Gene expression and morphological responses of Lolium perenne L. exposed to cadmium (Cd2+) and mercury (Hg2+).

Soil contamination with heavy metals is a major problem worldwide, due to the increasing impact mainly caused by anthropogenic activities. This research evaluated the phytoremediation capacity of, Lolium perenne for heavy metals such as cadmium (Cd2+) and mercury (Hg2+), and the effects of these metals on morphology, biomass production, and the changes on gene expression. Seeds of L. perenne were exposed to six concentrations of Cd2+ and Hg2+ in the range of 0 to 25 mg L-1, and two mixtures of Cd2+-Hg2. The Non-Observed Effect Level (NOEL) was established with dose response curves and the expression of specific genes was evaluated applying a commercially available quantitative reverse transcription (RT-qPCR) assay. There was no significant effect when exposing the seeds to Hg2+, for Cd2+ the maximum concentration was established in 0.1 mg L-1, and for the two concentrations of mixtures, there was a negative effect. An increase of expression of genes that regulate antioxidant activity and stress was found when the plant was exposed to heavy metals. Given the high tolerance to metals analyzed that was reflected both, the development of the plant and in its molecular response, these results highlight that L. perenne is a plant with phytoremediator potential.

Helicobacter pylori culture as a key tool for diagnosis in Colombia.

INTRODUCTION: The presence of H. pylori in the stomach is associated with gastric pathologies. However, its diagnosis through culture methods is challenging because of its complex nutritional requirements and microaerophilic conditions for optimal growth. The preferred method for rapid diagnosis of H. pylori is the Rapid Urease Test (RUT) from human biopsies, which relies on the high activity of the urease enzyme present in H. pylori. However, RUT cannot say much more information about H. pylori. This makes evident the need for bacterial culture to know essential information such as the strain type, the kind of infection present and the bacteria's antibiotic susceptibility. METHODOLOGY: Gastric biopsies from 347 patients were used for H. pylori isolation. We correlated the culture results with the RUT and histological grading used at Hospital Universitario Fundación SantaFe de Bogotá (HU-FSFB), Colombia. The concordance between techniques was determined by the Cohen's Kappa coefficient (K). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were also calculated. RESULTS: The culture standardization was successful, and it could be applied for diagnosis in the clinical practice. H. pylori was positive by culture in 88 (26.34%) patients. The concordance of RUT and culture was strong (K= 0.805), and between histology and culture was moderate (K= 0.763) as well as for the gold standard defined and culture (K= 0.80). CONCLUSIONS: We present evidence that RUT and histological methods will be better interpreted for diagnosis of H. pylori if combined with bacterial isolation in cholesterol enriched culture.

Structural Analysis of Variability and Interaction of the N-terminal of the Oncogenic Effector CagA of Helicobacter pylori with Phosphatidylserine.

Helicobacter pylori cytotoxin-associated gene A protein (CagA) has been associated with the increase in virulence and risk of cancer. It has been demonstrated that CagA's translocation is dependent on its interaction with phosphatidylserine. We evaluated the variability of the N-terminal CagA in 127 sequences reported in NCBI, by referring to molecular interaction forces with the phosphatidylserine and the docking of three mutations chosen from variations in specific positions. The major sites of conservation of the residues involved in CagA⁻Phosphatidylserine interaction were 617, 621 and 626 which had no amino acid variation. Position 636 had the lowest conservation score; mutations in this position were evaluated to observe the differences in intermolecular forces for the CagA⁻Phosphatidylserine complex. We evaluated the docking of three mutations: K636A, K636R and K636N. The crystal and mutation models presented a ΔG of -8.919907, -8.665261, -8.701923, -8.515097 Kcal/mol, respectively, while mutations K636A, K636R, K636N and the crystal structure presented 0, 3, 4 and 1 H-bonds, respectively. Likewise, the bulk effect of the ΔG and amount of H-bonds was estimated in all of the docking models. The type of mutation affected both the ΔG ( χ 2 ( 1 ) = 93.82 , p-value < 2.2 × 10 - 16 ) and the H-bonds ( χ 2 ( 1 ) = 91.93 , p-value < 2.2 × 10 - 16 ). Overall, 76.9% of the strains that exhibit the K636N mutation produced a severe pathology. The average H-bond count diminished when comparing the mutations with the crystal structure of all the docking models, which means that other molecular forces are involved in the CagA⁻Phosphatidylserine complex interaction.

