Farmacogenómica del tratamiento de primera línea en el cáncer gástrico: avances en la identificación de los biomarcadores genómicos de respuesta clínica / Pharmacogenomics of the first-line treatment for gastric cancer: advances in the identification of genomic biomarkers for clinical response to chemotherapy

Debido a la inespecificidad de los síntomas, el cáncer gástrico (CG) es diagnosticado frecuentemente en etapas avanzadas, lo que da cuenta de los altos índices de mortalidad debido a esta neoplasia a nivel mundial. El esquema de tratamiento adyuvante o neoadyuvante en los países occidentales incluye el uso de fluoropirimidinas citotóxicas y compuestos de platino formadores de aductos en el ADN. La respuesta clínica al tratamiento con estos fármacos depende principalmente de la sensibilidad del tumor, la cual a su vez está condicionada por el nivel de expresión de los blancos terapéuticos y de las enzimas de reparación del ADN. Sumado a esto, algunos polimorfismos de línea germinal en genes asociados al metabolismo y a la respuesta a estos fármacos, han mostrado asociación con respuestas pobres y con el desarrollo de eventos adversos, incluso con resultados fatales. La identificación de biomarcadores genómicos, en la forma de polimorfismos genéticos o la expresión diferencial de genes específicos asociados a la respuesta quimioterapeútica ha sido motivo de intensa investigación como base para la aplicación de la farmacogenómica en el establecimiento de una terapia farmacológica racional y personalizada del CG. Sin embargo, ante la eventual aplicación de la farmacogenómica en el ámbito clínico, es necesario establecer el valor pronóstico real de dichos biomarcadores mediante los estudios de asociación genotipo-fenotipo, así como su prevalencia en el contexto de cada población de pacientes. Estos aspectos son indispensables al evaluar la relación costo-efectividad de la introducción de los productos de la medicina genómica predictiva en el tratamiento del CG.

Gastric cancer (GC) is often diagnosed at later stages due to the lack of specificity of symptoms associated with the neoplasm, causing high mortality rates worldwide. The first line of adjuvant and neoadjuvant treatment includes cytotoxic fluoropyrimidines and platin-containing compounds which cause the formation of DNA adducts. The clinical outcome with these antineoplastic agents depends mainly on tumor sensitivity, which is conditioned by the expression level of the drug targets and the DNA-repair system enzymes. In addition, some germ line polymorphisms, in genes linked to drug metabolism and response to chemotherapy, have been associated with poor responses and the development of adverse effects, even with fatal outcomes in GC patients. The identification of genomic biomarkers, such as individual gene polymorphisms or differential expression patterns of specific genes, in a patient-by-patient context with potential clinical application is the main focus of current pharmacogenomic research, which aims at developing a rational and personalized therapy (i.e., a therapy that ensures maximum efficacy with no predictable side effects). However, because of the future application of genomic technologies in the clinical setting, it is necessary to establish the prognostic value of these genomic biomarkers with genotype-phenotype association studies and to evaluate their prevalence in the population under treatment. These issues are important for their cost-effectiveness evaluation, which determines the feasibility of using these medical genomic research products for GC treatment in the clinical setting.

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

Enzymes have been long used in man-made biochemical processes, from brewing and fermentation to current industrial production of fine chemicals. The ever-growing demand for enzymes in increasingly specific applications requires tailoring naturally occurring enzymes to the non-natural conditions found in industrial processes. Relationships between enzyme sequence, structure and activity are far from understood, thus hindering the capacity to design tailored biocatalysts. In the field of protein engineering, directed enzyme evolution is a powerful algorithm to generate and identify novel and improved enzymes through iterative rounds of mutagenesis and screening applying a specific evolutive pressure. In practice, critical checkpoints in directed evolution are: selection of the starting point, generation of the mutant library, development of the screening assay and analysis of the output of the screening campaign. Each step in directed evolution can be performed using conceptually and technically different approaches, all having inherent advantages and challenges. In this article, we present and discuss in a general overview, challenges of designing and performing a directed enzyme evolution campaign, current advances in methods, as well as highlighting some examples of its applications in industrially relevant enzymes.

Changes of Gene Expression after Bone Marrow Cell Transfusion in Rats with Monocrotaline-Induced Pulmonary Hypertension

Pulmonary artery hypertension (PAH) causes right ventricular failure and possibly even death by a progressive increase in pulmonary vascular resistance. Bone marrow-derived mesenchymal stem cell therapy has provided an alternative treatment for ailments of various organs by promoting cell regeneration at the site of pathology. The purpose of this study was to investigate changes of pulmonary haemodynamics, pathology and expressions of various genes, including ET (endothelin)-1, ET receptor A (ERA), endothelial nitric oxide synthase (NOS) 3, matrix metalloproteinase (MMP) 2, tissue inhibitor of matrix metalloproteinase (TIMP), interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in monocrotaline (MCT)-induced PAH rat models after bone marrow cell (BMC) transfusion. The rats were grouped as the control (C) group, monocrotaline (M) group, and BMC transfusion (B) group. M and B groups received subcutaneous (sc) injection of MCT (60 mg/kg). BMCs were transfused by intravenous injection at the tail 1 week after MCT injection in B group. Results showed that the average RV pressure significantly decreased in the B group compared with the M group. RV weight and the ratio of RH/LH+septum significantly decreased in the B group compared to the M group. Gene expressions of ET-1, ERA, NOS 3, MMP 2, TIMP, IL-6, and TNF-alpha significantly decreased in week 4 in the B group compared with the M group. In conclusion, BMC transfusion appears to improve survival rate, RVH, and mean RV pressure, and decreases gene expressions of ET-1, ERA, NOS 3, MMP 2, TIMP, IL-6, and TNF-alpha.

Exploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements / Explorando el metagenoma del suelo antártico como fuente de nuevas enzimas adaptadas al frío y elementos génicos móviles

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.

A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.

Fenótipos de betalactamases em Klebsiella pneumoniae de hospital de emergência de Porto Alegre / Phenotypes of beta-lactamases in Klebsiella pneumoniae from emergency hospital of Porto Alegre

INTRODUÇÃO: Mundialmente, o crescente aumento de infecções hospitalares gera a necessidade de estudos na área de resistência bacteriana. Uma das bactérias Gram-negativas de maior prevalência no ambiente nosocomial é a Klebsiella pneumoniae, capaz de expressar uma diversidade de enzimas de resistência, justificando seu monitoramento constante. OBJETIVO: Analisar a frequência fenotípica das enzimas betalactamases de espectro estendido (ESBL), cromossômicas (AmpC) plasmidial, metalobetalactamase (MBL) e Klebsiella pneumoniae carbapenemase (KPC) oriundas de hospital de emergência em Porto Alegre (RS). MATERIAL E MÉTODO: Foram incluídos no estudo 58 isolados bacterianos com sensibilidade diminuída a cefalosporinas de terceira geração e/ou a cefoxitina. Nestas, foram aplicados os testes de disco-difusão compatíveis para as respectivas betalactamases. RESULTADOS: Encontrou-se elevada frequência de ESBL (48,3 por cento), seguida por ESBL simultânea à AmpC plasmidial (15,5 por cento), além de duas cepas com carbapenemases. DISCUSSÃO: O elevado índice de ESBL e ESBL/AmpC conduz à necessidade de uso terapêutico de carbapenens. Consequentemente, o uso massivo desses fármacos gera pressão seletiva que favorece o aparecimento de cepas mais resistentes, como as produtoras de carbapenemases (KPC e/ou MBL). As carbapenemases são muito relevantes devido à limitação terapêutica inerente e por seu potencial de disseminação. A resistência a todos os fármacos betalactâmicos usados no screening por disco-difusão pode ser um "sinalizador" laboratorial para a presença desses relevantes mecanismos de resistência. CONCLUSÃO: A pesquisa de fenótipos de resistência em Klebsiella é ferramenta fácil e exequível nos laboratórios de microbiologia, além de ser fundamental para o conhecimento da epidemiologia local das betalactamases, possibilitando a adequação da terapia antimicrobiana mais conveniente.

INTRODUCTION: Worldwide, the incidence of nosocomial infections raises the studies of bacterial resistance. Klebsiella pneumoniae is a Gram-negative bacteria most prevalent in the nosocomial environment, capable of expressing a variety of resistance enzymes, justifying their continued monitoring. OBJECTIVE: We sought to determine the frequency of beta-lactamases ESBL, AmpC plasmid, MBL and KPC in K. pneumoniae strains from emergency hospital in Porto Alegre (RS). MATERIAL AND METHOD: Fifty-eight bacterial isolates K. pneumoniae with reduced susceptibility to third generation cephalosporins and/or cefoxitin were included in this study. Phenotypic detection of beta-lactamases was carried out by using specific tests for ESBL, AmpC plasmid, KPC and MBL. RESULTS: Were detected a high frequency of ESBL (48.3 percent), followed by the AmpC plasmid/ESBL (15.5 percent), and two carbapenemase strains. DISCUSSION: The high rates of ESBL and ESBL/AmpC leads to the therapeutic use of carbapenens. Consequently, the massive use of carbapenens creates selective pressure favoring the more resistant strains, such as carbapenemase producing (KPC and/or MBL). The carbapenemase is very relevant due to the inherent therapeutic limitation, as well as their potential to spread. The resistance to all beta-lactam drugs used in the screening by disk diffusion can be a "flag" laboratory for presence of relevant mechanisms of resistance. CONCLUSION: Research of resistance phenotypes in Klebsiella is easy and feasible tool in microbiology laboratories, and is fundamental to understanding the local epidemiology of beta-lactamases, allowing the appropriateness of antimicrobial therapy more convenient.

