AGCDA. Artículos de Investigación
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- PublicaciónAcceso abiertoParticipation of valine 171 in α-helix 5 of Bacillus thuringiensis Cry1Ab δ-endotoxin in translocation of toxin into Lymantria dispar midgut membranes(2010-10-01) Alzate, Oscar; Osorio, Cristina; Florez, Alvaro M.; Dean, Donald H.The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab δ-endotoxin-susceptible insect.
- PublicaciónAcceso abiertoBacteria del Lago Mono propone paradigmas adicionales a la Biología Moderna(2011-03) Florez, Alvaro M.; Quijano, Jairo; Orduz, Sergio
- PublicaciónAcceso abiertoCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity(2011-10) Hussain, Syed-Rehan A.; Florez, Alvaro M.; Osorio, Cristina; Dean, Donald H.; Alzate, OscarThe centrally located a-helix 5 of Bacillus thuringiensis d-endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against Manduca sexta. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (Tm= 59 °C) was more stable than H168Q (Tm= 57.5 °C) or than the wt (Tm= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (Kcom) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (kobs), consistent with its lower LC50 value. Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.
- PublicaciónAcceso abiertoTwo disulfide mutants in domain I of Bacillus thuringiensis Cry3Aa δ-endotoxin increase stability with no effect on toxicity(2012-05) Wu, Sheng Jiun; Florez, Alvaro M.; Homoelle, Bradley J.; Dean, Donald H.; Alzate, OscarTo 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.
- PublicaciónAcceso abiertoEnzymatic hydrolysis of molecules associated with bacterial quorum sensing using an acyl homoserine lactonase from a novel Bacillus thuringiensis strain(2014-01) Pedroza, Carmen Julia; Florez, Alvaro M.; Ruiz, Orlando S.; Orduz, SergioN-acyl homoserine lactones are key components of quorum sensing, the bacterial communication system. This communication mechanism regulates the expression of genes, including those involved in virulence and biofilm formation. This system can be interrupted by the action of enzymes that hydrolyze the signaling molecules. In this work, we studied the enzymatic properties of a recombinant AHL-lactonase from Bacillus thuringiensis strain 147-11516, using substrates with acyl chains of different length (C4-HSL, C6-HSL, C7-HSL, C8-HSL and C10-HSL), we also investigated the effect of pH (5.0–9.0), temperature (20–70 °C), concentration of monovalent, divalent and trivalent metals ions (0.2 and 2.0 mM) and EDTA. The results showed that the recombinant AHL-lactonase had biological activity in alkaline pH conditions (8.0) and high temperature (47 % of hydrolyzed substrate at 60 °C). The recombinant AHL-lactonase has activity on substrates with different acyl chain length. However, the activity of the recombinant enzyme was decreased in the two concentrations of all metal ions evaluated but was not inhibited by EDTA. The affinity of the enzyme for all substrates tested and its performance, in the evaluated conditions, suggest that the AHL-lactonase from B. thuringiensis strain 147-11516 could be used as a strategy for disruption of the Gram-negative bacteria communication system under normal and challenging conditions.
- PublicaciónAcceso abiertoDNA secondary structure formation by DNA shuffling of the conserved domains of the Cry protein of Bacillus thuringiensis(2017-12) Pinzón Reyes, Efraín-Hernando; Sierra, Daniel A.; Suárez Barrera, Miguel Orlando; Orduz, Sergio; Florez, Alvaro M.Background The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy (ΔG¯¯¯¯¯¯¯¯) were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G. Results The results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA. Conclusion These results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.
- PublicaciónAcceso abiertoEl helado elaborado de Musa paradisiaca y probióticos mejora los niveles séricos de calcio, hierro y potasio en una población infantil(2018) López Torres, Jesmar A.; Maldonado Sánchez, Geomar J.; Martínez Marciales, Karen-Piedad; Salas Osorio, Elaysa J.; Varela Rangel, Yasmin Y.Ice cream prepared with Musa paradisiaca and probiotic improves the serum levels of calcium, iron and potassium in a children population. Malnutrition affects society, mainly children with limited resources, with low levels of minerals being the direct consequence. An explanatory research was developed, with experimental design to evaluate the effectiveness of the consumption of an ice cream of Musa paradisiacasupplemented with probiotics in a child population of Cúcuta-Colombia. From 238 schoolchildren, a sample of 33 children aged 4 to 6 years with serum deficits of Ca, Fe and K participated and they were randomly divided into two groups. For 60 days the ice cream was supplied to Group A and Group B a placebo ice cream, registering weight, height, serum levels of Ca, Fe and K before, during and after the ice cream administration. There was a significant increase (p = 0.01) in the iron and calcium levels in Group A; with no significant changes for Group B. In both groups potassium was significantly increased (p <0.05). The body weight and height of the children was increased although there were no differences (p>0.05) between both groups. The consumption of banana-based ice cream supplemented with probiotics represents an alternative to address the deficit of minerals in children.