Genotipificación de cagA y de la región intermedia de vacA en cepas de Helicobacter pylori aisladas de pacientes adultos colombianos y su asociación con enfermedades gástricas / Genotyping of cagA and the intermediate region of vacA in strains of Helicobacter pylori isolated from Colombian adult patients and associations with gastric diseases

Resumen Objetivo: este estudio caracteriza la diversidad de los genes de virulencia cagA (gen asociado con la citotoxina A) y vacA (citotoxina vacuolizante) en pacientes colombianos para determinar posibles asociaciones entre estos 2 genes y la severidad de los hallazgos endoscópicos teniendo en cuenta todos los genotipos reportados para el gen vacA (s, m e i). Materiales y métodos: Helicobacter pylori fue detectado por cultivo y por métodos moleculares en biopsias de 62 pacientes. Los genotipos de cagA y vacA (m/i/s) se determinaron por reacción en cadena de la polimerasa (PCR) y secuenciación. Resultados: se aislaron 124 cepas de 62 pacientes; de estas, el 48,5% (n = 48) fueron vacA s2/m2/i2-cagA (-) presente en su mayoría en pacientes con gastritis folicular; mientras el 32,3% (n = 32) fueron vacA s1/m1/i1-cagA (+) presentes mayormente en pacientes con gastritis folicular, gastritis crónica y posible metaplasia. Se encontró una asociación significativa entre la presencia de cagA y el genotipo vacA s1/m1/i1 y la ausencia de cagA y el genotipo vacA s2/m2/i2 (p <0,001). No se encontró una asociación significativa entre la severidad de los hallazgos endoscópicos y el estatus cagA-vacA de las cepas. Conclusión: se encontró una baja prevalencia de cepas cagA (+), el estatus cagA-vacA no es un predictor de riesgo en la población estudiada y la presencia de infecciones heterogéneas sin tropismo sugieren la necesidad de tomar biopsias tanto del cuerpo como del antro del estómago en la práctica clínica rutinaria.

Abstract Objective: This study characterizes the diversity of cagA and vacA virulence genes in Colombian patients to determine possible associations between them and the severity of endoscopic findings. It considers all four genotypes reported for the vacA gene (s, m and i). Materials and methods: Helicobacter pylori was detected in biopsies of 62 patients through culturing and by molecular methods. Genotypes of cagA and vacA (m/i/s) were determined by PCR and sequencing. Results: One hundred twenty four strains from 62 patients were isolated. Of these, 48.5% (n = 48) were vacA s2/m2/i2 - cagA (-) which were mostly found in patients with follicular gastritis; 32.3% (n = 32) were vacA s1/m1/i1-cagA (+) which were mostly found in patients with follicular gastritis, chronic gastritis and possible metaplasia. Significant associations were found between the presence of cagA and the vacA s1/m1/i1 genotype and the absence of cagA and the vacA s2/m2/i2 genotype (p <0.001). No significant association was found between the severity of endoscopic findings and the cagA-vacA status of the strains. Conclusion: We found a low prevalence of cagA (+) strains, the cagA-vacA status is not a predictor of risk in this population. Moreover, the presence of heterogenous infections without tropism suggests a need for biopsies from both the corpus and the antrum of the stomach in routine clinical practice.

Adherencia a la guía de manejo de sepsis severa y choque séptico en pacientes mayores de 65 años que ingresan a UCI / Adherence to the guidelines for the management of severe sepsis and septic shock in patients over 65 years of age admitted to the ICU

Introduction: The use of guidelines for goal-oriented resuscitation in patients with severe sepsis and septic shock has a positive impact on multiple organ failure and mortality outcomes. However, in patients over 65, adherence to the guidelines may be less stringent because of considerations of lower functional, cardiac, pulmonary and renal reserve. This study compares adherence to the guidelines and compliance with resuscitation goals by the healthcare staff in populations over and under 65 years of age. Objective: To determine adherence to the guidelines by the healthcare staff in the treatment of severe sepsis and septic shock in the group over 65 years of age, compared with patients under 65. Materials and methods: Observational, analytical, cross-sectional study of patients under and over 65 years of age admitted to the Intensive Care Unit (ICU) after having been diagnosed with severe sepsis and septic shock. Frequency of adherence to the guidelines was determined and a comparison of the different resuscitation goals in relation to adherence to the guidelines was made. Results: Except for adherence to early initiation of antibiotic therapy, there was better adherence to the other goals in the patients under 65 when compared to patients over 65. Adherence to the guidelines declined progressively as the number of goals increased. Conclusions: There was a lower adherence to resuscitation guidelines in the group of patients over 65 with severe sepsis and septic shock when compared with the group of patients under 65.

Introducción: El uso de guías de reanimación por metas en los pacientes con sepsis severa y choque séptico, genera un impacto benéfico en los desenlaces de disfunción orgánica múltiple y de la mortalidad, sin embargo en los pacientes mayores de 65 años, la adherencia a las guías puede ser menor, por la menor reserva funcional, cardíaca, pulmonar y renal. En el presente estudio comparamos la adherencia a la guía y el cumplimiento de las metas de reanimación por parte del personal de salud en la población mayor y menor de 65 años. Objetivo: Determinar la adherencia a las guías para el tratamiento de la sepsis severa y choque séptico por parte del personal de salud en el grupo mayor de 65 años comparado con el grupo de pacientes menores de 65 años.. Materiales y métodos: Estudio observacional analítico de corte transversal de los pacientes menores y mayores de 65 años ingresados a la Unidad de Cuidados Intensivos (UCI), con diagnóstico de sepsis severa y choque séptico, se determinó la frecuencia de adherencia a la guía para cada grupo de edades y se compararon las diferentes metas de reanimación en relación a la adherencia. Resultados: Excepto por la adherencia del inicio temprano de la antibiótico terapia, las demás metas presentaron una mejor adherencia, en el grupo de pacientes menores de 65 años cuando se compararon con los pacientes mayores de 65 años, la adherencia para ambos grupos disminuyo progresivamente a medida que se consideraron mayor número de metas. Conclusiones: Existe una menor adherencia a las guías de reanimación, en el grupo de grupo de pacientes mayores de 65 años con sepsis severa y choque séptico cuando se compara con el grupo de pacientes menores de 65 años.

Virus and Mycoplasma pneumoniae prevalence in a selected pediatric population with acute asthma exacerbation.

OBJECTIVE: To determine the prevalence of viral and atypical bacteria Mycoplasma pneumoniae infection in children experiencing asthma exacerbation and compare positive and negative subjects with regard to exacerbation severity, need for hospitalization, and treatment. METHODS: One hundred sixty-nine asthmatic children aged 2-15 years old who were admitted to emergency rooms in Bogota, Colombia for acute asthma exacerbation were interviewed. Nasopharyngeal aspirates were taken for DNA and RNA extraction. M. pneumoniae and virus were detected by PCR using specific primers. RESULTS: The prevalence of M. pneumoniae and viral infection in the study population was 12.4% and 83.7%, respectively. All subjects positive for M. pneumoniae were also positive for viral infection. Rhinovirus was the most frequently detected viral agent. No significant differences in severity of asthma exacerbations or in need for hospitalization between the virus or M. pneumoniae positive and negative groups were observed. A significantly lower percentage of M. pneumoniae positive subjects had used inhaled steroids over the six months prior to asthma exacerbation compared to M. pneumoniae negative subjects (38.1% vs. 68.2%), suggesting that inhaled corticosteroids may have a protective effect against M. pneumoniae infections. CONCLUSIONS: The M. pneumoniae and virus prevalence found in this study were similar to those described in the literature. The 100% co-infection rate observed suggests that viral infection can predispose patients to M. pneumoniae infection, and that this interaction may trigger asthmatic exacerbation. Further studies should be done to confirm the protective effect of inhaled corticosteroids on M. pneumoniae infection in patients with asthma exacerbations.

[Genetic characterization of the interleukin 1 b polymorphisms -511, -31 y +3954 in a Colombian population with dyspepsia]. / Genotipificación de los polimorfismos -511, -31 y +3954 del gen de la interleucina-1 ß humana en una población colombiana con cuadro de dispepsia.