Biochemical comparison of two Hypostomus populations (Siluriformes, Loricariidae) from the Atlântico Stream of the upper Paraná River basin, Brazil

Two syntopic morphotypes of the genus Hypostomus - H. nigromaculatus and H. cf. nigromaculatus (Atlântico Stream, Paraná State) - were compared through the allozyme electrophoresis technique. Twelve enzymatic systems (AAT, ADH, EST, GCDH, G3PDH, GPI, IDH, LDH, MDH, ME, PGM and SOD) were analyzed, attributing the score of 20 loci, with a total of 30 alleles. Six loci were diagnostic (Aat-2, Gcdh-1, Gpi-A, Idh-1, Ldh-A and Mdh-A), indicating the presence of interjacent reproductive isolation. The occurrence of few polymorphic loci acknowledge two morphotypes, with heterozygosity values He = 0.0291 for H. nigromaculatus and He = 0.0346 for H. cf. nigromaculatus. F IS statistics demonstrated fixation of the alleles in the two morphotypes. Genetic identity (I) and distance (D) of Nei (1978) values were I = 0.6515 and D = 0.4285. The data indicate that these two morphotypes from the Atlântico Stream belong to different species.

Revisão sobre os valores do antígeno prostático específico e correlações com alterações da próstata / Revision about the values of the prostate-specific antigen and correlations with alterations of the prostate

Os autores fazem uma revisão bibliográfica sobre as principais causas de modificações nos valores normais do antígeno prostático específico (Prostate Specific Antigen - PSA) e suas correlações com patologias da próstata e outras causas de variações desta proteína

Preliminary evaluation of the genetic relatedness of three species of the subgenus Dendromyia Theobald and other species of the genus Wyeomyia Theobald (Diptera: Culicidae)

An eletrophoretic analysis of three species of the subgenus Dendromyia (Wyeomyia luteoventralis, Wy. ypsipola and Wy. testei) and three species belonging to different groups in the genus Wyeomyia (Wy. negrensis, Wy. mystes and Wy. confusa) was performed. Eight enzyme loci were analysed. High values of genetic identity were detected among the species of the subgenus Dendromyia: Wy. luteoventralis, Wy. ypsipola and Wy. testei (mean value 0.63). On the other hand, low values of genetic identity were observed among Wy. negrensis, Wy. mystes and Wy. confusa (mean value 0.23), suggesting that they belong, at least, to distinct subgenera within the Genus Wyeomyia. The UPGMA phenogram revealed the grouping of the Dendromyia species, while the others clustered at lower identity levels.

Enzyme polymorphism on the metabolic-demethylation of dextromethorphan in a Southamerican population

The polymorphic oxidative metabolism of debrisoquine and sparteine were discovered in the seventies by Mahgoub and Eichelbaum. Since then, many other therapeutic substances were added and one of these drugs is dextromethorphan. The object of this investigation was to ascertain the distribution of the oxidative phenotype of dextromethorphan in the Uruguayan population. The drug and its metabolite, dextrorphan, were quantified in the urine of 165 healthy volunteers by a modificacion of an HPLC method by Chen et al. The metabolic ratio was calculated and frequency distribution histograms were drawn. By inspection of the histogram two antimodes can be assigned which determine three sub-populations: on one side the fast extensive metabolizers (n = 30, 18.2 per cent), in the middle the extensive metabolizers (n = 123, 74.5 per cent) and on the other extreme of the histogram the slow metabolizers (n = 12, 7.3 per cent). No other studies have confirmed thus far this trimodal distribution. This research will be continued by genotyping the populations studied in order to confirm these findings and to elucidate the underlying genetic mechanisms of the polymorphism.

Human red cell enzymes distribution in a population sample from bambui (Minas Gerais)

Trezentos e noventa indivíduos de uma populaçäo tri-híbrida brasileira foram estudados em relaçäo a 4 sistemas genéticos (ACP1, ESA, PEPA and ADA). Somente os alelos comuns foram detectados nesta populaçäo, com exceçäo do sistema ESA que apresentou frequencia polimórfica numa variante rara. Esta variante pode ser classificada como sendo ESA*Dmaci (Neel et al., 1977) or ESA*C (Tashian, 1965). Säo apresentadas as freqüências gênicas e fenotípicas e estas säo encontradas em distribuiçäo semelhante a de outras populaçöes descritas