJHvB; and CTQ2016-79138-R, http:// www.mineco.gob.es/portal/site/mineco/?lang_ choosen=en, funding received by: MFL VG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer ReviewedPostprint (published version

A Study on Binary Asteriod System Deflection

The study of asteroids, its composition and trajectories, has been a persistent interest in the space exploration community. In addition, they are also perceived as a great threat to life on Earth, considering the possibility of an impact with our planet. A considerable portion, around 15%, of the asteroid population are believed to be part of a double or triple asteroid system

Structural changes in intestinal enteroendocrine cells after ileal interposition in normal rats

INTRODUCTION: No therapeutic approach has significantly impacted the progression of diabetes. As early improvement of glicaemic control is observed after bariatric surgeries, there is currently a search for surgical procedures that can promote euglycemia also in non-obese patients. Glicaemic control can be achieved by increasing the blood concentration of GLP-1, a hormone produced by L cells that are more densely concentrated in the terminal ileum. The interposition of ileal segment to a more anterior region (proximal jejunum) can promote a greater stimulation of the L cells by poorly digested food, increasing the production of GLP-1 and reflecting on glicaemic control.
AIMS: To investigate long-term histological modifications of intestinal mucosa of rats submitted to interposition of ileum segment to a proximal region (jejunum).
METHODS: Forty 8-week old male Wistar-EPM1 rats (Rattus norvegicus albinus) were randomly distributed into 3 groups: the Interposition Group (IG) was subjected to ileal interposition, the Sham Group (SG) was subjected to sham operations, and the Control Group (CG) was not subjected to surgery. All animals were followed until the 60th postoperative day (8 postoperative week) when they were euthanized. Segments of jejunum and ileum from all groups were collected and analyzed by optical microscopy and immunohistochemistry.
RESULTS: No structural nor histological changes in intestinal L cells in the interposed intestinal segment and other intestinal segments were noted after ileal interposition surgery. 
CONCLUSION: As L cells endocrine characteristics were likely maintained, the use of metabolic surgical techniques for the treatment of metabolic diseases, especially diabetes, seems to be justified

Ballast Treated with Asphalt Emulsion, Megarok and San Jose. AASTHO Standards

In the research work, the results obtained at the laboratory level of the lending material (ballast) treated with asphalt emulsion from the quarries "Megarok and San José" are compared, applying the AASTHO standards. The quarries are located in the Picoazá-La Sequita parish of the Portoviejo canton. Samples from both quarries were evaluated in the laboratory and it was possible to carry out tests of natural humidity, Atterberg, granulometric, compaction, and soil bearing capacity (CBR). Based on the results obtained, they were compared by disclosing which of the quarries meets the appropriate conditions within the construction standards and techniques, in order to stabilize the material with asphalt emulsion; in addition to comparing it with the specifications of the AASTHO standards and the MTOP standard. The result was that the materials extracted from the quarries do not meet the technical specifications; but when mixed with the asphalt emulsion, the Megarok material fulfills as type A base

Got her mummy´s eyes: Eye color investigation using IrisPlex – preliminary study

Poster apresentado no 9th ISABS Conference on Forensic and Anthropologic Genetics,22 a 26 de junho de 2015, ilha de Brač, CroáciaSNP phenotypic markers are being studied by several groups worldwide. As a result, phenotypic loci such as the ones responsible for human eye, hair and skin color are starting to get known. These may provide information for identification of unknown sample donors in criminal casework when there are no suspects. This kind of information may be even more relevant when the sample donor possesses phenotypic characteristics which distinguish him from the population in which he is inserted. Nevertheless, this approach may be used in similar scientific areas. In this work, a DNA sample from a mummified corpse with historical and scientific interest was studied in order to discover more information about who this corps belonged to. One of the tools used in this investigation was IrisPlex, designed for eye color prediction. This study presents one example on how this methodologies may be useful, not only in forensic investigation but also in areas such as physical anthropology.N/

Flavobacterium plurextorum sp. nov. isolated from farmed rainbow trout (Oncorhyncus mykiss)

Five strains (1126-1H-08T, 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 mm wide and 3 mm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25uC. They grew in a temperature range between 15uC to 30uC, but they did not grow at 37uCor 42uC. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08T exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678T and Flavobacterium pectinovorum DSM 6368T (98.5% and 97.9% sequence similarity, respectively). DNA–DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H- 08T, selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678T and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08T was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C15:0 and C15:0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126- 1H-08T ( = CECT 7844T = CCUG 60112T)

Identification by MALDI-TOF MS of Sporothrix brasiliensis isolated from a subconjunctival infiltrative lesion in an immunocompetent patient

Sporotrichosis is a globally distributed subcutaneous fungal infection caused by dimorphic fungi belonging to the Sporothrix species complex that affects the skin of limbs predominantly, but not exclusively. A rare case of ocular sporotrichosis in an immunocompetent Brazilian patient from the countryside of Rio de Janeiro State is reported. A 68-year-old woman presented with a subconjunctival infiltrative lesion in the right eye with pre-auricular lymphadenopathy of onset 4 months ago that evolved to suppurative nodular lesions on the eyelids. Conjunctival secretion was evaluated by histopathological examination and inoculated on Sabouraud Dextrose Agar (SDA). Histopathology showed oval bodies within giant cells and other mononucleated histiocytes. Fungus grown on SDA was identified as Sporothrix sp. by morphological observations. The isolated strain was finally identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) associated with an in-house database enriched with reference Sporothrix complex spectra. The strain presented a MALDI spectrum with the ion peaks of the molecular mass profile of S. brasiliensis. The patient was adequately treated with amphotericin B subsequently replaced by itraconazole. Due to scars left by the suppurative process, the patient presented poor final visual acuity. The present work presents an overview of ocular sporotrichosis and discusses the diagnostic difficulty that can lead to visual sequelae in these cases.Financial support for this work by Fundação de Amparo à Pesquisa do Rio de Janeiro [FAPERJ] (Grants: INST E-26/010.001784/2016; JCNE E-26/203.301/2017), by Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq] (Grant Proc. 409227/2016-1). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES). MALDI-TOF MS analyses were partially developed using equipment funded by CONICYT/Chile through the project Fondequip EQM160054 2016. The Universidad de La Frontera (Temuco, Chile) partially funded this work through the Project DIUFRO PIA19-0001. Furthermore, this study was also supported by FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio