Comprehensive characterization of Mycobacterium tuberculosis strains after acquisition of isoniazid resistance

Includes bibliographical references.2016 Fall.Despite the global efforts to reduce tuberculosis (TB) rates, the emergence of drug resistant TB has not allowed effective control of this disease. In the last decade, there were roughly 10 million new TB cases per year and isoniazid resistant (INHr) TB accounted for 9.5% of these cases around the world. In 2012, United States had an interruption in the supply of isoniazid (INH), which increased the likelihood of INH resistance rates. Although INH resistance in Mycobacterium tuberculosis (Mtb) is multigenic, mutations in the catalase-peroxidase (katG) gene predominate amongst INHr Mtb strains. The characterization of the Mtb proteome before and after acquiring INH resistance remains understudied. Additionally, the effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. The purpose of this work is to describe a complete biochemical and immunological characterization of the INHr acquisition in Mtb. In this way, a global exploration of the protein and mycolic acids differences in Mtb cultures, as well as differences in the immune response and bacterial virulence in the mouse model comparing clonal susceptible and INHr pairs of Mtb were evaluated. After this, common trends were analyzed and the findings were interpreted in the context of bacterial metabolism and host-interaction. For this work, two clonal clinical Mtb strains and one laboratory clonal pair of the H37Rv strain with different susceptibility profiles to INH were studied. The H37Rv INHr strain was isolated from a mouse that was exposed to INH in the lab and developed the same katG mutation that one of the clinical INHr strain has (V1A). In all cases, the first strain was susceptible to all tested drugs (mostly known as the INHs strain in this dissertation) while the second strain was resistant only to INH (named INHr throughout this work). The clinical pairs were confirmed as clonal pairs of the Beijing and T genotype respectively by spoligotyping and restriction fragment polymorphism analysis that uses the patterns given by the distribution of the insertion sequence (IS)-6110. Previous whole genome sequencing analysis of the clinical clonal pairs showed a katG mutation and the presence of some additional non-synonymous polymorphisms in the INHr strains. After the proteomic analysis, a katG PCR sequencing confirmed two mutations in katG for the T INHr pair (V1A and E3V) while the L101R mutation previously identified for the Beijing INHr was not confirmed. This mutation was highly unstable and the Beijing INHr might have reversed its phenotype after the absence of INH during in vitro growth. Therefore, the analysis with the Beijing clonal pair is only presented in chapter II. Protein comparison of secreted and cellular fractions (membrane, cytosol and cell wall) between clinical and lab clonal pairs of Mtb before and after acquisition of INH resistance revealed at least 25 commonly altered proteins looking at the same cellular fractions. These proteins were involved in ATP synthase machinery, lipid metabolism, regulatory events, virulence, detoxification and adaptation processes. Western blot analysis supported some of our findings, particularly the lower level of bacterial enzyme KatG in the INHr strains. Mycolic acid (MA) analysis in these clonal pairs did not reveal a common trend in these molecules for INHr strains but generated supporting information about an alternative fatty acid biosynthetic pathway in the clinical INHr strain. These analyses are further described in chapter III. Additionally, differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of KatG were evaluated in the C57BL/6 mouse model. The results in the mouse study support the idea of the individual effect of specific located mutations in the katG gene together with the associated changes in the bacterial proteome induce differences in the Mtb virulence and pathogenicity. In addition, the in vivo results also suggest the contribution of innate immune response via TLR-2 in the clearance of the INHr-attenuated Mtb strains. Further details of this work are described in chapter IV. This work provides a better understanding of new compensatory mechanisms in Mtb after INH resistance acquisition providing novel information that could be used to address alternative combined therapies as well as the identification of new drug targets in INHr strains. The results presented here also contribute to the generation of new hypothesis regarding RNA decay in Mtb and the need to evaluate if the observed biochemical differences are also associated with the bacterial exposure to the first line drug therapy that occurred in the patient. After the results obtained in this study, a subsequent biochemical analysis of Mtb strains obtained from patients before and after drug treatment is proposed to improve the description of the evolution of the acquired drug resistant phenomena observed in TB cases that limit the global disease control and hence its eradication (chapter V)

The Physiology of Mycobacterium tuberculosis in the Context of Drug Resistance: A System Biology Perspective

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), is the main cause of death due to an infectious disease. After more than 100 years of the discovery of Mtb, clinicians still face difficulties finding an effective treatment for the increasing number of drug-resistant cases. The difficulties in the clinical setting can be related to the slow pace at which the understanding of the physiology of this bacterium has occurred. Mtb is distinct from other microorganisms not only due to its slow growth and difficulties to study in the laboratory, but also due to its inherent physiology such as its complex cell envelope and its metabolic pathways. Understanding the physiology of drug susceptible and resistant Mtb strains is crucial for the design of an effective chemotherapy against TB. This chapter will review the mycobacterial cell envelope and major physiological pathways together with recent discoveries in Mtb drug resistance through different “omics” disciplines

Do investors in Spain react to news on sustainability and corporate social responsibility?

We analyse whether sustainability and corporate social responsibility-related news affects returns of stocks traded on the Spanish stock market. We used event methodology and an approach consistent with the active management of investment portfolios. Results show that in the short term, investors do not consider these news items to be relevant, and they therefore have no effect on the price of the stocks analysed. This result holds when the study is conditioned to the type of news (positive or negative) and whether or not the stocks belong to an index formed following socially responsible investment criteri

Multidrug-Resistant Mycobacterium tuberculosis, Southwestern Colombia

Using spoligotyping, we identified 13 genotypes and 17 orphan types among 160 Mycobacterium tuberculosis isolates from patients in Valle del Cauca, Colombia. The Beijing genotype represented 15.6% of the isolates and was correlated with multidrug-resistant tuberculosis, female sex of the patients, and residence in Buenaventura and may represent a new public health threat

Secreted phospholipase A2-IIA modulates transdifferentiation of cardiac fibroblast through EGFR transactivation: An inflammation–fibrosis link

Producción CientíficaSecreted phospholipase A2-IIA (sPLA2-IIA) is a pro-inflammatory protein associated with cardiovascular disorders, whose functions and underlying mechanisms in cardiac remodelling are still under investigation. We herein study the role of sPLA2-IIA in cardiac fibroblast (CFs)-to-myofibroblast differentiation and fibrosis, two major features involved in cardiac remodelling, and also explore potential mechanisms involved. In a mice model of dilated cardiomyopathy (DCM) after autoimmune myocarditis, serum and cardiac sPLA2-IIA protein expression were found to be increased, together with elevated cardiac levels of the cross-linking enzyme lysyl oxidase (LOX) and reactive oxygen species (ROS) accumulation. Exogenous sPLA2-IIA treatment induced proliferation and differentiation of adult rat CFs. Molecular studies demonstrated that sPLA2-IIA promoted Src phosphorylation, shedding of the membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) ectodomain and EGFR phosphorylation, which triggered phosphorylation of ERK, P70S6K and rS6. This was also accompanied by an up-regulated expression of the bone morphogenic protein (BMP)-1, LOX and collagen I. ROS accumulation were also found to be increased in sPLA2-IIA-treated CFs. The presence of inhibitors of the Src/ADAMs-dependent HB-EGF shedding/EGFR pathway abolished the CF phenotype induced by sPLA2-IIA. In conclusion, sPLA2-IIA may promote myofibroblast differentiation through its ability to modulate EGFR transactivation and signalling as key mechanisms that underlie its biological and pro-fibrotic effects.Ministerio de Economía, Industria y Competitividad (grants SAF2012-34460 and SAF2016-81063)Instituto de Salud Carlos III (grant PI18/010257729

Development and Validation of a Model to Predict Severe Hospital-Acquired Acute Kidney Injury in Non-Critically Ill Patients

Lesión renal aguda; Registros electrónicos de datos de salud; Adquirido en el hospitalLesió renal aguda; Registres electrònics de dades de salut; Adquirit a l'HospitalAcute kidney injury; Electronic health data records; Hospital-acquiredBackground. The current models developed to predict hospital-acquired AKI (HA-AKI) in non-critically ill fail to identify the patients at risk of severe HA-AKI stage 3. Objective. To develop and externally validate a model to predict the individual probability of developing HA-AKI stage 3 through the integration of electronic health databases. Methods. Study set: 165,893 non-critically ill hospitalized patients. Using stepwise logistic regression analyses, including demography, chronic comorbidities, and exposure to risk factors prior to AKI detection, we developed a multivariate model to predict HA-AKI stage 3. This model was then externally validated in 43,569 non-critical patients admitted to the validation center. Results. The incidence of HA-AKI stage 3 in the study set was 0.6%. Among chronic comorbidities, the highest odds ratios were conferred by ischemic heart disease, ischemic cerebrovascular disease, chronic congestive heart failure, chronic obstructive pulmonary disease, chronic kidney disease and liver disease. Among acute complications, the highest odd ratios were associated with acute respiratory failure, major surgery and exposure to nephrotoxic drugs. The model showed an AUC of 0.906 (95% CI 0.904 to 0.908), a sensitivity of 89.1 (95% CI 87.0–91.0) and a specificity of 80.5 (95% CI 80.2–80.7) to predict HA-AKI stage 3, but tended to overestimate the risk at low-risk categories with an adequate goodness-of-fit for all risk categories (Chi2: 16.4, p: 0.034). In the validation set, incidence of HA-AKI stage 3 was 0.62%. The model showed an AUC of 0.861 (95% CI 0.859–0.863), a sensitivity of 83.0 (95% CI 80.5–85.3) and a specificity of 76.5 (95% CI 76.2–76.8) to predict HA-AKI stage 3 with an adequate goodness of fit for all risk categories (Chi2: 15.42, p: 0.052). Conclusions. Our study provides a model that can be used in clinical practice to obtain an accurate dynamic assessment of the individual risk of HA-AKI stage 3 along the hospital stay period in non-critically ill patients.This research received no external funding

Social and work profile of the Health Administration professionals from Universidad de Antioquia, 1999-2008

ABSTRACT: To characterize the social and work profiles of the professionals from Universidad de Antioquia (University of Antioquia) who hold a degree in Health Administration. Methodology: A cross-sectional study based on primary data obtained by surveying 356 graduate professionals between 1999 and 2008. Descriptive and inferential statistical analysis techniques were applied. Depending on the nature of the variables, absolute values, ratios, and summary measures of central tendency were reported. Results: It was a group of young professionals with an average age of 32.5 years. They were mainly women with professional work experience after graduation. They were employed at the time of the survey, but 12.1% of them reported being unemployed. The main sector of the economy where they work is services, followed by construction. Their most common position is that of administrator, followed by operational positions. In most cases, their income doesn´t exceed 4 legal minimum wages. Discussion: some findings are consistent with previous studies conducted in 2001 and 2004. Other variables contrast with these studies and with other national references. We conclude that the conditions of job insecurity are not inherent to the profession itself but to the labor market characteristics of the current capitalist world.RESUMEN: Caracterizar el perfil socio-laboral de los profesionales egresados del programa Administración en Salud de la Universidad de Antioquia. Metodología: estudio descriptivo de corte transversal basado en información primaria mediante encuesta a 356 profesionales egresados del programa entre 1999 y 2008. Se aplicaron técnicas de análisis estadístico descriptivo e inferencial. Según la naturaleza de las variables, se reportan valores absolutos, proporciones, medidas de resumen y tendencia central. Resultados: se trata de un grupo de profesionales jóvenes con edad promedio de 32,5 años, principalmente mujeres, en su mayoría con experiencia laboral profesional después de haberse graduado y con vínculo laboral al momento de la encuesta. El 12,1% reportó estar desempleado. El principal sector de la economía donde laboran es el de servicios, seguido de la construcción. El cargo desempeñado durante su historia laboral más frecuente es el administrativo seguido del operativo. Para la mayoría, sus ingresos salariales no superan los 4 Salarios Mínimos Legales Vigentes (SMLV). Discusión: algunos hallazgos se corresponden con estudios previos de 2001 y 2004. Otras variables contrastan con esos estudios y con otros referentes nacionales. Se concluye que las condiciones de precariedad laboral no son inherentes a la profesión en sí misma sino a las características del mercado laboral del mundo capitalista actual

Scalable bio marker combinations for early stroke diagnosis: A systematic review

Background: Acute stroke treatment is a time-critical process in which every minute counts. Laboratory biomarkers are needed to aid clinical decisions in the diagnosis. Although imaging is critical for this process, these biomarkers may provide additional information to distinguish actual stroke from its mimics and monitor patient condition and the effect of potential neuroprotective strategies. For such biomarkers to be effectively scalable to public health in any economic setting, these must be cost-effective and non-invasive. We hypothesized that blood-based combinations (panels) of proteins might be the key to this approach and explored this possibility through a systematic review. Methods: We followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines for systematic review. Initially, the broader search for biomarkers for early stroke diagnosis yielded 704 hits, and five were added manually. We then narrowed the search to combinations (panels) of the protein markers obtained from the blood. Results: Twelve articles dealing with blood-based panels of protein biomarkers for stroke were included in the systematic review. We observed that NR2 peptide (antibody against the NR2 fragment) and glial fibrillary acidic protein (GFAP) are brain-specific markers related to stroke. Von Willebrand factor (vWF), matrix metalloproteinase 9 (MMP-9), and S100β have been widely used as biomarkers, whereas others such as the ischemia-modified albumin (IMA) index, antithrombin III (AT-III), and fibrinogen have not been evaluated in combination. We herein propose the following new combination of biomarkers for future validation: panel 1 (NR2 + GFAP + MMP-9 + vWF + S100β), panel 2 (NR2 + GFAP + MMP-9 + vWF + IMA index), and panel 3 (NR2 + GFAP + AT-III + fibrinogen). Conclusions: More research is needed to validate, identify, and introduce these panels of biomarkers into medical practice for stroke recurrence and diagnosis in a scalable manner. The evidence indicates that the most promising approach is to combine different blood-based proteins to provide diagnostic precision for health interventions. Through our systematic review, we suggest three novel biomarker panels based on the results in the literature and an interpretation based on stroke pathophysiology

Population structure among Mycobacterium tuberculosis Isolates from pulmonary tuberculosis patients in Colombia

Background: Phylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world. Principal Findings: A total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1). Conclusions: This study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellı ´n and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this sublineage

La Epigrafía griega y latina en la enseñanza de las materias de Filología Clásica: aplicación de nuevas metodologías y nuevas tecnologías (II)

El proyecto PIMCD 2017/18-42 es continuación de dos proyectos anteriores, PIMCD 2011/12-314 y PIMCD 2016/17-5. Su finalidad es familiarizar a los alumnos con diversas cuestiones de la epigrafía griega y latina mediante una serie de unidades didácticas expuestas en un Seminario abierto, cuyas sesiones se celebran en la Facultad de Filología de la UCM