INTRODUCTION: The human interleukin-1b gen (IL-1 b) polymorphisms such as -511, -31 and +3954 have been associated with the presence of gastric cancer, due to the inhibitor effect that this protein has on acid secretion in the stomach. This facility can enhance the colonization and infection by agents like Helicobactor. pylori and the genesis of preneoplastic states that can lead to cancer development. OBJECTIVE: Three polymorphisms of IL-1ß (+3954, -511 and -31) will be genetically characterized and their frequencies established in a population of patients with gastric symptoms. MATERIALS AND METHODS: Gastric antrum biopsies were obtained from 111 patients that showed signs of gastric disorder. A PCR was done to detect the H. pylori presence; a PCR using designed primers for specific regions was done to define the three polymorphic regions of IL-1ß, and a RFLP was carried out using Aval, Alul and TaqI for the position -511, -231 and +3954 for each case. RESULTS: Helicobacter pylori was detected in 59.5% of the evaluated gastric while the histopathology study revealed that 82.9% of patients had some pathology. Characterization of polymorphic regions of IL-1ß gen were joined to RFLP typing evidenced that all described genotypes were present in the study population. However, patients with benign pathologies infected with H. pylori had a high frequency of the CC genotype (28.6%) in the -31 polymorphic regions. CONCLUSION: No significant differences were found between the genotype frequencies of the H. pylori-infected and the non-infected populations with one exception. The CC genotype in the -31 polymorphic region was associated with benign pathologies.

Genotificación de los polimorfismos -511, -31 y +3954 del gen de la interleucina 1 beta humana en una población colombiana con cuadro de dispepsia / Genetic characterization of the interleukin 1 beta polymorphisms -511, -31 y +3954 in a Colombian population with dyspepsia

Introducción. Varios estudios sugieren que algunos polimorfismos del gen de la interleucina-1beta humana (IL-1beta), como -511, -31 y +3954, están asociados al cáncer gástrico, debido al efecto inhibidor que esta citocina tiene sobre la secreción ácida del estómago, lo cual facilita la colonización e infección por agentes como Helicobacter pylori, así como la génesis de estados preneoplásicos que pueden conducir al desarrollo de cáncer. Objetivo. Genotipificar los polimorfismos +3954,-511 y -31 de la IL-1beta y establecer sus frecuencias en una población de pacientes con diferente sintomatología gástrica. Materiales y métodos. Se analizaron 111 biopsias del antro gástrico obtenidas de pacientes con sintomatología de alguna alteración gástrica. La detección de H. pylori en las muestras se realizó mediante PCR empleando iniciadores específicos para cada región y la genotipificación de las regiones polimórficas de la IL-1beta se realizó por RFLP empleando las enzimas Aval, Alul y Taql para -511, -31 y +3954, respectivamente. Resultados. Se detectó H. pylori en 59,5% de las biopsias gástricas, mientras que el estudio histopatológico reveló que 82,9% de los pacientes padecía alguna enfermedad. La caracterización de las regiones polimórficas del gen de la IL-1beta, seguida de la tipificación por RFLP, permitió evidenciar los tres posibles genotipos de cada uno de los polimorfismos en la población. En los pacientes infectados por H. pylori se encontró con mayor frecuencia (28,6%) el genotipo CC en la región polimórfica -31. Conclusión. No se encontraron diferencias significativas en los genotipos de los individuos infectados y los no infectados por H. pylori, a excepción del genotipo CC en la región polimórfica -31, el cual se encontró con mayor frecuencia en los pacientes con enfermedades benignas.

Introduction. The human interleukin-1beta gen (IL-1 beta) polymorphisms such as -511, -31 and +3954 have been associated with the presence of gastric cancer, due to the inhibitor effect that this protein has on acid secretion in the stomach. Thisfacility can enhance the colonization and infection by agents like Helicobactor. pylori and the genesis of preneoplastic states that can lead to cancer development. Objective. Three polymorphisms of IL-1beta (+3954, -511 and -31) will be genetically characterized and their frequencies established in a population of patients with gastric symptoms. Materials and methods. Gastric antrum biopsies were obtained from 111 patients that showed signs of gastric disorder. A PCR was done to detect the H. pylori presence; a PCR using designed primers for specific regions was done to define the three polymorphic regions of IL-1beta, and a RFLP was carried out using Aval, Alul and TaqI for the position -511, -231 and +3954 for each case. Results. Helicobacter pylori was detected in 59.5% of the evaluated gastric while the histopathology study revealed that 82.9% of patients had some pathology. Characterization of polymorphic regions of IL-1beta gen were joined to RFLP typing evidenced that all descfribed genotypes were present in the study population. However, patients with benign pathologies infected with H. pylori had a high frequency of the CC genotype (28.6%) in the -31 polymorphic regions.Conclusion. No significant differences were found between the genotype frequenciess of the H. pylori-infected and the non-infected populations with one exception. The CC genotype in the -31 polymorphic region was associated with benign pathologies.

