Immature rat testis sustained long-term development using an integrative model

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

Resonant Recognition Model Study for Interactions between SARS CoV 2 and Human Proteins

This study was devoted to the Resonant Recognition Model (RRM) analysis of SARS-CoV-2 proteins and their possible interaction with other human proteins, specifically, SARS CoV replicases and methyl transferases, were tested, via RRM analysis, for possible interactions with host CD4 T receptor proteins and prohibitins which participate in human organism response to viral infections. The following protein sequences were studied: twenty human SARS coronavirus methyltransferase proteins, eight replicase proteins, twenty-one prohibitin proteins, and eleven CD4 -T-cell surface antigens T4 proteins. Results revealed RRM peaks at f1=0.07349 and f2=0.2839. The peak at f1 was also common for interaction between SARS-CoV-2 methyl transferases and human prohibitins, where opposite phase suggest binding between these proteins during viral infection. This interaction was not supported for viral methyltransferase and human CD4 receptors (72.4 o phase shift). Viral replicases exhibited opposite phase interaction with both prohibitins and CD4 receptors. Overall, RRM revealed common RRM frequencies for both replicases and methyl transferases, and added plausibility to interactions between SARSCoV2 methyl transferase and human prohibitin, as well as between SARS Cov2 replicase and human prohibitin and CD4 T-cell receptors(AU)

Este estudio se dedicó al análisis mediante el Modelo de Reconocimiento Resonante (RRM) de las proteínas del SARS-CoV-2 y su posible interacción con otras proteínas humanas, específicamente, fueron analizadas las replicasas de SARS CoV y las metiltransferasas, mediante análisis RRM, para detectar posibles interacciones con las Proteínas del receptor CD4 T y las prohibitinas humanas, las cuales participan en la respuesta del organismo humano a las infecciones virales. Se estudiaron las siguientes secuencias de proteínas: veinte proteínas metiltransferasas del coronavirus del SARS humano, ocho replicasas, veintiuna prohibitinas y once proteínas T4 de antígenos de superficie de células T CD4. Los resultados revelaron picos de RRM en f1 = 0.07349 y f2 = 0.2839. El pico en f1 también fue común para la interacción entre las metiltransferasas del SARS-CoV-2 y las prohibitinas humanas, donde la fase opuesta sugiere la unión entre estas proteínas durante la infección viral. Esta interacción no fue apoyada para la metiltransferasa viral y los receptores CD4 humanos (cambio de fase de 72,4 o). Las réplicas virales exhibieron una interacción de fase opuesta tanto con las prohibitinas como con los receptores CD4. En general, el análisis de RRM reveló frecuencias comunes de RRM para replicasas y metiltransferasas, y apoyó plausibilidad de las interacciones entre la metiltransferasa de SARSCoV2 y la prohibitina humana, así como entre la replicasa de Cov2 del SARS con la prohibitina humana y los receptores de células T CD4(AU)

Role of aminoglycoside-modifying enzymes and 16S rRNA methylase (ArmA) in resistance of Acinetobacter baumannii clinical isolates against aminoglycosides

Abstract INTRODUCTION: This study aimed to determine the role of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase (ArmA) in Acinetobacter baumannii clinical isolates. METHODS: We collected 100 clinical isolates of A. baumannii and identified and confirmed them using microbiological tests and assessment of the OXA-51 gene. Antibiotic susceptibility testing was carried out using disk agar diffusion and micro-broth dilution methods. The presence of AME genes and ArmA was detected by PCR and multiplex PCR. RESULTS: The most and least effective antibiotics in this study were netilmicin and ciprofloxacin with 68% and 100% resistance rates, respectively. According to the minimum inhibitory concentration test, 94% of the isolates were resistant to gentamicin, tobramycin, and streptomycin, while the highest susceptibility (20%) was observed against netilmicin. The proportion of strains harboring the aminoglycoside resistance genes was as follows: APH(3′)-VIa (aphA6) (77%), ANT(2")-Ia (aadB) (73%), ANT(3")-Ia (aadA1) (33%), AAC(6′)-Ib (aacA4) (33%), ArmA (22%), and AAC(3)-IIa (aacC2) (19%). Among the 22 gene profiles detected in this study, the most prevalent profiles included APH(3′)-VIa + ANT(2")-Ia (39 isolates, 100% of which were kanamycin-resistant), and AAC(3)-IIa + AAC(6′)-Ib + ANT(3")-Ia + APH(3′)-VIa + ANT(2")-Ia (14 isolates, all of which were resistant to gentamicin, kanamycin, and streptomycin). CONCLUSIONS: High minimum inhibitory concentration of aminoglycosides in isolates with the simultaneous presence of AME- and ArmA-encoding genes indicated the importance of these genes in resistance to aminoglycosides. However, control of their spread could be effective in the treatment of infections caused by A. baumannii.

