Andrés Julián Gutiérrez-Escobar

Toxoplasma gondii: Molecular cloning and characterization of a nitric oxide synthase-like protein

Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of l-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21 nmol/min/ml nitrites per microgram of protein and followed Michaelis-Menten kinetics, with a K(m) for L-arginine of 2.3mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from L-arginine as substrate.

Phylogenomics of Colombian Helicobacter pylori isolates

BackgroundDuring the Spanish colonisation of South America, African slaves and Europeans arrived in the continent with their corresponding load of pathogens, including Helicobacter pylori. Colombian strains have been clustered with the hpEurope population and with the hspWestAfrica subpopulation in multilocus sequence typing (MLST) studies. However, ancestry studies have revealed the presence of population components specific to H. pylori in Colombia. The aim of this study was to perform a thorough phylogenomic analysis to describe the evolution of the Colombian urban H. pylori isolates.ResultsA total of 115 genomes of H. pylori were sequenced with Illumina technology from H. pylori isolates obtained in Colombia in a region of high risk for gastric cancer. The genomes were assembled, annotated and underwent phylogenomic analysis with 36 reference strains. Additionally, population differentiation analyses were performed for two bacterial genes. The phylogenetic tree revealed clustering of the Colombian strains with hspWestAfrica and hpEurope, along with three clades formed exclusively by Colombian strains, suggesting the presence of independent evolutionary lines for Colombia. Additionally, the nucleotide diversity of horB and vacA genes from Colombian isolates was lower than in the reference strains and showed a significant genetic differentiation supporting the hypothesis of independent clades with recent evolution.ConclusionsThe presence of specific lineages suggest the existence of an hspColombia subtype that emerged from a small and relatively isolated ancestral population that accompanied crossbreeding of human population in Colombia.

Eficacia del Método de Inmunocromatografía en Heces para el Diagnóstico de Helicobacter Pylori en Pacientes con Dispepsia: Evaluación Preliminar

Background: Helicobacter pylori infection is considered one of the most important emerging diseases of this century, which is closely related with gastroduodenal tract diseases and as a predisposing factor for gastric carcinoma. For diagnosis purpose, it have been used invasive and noninvasive tests, among the latter the fecal immunochromatography, which qualitatively detect pathogen’s antigens. Objective: The aim of this study was to determine the effectiveness of this method for diagnosis of infection with H. pylori in dyspeptic patients from a hospital in Cundinamarca. Methods: We conducted a preliminary study to evaluate a diagnostic test, for which we used patients with dyspepsia history and gastric biopsy report in their medical record. Stool samples were evaluated and through the rapid test SD Bio Line H. pylori Ag™, we determined the presence of fecal H. pylori antigens. Results: We evaluated a total of 33 patients: 48.4% men and 51.5% women, the sensitivity was 30% and specificity of 84.62%. The positive predictive value calculated was 75% and the negative predictive value was 44%. Conclusion: The specificity calculated for the test is comparable to that found in the literature; however, due to the reliability index’s interval showed, it would take more evidence to increase the reliability of the results shown here.

Rapid evolution of the Helicobacter pylori AlpA adhesin in a high gastric cancer risk region from Colombia

To be able to survive, Helicobacter pylori must adhere to the gastric epithelial cells of its human host. For this purpose, the bacterium employs an array of adhesins, for example, AlpA. The adhesin AlpA has been proposed as a major adhesin because of its critical role in human stomach colonization. Therefore, understanding how AlpA evolved could be important for the development of new diagnostic strategies. However, the genetic variation and microevolutionary patterns of alpA have not been described in Colombia. The study aim was to describe the variation patterns and microevolutionary process of alpA in Colombian clinical isolates of H. pylori. The existing polymorphisms, which are deviations from the neutral model of molecular evolution, and the genetic differentiation of the alpA gene from Colombian clinical isolates of H. pylori were determined. The analysis shows that gene conversion and purifying selection have shaped the evolution of three different variants of alpA in Colombia.

Interactome Analysis of Microtubule-targeting Agents Reveals Cytotoxicity Bases in Normal Cells

Cancer causes millions of deaths annually and microtubule-targeting agents (MTAs) are the most commonly-used anti-cancer drugs. However, the high toxicity of MTAs on normal cells raises great concern. Due to the non-selectivity of MTA targets, we analyzed the interaction network in a non-cancerous human cell. Subnetworks of fourteen MTAs were reconstructed and the merged network was compared against a randomized network to evaluate the functional richness. We found that 71.4% of the MTA interactome nodes are shared, which affects cellular processes such as apoptosis, cell differentiation, cell cycle control, stress response, and regulation of energy metabolism. Additionally, possible secondary targets were identified as client proteins of interphase microtubules. MTAs affect apoptosis signaling pathways by interacting with client proteins of interphase microtubules, suggesting that their primary targets are non-tumor cells. The paclitaxel and doxorubicin networks share essential topological axes, suggesting synergistic effects. This may explain the exacerbated toxicity observed when paclitaxel and doxorubicin are used in combination for cancer treatment.

Draft Genome Sequence of a Helicobacter pylori Strain Isolated from a Patient with Diffuse Gastritis from a Region of High Cancer Risk in Colombia

ABSTRACT The draft genome sequence of one Colombian Helicobacter pylori strain is presented. This strain was isolated from a patient with diffuse gastritis from Tibaná, Boyacá, a region with high gastric cancer risk.