Caracterización molecular y bioinformática de la proteína CagA de Helicobacter pylori a partir de biopsias gástricas de pacientes colombianos / Molecular and bioinformatic characterization of Helicobacter pylori CagA protein using gastric biopsies of Colombian patients

El gen cagA de Helicobacter pylori codifica para la proteína CagA considerada uno de los factores de virulencia cuya presencia se asocia a un mayor riesgo de padecer enfermedades gástricas severas. El presente estudio planteó como objetivo el diseño de una estrategia molecular y bioinformática útil en la determinación de la presencia de secuencias repetitivas que pueden contener uno o más motivos de fosforilación (EPIYA). Se amplificó y secuenció la región variable de cagA en muestras H. pylori CagA positivas. Se realizó una búsqueda y selección de herramientas bioinformáticas que permitieran establecer las características de los motivos EPIYA. La presencia de motivos tipo EPIYA-A y EPIYA-B, seguido por una a dos repeticiones de EPIYA-C, similares a los reportados para países de Occidente, fueron encontrados. De las aplicaciones bioinformáticas evaluadas, solo un conjunto de herramientas demostró ser útil en la caracterización de las unidades de repetición en la proteína CagA.

Helicobacter pylori CagA protein, the cagA gen product, has been considered as a virulence factor associated with a considerable increase risk for develops severe gastric illness. The purpose of this research was to design a molecular and bioinformatics strategy that allowed the establishment of phosphorylation status of the tyrosine residue of the CagA protein. The amplification and sequencing of the variable fragment region of cagA in the positive CagA samples were used to do the bioinformatics analysis in order to establish the characteristics of the EPIYA motifs. The presence of the EPIYA-A and EPIYA-B motifs, followed by one or two EPIYA-C repetitions, similar to those reported previously for occidental countries were set up. From the different bioinformatics applications that were employed only one group of tools proved to be useful to characterize the repeated units presents in the CagA protein.

Deteccion de Helicobacter pylori y caracterizacion del la region -31 del gen de la Interleucina 1-ß humana en pacientes de una poblacion colombiana con enfermedades gastroduodenales / Helicobacter pylori detection and human Interleukin 1-ß genotyping in Colombian patients affected by gastroduodenal diseases

El prop¨®sito de este estudio fue investigar la presencia de Helicobacter pylori, realizar la caracterizaci¨®n del polimorfismo -31 del gen de la interleucina 1-¦Â humana y establecer si existe asociaci¨®n entre la presentaci¨®n de alguno de los genotipos y la severidad de las manifestaciones cl¨ªnicas en una poblaci¨®n colombiana. Se analizaron biopsias g¨¢stricas provenientes de 71 pacientes, mediante histopatolog¨ªa y m¨¦todos moleculares (PCR y PCR-RFLP). Los resultados mostraron que el 97,2% de los pacientes presentaba alteraciones de la mucosa g¨¢strica. La presencia de H. pylori se demostr¨® en 44 pacientes y la frecuencia de los genotipos de IL-1B: TT, CT y CC fue de 12,7%, 60,6% y 26,8% respectivamente. No se encontr¨® asociaci¨®n entre los genotipos de la regi¨®n polim¨®rfica -31 del gen IL-1B y el grado de alteraci¨®n de la mucosa g¨¢strica en la poblaci¨®n estudiada.