Spatio-temporal expression profile of Mettl3/Mettl14 during mouse retina development / Perfil de expresión espacio-temporal de Mettl3 / Mettl14 durante el desarrollo de la retina del ratón

SUMMARY: The Mettl3/Mettl14 methyltransferase complex installs the most ubiquitous internal mRNA modification- N6-methyladenosine (m6A). The vertebrate retina development is a multi-step process that requires fine-tuning of multiple cellular events, but very little is known about the potential function of Mettl3 and Mettl14 in this process. In this study, we demonstrated the spatio-temporal expression of Mettl3 and Mettl14 during retina development in mouse by quantitative PCR and immunofluorescence staining. We found that these two components of methyltransferase complex could be detected from the beginning of retina development; and the expression of Mettl3 and Mettl14 were gradually restricted to inner nuclear layer (INL) and ganglion cell layer (GCL); Double labeling showed that Mettl3 and Mettl14 had similar expression patterns in mature retinal INL and GCL. Overall, our spatio-temporal expression data provided the foundation for future research on the function of m6A modification in the retina development.

RESUMEN: El complejo Mettl3 / Mettl14 metiltransferasa establece la modificación interna más significativa de ARNm: N6- metiladenosina (m6A). El desarrollo de la retina de los vertebrados es un proceso de varios pasos que requiere múltiples eventos celulares; existe muy poca información sobre la función potencial de Mettl3 y Mettl14 en este proceso. En este estudio, demostramos la expresión espacio-temporal de Mettl3 y Mettl14 durante el desarrollo de la retina en ratón mediante PCR cuantitativa y tinción de inmunofluorescencia. Descubrimos que estos dos componentes del complejo de metiltransferasa podían ser detectados desde el comienzo del desarrollo de la retina; la expresión de Mettl3 y Mettl14 se restringió gradualmente a la capa nuclear interna (INL) y la capa de células ganglionares (GCL); se observó que Mettl3 y Mettl14 tenían patrones de expresión similares en INL y GCL retinianos maduros. En general, nuestros datos de expresión espacio-temporal proporcionan información para futuras investigaciones sobre la función de la modificación de m6A en el desarrollo de la retina.

Nicotinamide activates latent HIV-1 ex vivo in ART suppressed individuals, revealing higher potency than the association of two methyltransferase inhibitors, chaetocin and BIX01294

ABSTRACT Background: Latent HIV-1 is a major hurdle in obtaining HIV-1 sustained virological remission (SVR). Here we explored histone deacetylation inhibition property of nicotinamide (NAM; n = 17) for the first time in comparison to a combination of methyltransferase inhibitors (MTIs; Chaetocin and BIX01294; n = 25) to reactivate latent HIV ex vivo in CD8-depleted PBMCs from antiretroviral treated aviremic individuals. Results: NAM reactivated HIV-1 from 13/17 (76.4%) samples compared to 20/25 (80.0%) using MTIs with mean viral load (VLs) of 4.32 and 3.22 log10 RNA copies/mL, respectively (p = 0.004). Mean purging time after NAM and MTIs stimulation was 5.1 and 6.75 days, respectively (p = 0.73). Viral purging in autologous cultures exhibited blunted HIV recovery with fluctuating VLs followed by a complete viral extinction when expanded in allogenic system. Electron microscopy from five supernatants revealed anomalous viral particles, with lack of complete viral genomes when characterized by ultradeep sequencing through metagenomics approach (n = 4). Conclusion: NAM alone was more potent HIV-1 activator than combination of MTIs, with potential of clinical use.