- PublicaciónAcceso abiertoHuman predecidual stromal cells have distinctive characteristics of pericytes : Cell contractility, chemotactic activity, and expression of pericyte markers and angiogenic factors(2018-01) Muñoz Fernández, Raquel; De la Mata, Claudia; Prados, Alejandro; Perea, Ana; Ruiz Magaña, María José; Llorca, Tatiana; Fernández Rubio, Pablo; Blanco Muñoz, Osmany; Abadía Molina, Ana C.; Olivares, Enrique G.Introduction Human decidual stromal cells (DSCs) play a key role in maternal–fetal interactions. Precursors of DSCs (preDSCs) localize around vessels in both the endometrium and decidua. Previous studies suggested a relationship between preDSCs and pericytes because these cells share a perivascular location, alpha smooth muscle actin (α-SM actin) expression and the ability to contract under the effects of cytokines. Methods To further study this relationship, we established 15 human preDSC lines and 3 preDSC clones. The preDSC lines and clones were tested by flow cytometry with a panel of 29 monoclonal antibodies, 14 of which are pericyte markers. The expression of angiogenic factors was determined by RT-PCR, chemotactic activity was studied with the migration assay, and cell contractility was evaluated with the collagen cell contraction assay. Confocal microscopy was used to study decidual sections. Results Under the effect of progesterone and cAMP, these lines decidualized in vitro: the cells became rounder and secreted prolactin, a marker of physiological DSC differentiation (decidualization). The antigen phenotype of these preDSC lines and clones was fully compatible with that reported for pericytes. PreDSC lines displayed pericyte characteristics: they expressed angiogenic factors and showed chemotactic and cytokine-induced contractile activity. Confocal microscopic examination of decidual sections revealed the expression of antigens detected in preDSC lines: α-SM actin colocalized with CD146, CD140b, MFG-E8, nestin, and STRO-1 (all of which are pericyte markers) in cells located around the vessels, a distinctive location of preDSCs and pericytes. Discussion Taken together, our results show that preDSCs are pericyte-like cells.
- PublicaciónAcceso abiertoSoftepigen : Primers design web-based tool for MS-HRM technique(2018-08-29) Pinzón, Efraín; Rondón Villarreal, Paola; Álvarez, William A.; Hernández, Hernan GuillermoPolymerase Chain Reaction (PCR) based techniques for DNA methylation techniques includes MS-HRM technique. Methylation Sensitive High-Resolution Melting (MS-HRM) primer-design requires a set of necessary recommendations for such DNA methylation assessment. However, there were not any available software that allows an automatic design of this kind primers. We present Softepigen, the first complete MS-HRM primer design software. Softepigen allows to search for primers in a genomic region following Wojdacz's recommendations and targets primer binding regions with high linguistic complexity sequences that increase the specificity of the converted sequence of the human genome. We performed in-silico PCR analysis through BiSearch ePCR tool to validate the specificity of the of the primers designed using Softepigen. Softepigen for MS-HRM performance in our genomic regions of interest show satisfactory specificity measurements, and we implemented it for freely available use in web-based interface in www.soft-epigen.com.
- PublicaciónAcceso abiertoToxic activity, molecular modeling and docking simulations of Bacillus thuringiensis Cry11 toxin variants obtained via DNA shuffling(2018-10-17) Suárez Barrera, Miguel Orlando; Florez, Alvaro M.; Morales, Gloria M.; Rivera, Karen Viviana; Orduz, Sergio; Ochoa, Rodrigo; Guerra, Diego; Muskus, CarlosThe Cry11 family belongs to a large group of d-endotoxins that share three distinct structural domains. Among the dipteran-active toxins referred to as three-domain Cry11 toxins, the Cry11Aa protein from Bacillus thuringiensis subsp. israelensis (Bti) has been the most extensively studied. Despite the potential of Bti as an effective biological control agent, the understanding of Cry11 toxins remains incomplete. In this study, five Cry11 variants obtained via DNA shuffling displayed toxic activity against Aedes aegypti and Culex quinquefasciatus. Three of these Cry11 variants (8, 23, and 79) were characterized via 3D modeling and analysis of docking with ALP1. The relevant mutations in these variants, such as deletions, insertions and point mutations, are discussed in relation to their structural domains, toxic activities and toxin-receptor interactions. Importantly, deletion of the N-terminal segment in domain I was not associated with any change in toxic activity, and domain III exhibited higher sequence variability than domains I and II. Variant 8 exhibited up to 3.78- and 6.09-fold higher toxicity to A. aegypti than Cry11Bb and Cry11Aa, respectively. Importantly, variant 79 showed an a-helix conformation at the C-terminus and formed crystals retaining toxic activity. These findings indicate that five Cry11 variants were preferentially reassembled from the cry11Aa gene during DNA shuffling. The mutations described in loop 2 and loop 3 of domain II provide valuable information regarding the activity of Cry11 toxins against A. aegypti and C. quinquefasciatus larvae and reveal new insights into the application of directed evolution strategies to study the genetic variability of specific domains in cry11 family genes.
- PublicaciónAcceso abiertoIdentification of potential natural neuroprotective molecules for Parkinson's disease by using chemoinformatics and molecular docking(2020-01-01) Rondón Villarreal, Paola; Contreras López, William-OmarParkinson’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.
- PublicaciónAcceso abiertoGeneration of Cry11 Variants of Bacillus thuringiensis by Heuristic Computational Modeling(2020-07-27) Pinzón-Reyes, Efraín Hernando; Sierra-Bueno, Daniel Alfonso; Suarez-Barrera, Miguel Orlando; Rueda-Forero, Nohora Juliana; BiomolDirected 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) Rueda-Forero, Nora Juliana; Bedoya, Astrid; Goyeneche-Patiño, Diego A.; BiomolLa 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.