Detección de Mycoplasma pneumoniae mediante PCR-hibridación in vitro en niños con infección respiratoria / Mycoplasma pneumoniae detection using PCR-in vitro hybridization in children with respiratory infection

Antecedentes: Las infecciones respiratorias agudas (IRA) son una de las causas más frecuentes de enfermedades en el mundo y pueden asociarse a complicaciones graves, entre las que la neumonía ocupa un lugar prioritario; diversos agentes virales y bacterianos se implican en su desarrollo. Recientemente Mycoplasma pneumoniae ha adquirido gran importancia ya que estudios epidemiológicos sugieren un aumento en la incidencia de enfermedades respiratorias causadas por este patógeno. Sin embargo, los laboratorios clínicos enfrentan varias dificultades para su detección haciendo necesario el estudio de nuevas técnicas moleculares. Objetivo: Detección de M. pneumoniae mediante PCR-hibridación in vitro en niños con infección respiratoria. Métodos: Utilizando la técnica de PCR-hibridación in vitro se analizaron 36 hisopados orofaríngeos de niños entre los 0 y 9 años de edad con infección respiratoria; 36% presentaron neumonía y el 30% bronconeumonía. Resultados: La implementacion y utilización de la técnica de PCR-hibridación in vitro para la detección de M. pneumoniae permitió detectar su ADN en el 67% de los pacientes estudiados. De éstos, el 33% tenía diagnóstico de neumonía. Conclusión: La técnica de PCR-hibridación in vitro demostró ser útil en la detección de M. pneumoniae en las muestras analizadas. Los resultados de este estudio evidencian que la presencia de ácidos nucleicos de este patógeno es frecuente en las muestras respiratorias analizadas y sugiere que el microorganismo puede ser un agente etiológico frecuente de neumonías atípicas y de otras patologías respiratorias.

Background: Acute respiratory infection is considered one of the main causes of morbidity worldwide; it can be associated to serious complications. A great number of viral and bacterial agents have been implicated in the development of the disease. Recently, importance has been given to Mycoplasma pneumoniae as a respiratory pathogen, since epidemiologic studies suggest a raise in the incidence of diseases due to this microorganism. Nevertheless, nowadays clinical laboratories deal with a variety of difficulties in the diagnosis of this agent, raising the need for other molecular methods for its detection. Objective: The detection of M. pneumoniae in children with respiratory infection by a PCR-in vitro hybridization technique. Methods: 36 oropharyngeal swabs from children between 0 and 9 years of age with respiratory infection were analyzed by PCR-in vitro hybridization; 36% had pneumonia and 30% bronchopneumonia. Results: The implementation of the PCR-in vitro hybridization for the detection of M. pneumoniae in patients with respiratory infection allowed the detection of the pathogen's DNA in 67% of the patients studied. Of these, 33% had pneumonia. Conclusion: PCR-in vitro hybridization is useful for the detection of M. pneumoniae. Our results show the frequent presence of nucleic acids of M. pneumoniae in the samples studied and suggest this microorganism can be a frequent causal agent of respiratory infection.

Niveles séricos de zinc, hierro y cobre en tres grupos de escolares colombianos: relación con algunos parámetros antropométricos

La deficiencia de micronutrientes es frecuente en países en desarrollo. El objetivo del estudio fué comparar los niveles séricos de zinc, hierro y cobre en tres grupos de escolares (8 a 9 años) y correlacionarlos con algunas medidas antropométricas. El grupo 1 estuvo constituido por 26 escolares de un colegio privado de Santafé de Bogotá, el 2 por 21 niños de instituciones públicas de una zona rural, cercana a esta ciudad y el 3 por 28 sujetos de una escuela pública de la capital. Los micronutrientes se midieron por espectrometria de emisión atómica con plasma acoplado por inducción. El grupo 1 presentó talla (126.2+/-3.6) mayor al 2 (122.6+/-4.8 cm) y al 3 (122.1 +/-5.0 cm). Las concentraciones de zinc del grupo 1 (67+/-9 ug/dl) fueron mayores al 2 (59+/-11 ug/dl) y al 3 (50+/-13ug/dl). El grupo 3 presentó sidermia menor (71+/-21ug/dl) al 1 (90+/-30ug/dl). El grupo 1 evidenció niveles de cobre (100+/-19Ug/dl) mayores al 2 (77+/-13ug/dl) y al 3 (82+/-20ug/dl). Los resultados suguieren que los niños de los grupos 2 y 3 están expuestos a posibles condicienes medioambientales que inciden desfavorablemente en su crecimiento corporal y que son los niños del grupo 2 los que presntan el menor acceso a los micronutrientes zinc y hierro.