Variantes genéticas de la tiopurina metiltransferasa e incidencia de eventos adversos en pacientes con indicación de azatioprina / Genetic polymorphisms of thiopurine methyltransferase and incidence of adverse events in patients with medical indication of azathioprine

La azatioprina es una tiopurina que presenta rango terapéutico estrecho y marcada toxicidad hematológica y hepática. La tiopurina S-metiltransferasa es una enzima que metaboliza ese grupo de drogas. Mutaciones en el gen que codifica dicha enzima aumentan el riesgo de presentar eventos adversos, por lo que su estudio farmacogenético permite contar con información para el diseño de la estrategia terapéutica. Sin embargo, su utilidad en el medio local no está completamente establecida. Fueron incluidos 45 sujetos (13 hombres) con indicación de azatioprina. Se determinó la presencia de las mutaciones *2, *3A, *3B y *3C de TMPT por PCR-RFLP y se analizó la relación entre el genotipo y la incidencia de eventos adversos relacionados al fármaco. Nueve portaban al menos un alelo no funcional, uno de ellos con genotipo *3A/*3A. Se detectó toxicidad en 3 de los 18 que iniciaron tratamiento con azatioprina: 2 pacientes con genotipo normal presentaron eventos adversos leves, y el único evento adverso de gravedad (aplasia medular) ocurrió en el sujeto con genotipo homocigota mutado. El único que presentó genotipo homocigota mutado desarrolló el más grave de los eventos adversos registrados, a pesar de estar en tratamiento con dosis bajas de azatioprina. Por este motivo, la determinación del genotipo de la tiopurina metiltransferasa pareciera ser de utilidad, pero no reemplaza la necesidad de seguimiento clínico y bioquímico en pacientes en tratamiento con tiopurinas.

Azathioprine is a thiopurine which has a narrow therapeutic index and marked hematological and hepatic toxicity. Thiopurine s-methyltransferase is an enzyme involved in the metabolism of thiopurines. Mutations in the gene that encodes the enzyme may augment the risk of adverse events. For that reason, pharmacogenetic determinations prior to the initiation of therapy can provide useful information for the future therapeutic strategy. Nevertheless, its utility in the local environment is not completely established. Forty-five subjects (13 men) who had been prescribed azathioprine were included. The presence of *2, *3A, *3B and *3C mutations were determined by PCR-RFLP, and the relationship between genotype and incidence of adverse events related to the drug was analyzed. Nine carried at least one non-functional allele, one of them with *3A/*3A genotype. Among the eighteen patients who initiated treatment with azathioprine, toxicity was detected in 3 cases: 2 mild events were observed in patients with normal genotype, and the only serious event (bone marrow suppression) occurred in the individual with homozygous mutant genotype. The only homozygous mutant patient developed the most severe of the registered events, in spite of being under treatment with low doses of azathioprine. This is the reason why enzymatic determination could be of utility, even though it does not replace clinical and biochemical follow-up in patients under thiopurine treatment.

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Polimorfismos del gen Arsénico 3 Metiltransferasa (As3MT) y la eficiencia urinaria del metabolismo del arsénico en una población del norte de México / Polymorphisms of the Arsenite Methyltransferase (As3MT) gene and urinary efficiency of arsenic metabolism in a population in northern Mexico

RESUMEN El objetivo del estudio fue evaluar la relación de los polimorfismos rs11191439 y rs3740393 del gen Arsénico 3 Metiltransferasa (As3MT) con los perfiles de excreción urinaria de arsénico, en una población del norte de México. Se realizó un estudio transversal con 102 participantes de 14 a 75 años en la Comarca Lagunera, México. Se determinó la especiación de los metabolitos urinarios de arsénico y extracción de ADN, el análisis y amplificación de los polimorfismos se realizó por PCR tiempo real. Se realizó un análisis descriptivo y se evaluó las diferencias en las concentraciones de arsénico urinario por sexo y genotipo. La mediana de concentración de arsénico en el agua fue de 82 μg/L, los niveles de excreción urinaria del ácido dimetilarsínico (DMA) fueron más altos en mujeres que en los hombres. Los portadores de la variante genética CC del gen As3MT (rs3740393) presentaron concentraciones urinarias superiores de ácido monometilarsónico (p=0,01) y DMA (p=0,05).

ABSTRACT The aim of this study was to assess the relationship of the rs11191439 and rs3740393 polymorphisms of the Arsenite Methyltransferase (As3MT) gene with the arsenic urinary excretion profiles in a population in northern Mexico. A crosssectional study was conducted with 102 participants between the ages of 14 and 75 in Comarca Lagunera, Mexico. Speciation of the arsenic urinary metabolites was determined, and DNA was extracted; analysis and amplification of the polymorphisms was made by PCR in real time. A descriptive analysis was carried out and the differences in the urinary arsenic concentrations were assessed according to sex and genotype. The median concentration of arsenic in water was 82 μg/L; the levels of urinary excretion of dimethylarsinic acid (DMA) were higher in women than in the men. The carriers of the CC genetic variant of the As3MT (rs3740393) gene showed higher urinary concentrations of methylarsinic acid (p=0.01) and DMA (p=0.05).

