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- PublicaciónAcceso abiertoGeneration of Cry11 Variants of Bacillus thuringiensis by Heuristic Computational Modeling(2020-07-27)Directed evolution methods mimic in vitro Darwinian evolution, inducing random mutations and selective pressure in genes to obtain proteins with enhanced characteristics. These techniques are developed using trial-and-error testing at an experimental level with a high degree of uncertainty. Therefore, in silico modeling of directed evolution is required to support experimental assays. Several in silico approaches have reproduced directed evolution, using statistical, thermodynamic, and kinetic models in an attempt to recreate experimental conditions. Likewise, optimization techniques using heuristic models have been used to understand and find the best scenarios of directed evolution. Our study uses an in silico model named HeurIstics DirecteD EvolutioN, which is based on a genetic algorithm designed to generate chimeric libraries from 2 parental genes, cry11Aa and cry11Ba, of Bacillus thuringiensis. These genes encode crystal-shaped δ-endotoxins with 3 conserved domains. Cry11 toxins are of biotechnological interest because they have shown to be effective as biopesticides for disease-spreading vectors. With our heuristic model, we considered experimental parameters such as DNA fragmentation length, number of generations or simulation cycles, and mutation rate, to get characteristics of Cry11 chimeric libraries such as percentage of population identity, truncation of variants obtained from the presence of internal stop codons, percentage of thermodynamic diversity, and stability of variants. Our study allowed us to focus on experimental conditions that may be useful for the design of in vitro and in silico experiments of directed evolution with Cry toxins of 3 conserved domains. Furthermore, we obtained in silico libraries of Cry11 variants, in which structural characteristics of wild Cry families were observed in a review of a sample of in silico sequences. We consider that future studies could use our in silico libraries and heuristic computational models, as the one suggested here, to support in vitro experiments of directed evolution.
- PublicaciónAcceso abiertoDeterminación de marcadores moleculares para la hepatitis B, mediante secuenciación profunda del genoma viral y la expresión de miARNs en muestras obtenidas de bancos de sangre en Colombia(Universidad de Antioquia, 2021-06-29)La hepatitis B (HB), enfermedad producida por el Virus de la hepatitis B (VHB), se ha establecido como un problema de salud pública mundial. Entender con mayor detalle la interacción entre el virus y el huésped mediante el análisis profundo del genoma del VHB y el análisis de la expresión de micro ARNs (miARNs) permitirá abordar la complejidad y diversidad de la infección, generando posibles marcadores moleculares importantes en la detección, definición de fenotipos clínicos o tratamiento de la enfermedad. Con el fin de abordar este objetivo, el análisis genómico mediante secuenciación profunda de las poblaciones virales identificadas en las muestras, así como la caracterización de los miARN presentes en individuos, soportados en estrategias de secuenciación de próxima generación, permitirá desarrollar por primera vez un estudio que evidencie la diversidad viral, las mutaciones sub representadas para los genotipos F, A y la respuesta generada a la infección viral.
- PublicaciónAcceso abiertoIdentification of potential natural neuroprotective molecules for Parkinson's disease by using chemoinformatics and molecular docking(2020-01-01)Parkinson’s disease is a progressive nervous system disorder characterized by motor, cognitive, sensory, psychiatric, and autonomic disturbances. While there is currently no cure for Parkinson’s Disease, medication can offer relief from its symptoms for many years. Although these medications are considered safe, they can present acute or chronic side effects and can become less effective over time. Thus, new medications are highly needed. In this regard, α-synuclein is a protein of great interest to Parkinson’s researchers because it is a major constituent of Lewy bodies, which are protein clumps being the pathological hallmark of Parkinson’s disease. However, current medications are not focused on the inhibition of α-synuclein oligomerization, and therefore, therapeutics preventing the formation of these bodies through the inhibition of α-synuclein oligomerization may play a role in the fight against this and other synucleinopathies. In this study, we used chemoinformatics tools and molecular docking simulations to analyze molecules that have been experimentally tested and bound to α-synuclein, causing neuroprotective or neurotoxic activity, and whose results have been used to select potential natural neuroprotective molecules. We identified 6 potential natural neuroprotective molecules that are similar in their chemical structure to neuroprotective molecules and have a high number of hydrogen bonds with α-synuclein. We expect that these molecules may lead to the design or discovery of new effective treatments for Parkinson’s disease.
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- PublicaciónAcceso abiertoTwo disulfide mutants in domain I of Bacillus thuringiensis Cry3Aa δ-endotoxin increase stability with no effect on toxicity(2012-05)To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteinefree Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indicates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10˚C to 14˚C compared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double mutations M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.