Caracterização de 16s rRNA metiltransferases produzidas por bacilos gram-negativos isolados em diversos hospitais do Estado de São Paulo / Characterization of 16s rRNA methyltransferases produced by gram-negative bacilli isolated in several hospitals in the State of São Paulo

Os aminoglicosídeos (AGs) são antimicrobianos utilizados no tratamento de infecções graves causadas por bacilos Gram-negativos (BGNs), e são umas das poucas opções terapêuticas contra microrganismos multirresistentes no ambiente hospitalar. Contudo, há diversos relatos da produção de enzimas...(AU)

Leucemia linfoblástica aguda infantil: una aproximación genómica / Acute lymphoblastic leukemia: a genomic perspective

Resumen: En paralelo al proyecto de la secuenciación del genoma humano, se han desarrollado varias plataformas tecnológicas que están permitiendo ganar conocimiento sobre la estructura del genoma de las entidades humanas, así como evaluar su utilidad en el abordaje clínico del paciente. En la leucemia linfoblástica aguda (LLA), el cáncer infantil más común, las herramientas genómicas prometen ser útiles para detectar a los pacientes con alto riesgo de recaída, ya sea al diagnóstico o durante el tratamiento (enfermedad mínima residual), además de que permiten identificar los casos en riesgo de presentar reacciones adversas a los tratamientos antineoplásicos y ofrecer una medicina personalizada con esquemas terapéuticos diseñados a la medida del paciente. Un ejemplo claro de esto último es la identificación de polimorfismos de un solo nucleótido (SNPs) en el gen de la tiopurina metil transferasa (TPMT), donde la presencia de dos alelos nulos (homocigotos o heterocigotos compuestos) indica la necesidad de reducir la dosis de la mercaptopurina hasta en un 90% para evitar efectos tóxicos que pueden conducir a la muerte del paciente. En esta revisión se proporciona una visión global de la genómica de la LLA, describiendo algunas estrategias que contribuyen a la identificación de biomarcadores con potencial utilidad en la práctica clínica.

Abstract: In parallel to the human genome sequencing project, several technological platforms have been developed that let us gain insight into the genome structure of human entities, as well as evaluate their usefulness in the clinical approach of the patient. Thus, in acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, genomic tools promise to be useful to detect patients at high risk of relapse, either at diagnosis or during treatment (minimal residual disease), and they also increase the possibility to identify cases at risk of adverse reactions to chemotherapy. Therefore, the physician could offer patient-tailored therapeutic schemes. A clear example of the useful genomic tools is the identification of single nucleotide polymorphisms (SNPs) in the thiopurine methyl transferase (TPMT) gene, where the presence of two null alleles (homozygous or compound heterozygous) indicates the need to reduce the dose of mercaptopurine by up to 90% to avoid toxic effects which could lead to the death of the patient. In this review, we provide an overview of the genomic perspective of ALL, describing some strategies that contribute to the identification of biomarkers with potential clinical application.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Combined therapy of Ulmo honey (Eucryphia cordifolia) and ascorbic acid to treat venous ulcers / Terapia combinada com mel de Ulmo (Eucryphia cordifolia) e ácido ascórbico para úlceras venosas / Terapia combinada con miel de Ulmo (Eucryphia cordifolia) y ácido ascórbico en úlceras venosas

OBJECTIVE: to assess the clinical effect of topical treatment using Ulmo honey associated with oral ascorbic acid in patients with venous ulcers. METHOD: longitudinal and descriptive quantitative study. During one year, 18 patients were assessed who were clinically diagnosed with venous ulcer in different stages, male and female, adult, with a mean injury time of 13 months. Ulmo honey was topically applied daily. The dressing was applied in accordance with the technical standard for advanced dressings, combined with the daily oral consumptions of 500 mg of ascorbic acid. The monitoring instrument is the assessment table of venous ulcers. RESULTS: full healing was achieved in 100% of the venous ulcers. No signs of complications were observed, such as allergies or infection. CONCLUSION: the proposed treatment showed excellent clinical results for the healing of venous ulcers. The honey demonstrated debriding and non-adherent properties, was easy to apply and remove and was well accepted by the users. The described results generated a research line on chronic wound treatment. .

OBJETIVO: avaliar o efeito clínico de tratamento tópico com mel de Ulmo associado à administração oral de ácido ascórbico em pacientes portadores de úlceras venosas. MÉTODO: estudo quantitativo descritivo longitudinal. Um total de 18 pacientes adultos, ambos os sexos, clinicamente diagnosticados com úlcera venosa em diferentes estágios e com duração de 13 meses em média, foram avaliados pelo período de um ano. A aplicação tópica diária de mel de Ulmo foi realizada de acordo com a norma técnica de tratamento avançado combinada com o consumo diário de 500 mg de ácido ascórbico. O instrumento usado para monitoramento foi a tabela de avaliação de úlceras venosas. RESULTADOS: cicatrização completa foi observada em 100% das úlceras venosas. Não foram observados sinais de complicação tais como alergias ou infecção. CONCLUSÃO: o tratamento proposto apresentou resultados clínicos excelentes na cicatrização das úlceras venosas. Além de favorecer o debridamento, o mel não é aderente, é fácil de aplicar e remover, e é de fácil aceitação por parte dos usuários. Os resultados descritos geraram uma linha investigativa no tratamento de feridas crônicas. .

OBJETIVO: evaluar el efecto clínico del tratamiento con miel de Ulmo tópico asociado a ácido ascórbico oral en pacientes portadores de úlceras venosas. MÉTODO: estudio cuantitativo descriptivo longitudinal. Durante el período de un año se evaluaron 18 pacientes diagnosticados clínicamente de úlcera venosa en sus diferentes estadios, de ambos sexos, adultos, con 13 meses promedio de antigüedad de la lesión. Se realizó la aplicación tópica diaria de miel de Ulmo con curación según la norma técnica de curaciones avanzadas, combinada con el consumo oral diario de 500 mg de ácido ascórbico. El instrumento de seguimiento es la tabla de valoración de úlceras venosas. RESULTADOS: se logró la cicatrización total en el 100% de las úlceras venosas. No se observaron signos de complicación, tales como alergias o infección. CONCLUSIÓN: el tratamiento propuesto mostró excelentes resultados clínicos en la cicatrización de úlceras venosas, presentando la miel propiedades debridantes, no adherentes, fácil de aplicar, remover y aceptación del usuario. Los resultados descritos generaron una línea investigativa en el tratamiento de heridas crónicas. .

Impairing methylations at ribosome RNA, a point mutation-dependent strategy for aminoglycoside resistance: The rsmG case / Mutaciones en genes modificadores de ARN ribosómico y la resistencia a aminoglucósidos: el caso del gen rsmG

Introduction: Aminoglycosides like streptomycin are well-known for binding at specific regions of ribosome RNA and then acting as translation inhibitors. Nowadays, several pathogens have been detected to acquire an undefined strategy involving mutation at non structural ribosome genes like those acting as RNA methylases. rsmG is one of those genes which encodes an AdoMet-dependent methyltransferase responsible for the synthesis of m 7 G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m 7 G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. Objectives: After taking into account genetic information indicating that some clinical isolates of human pathogens show streptomycin resistance associated with mutations at rsmG , we decided to explore new hot spots for mutation capable of impairing the RsmG in vivo function and of promoting low-level streptomycin resistance. Materials and methods: To gain insights into the molecular and genetic mechanism of acquiring this aminoglycoside resistance phenotype and the emergence of high-level streptomycin resistance in rsmG mutants, we mutated Escherichia coli rsmG and also performed a genotyping study on rpsL from several isolates showing the ability to grow at higher streptomycin concentrations than parental strains. Results: We found that the mutations at rpsL were preferentially present in these mutants, and we observed a clear synergy between rsmG and rpsL genes to induce streptomycin resistance. Conclusion: We contribute to understand a common mechanism that is probably transferable to other ribosome RNA methylase genes responsible for modifications at central sites for ribosome function.

Introducción. Los aminoglucósidos son moléculas antibióticas capaces de inhibir la síntesis de proteínas bacterianas tras su unión al ribosoma procariota. La resistencia a aminoglucósidos está clásicamente asociada a mutaciones en genes estructurales del ribosoma bacteriano; sin embargo, varios estudios recientes han demostrado, de forma recurrente, la presencia de un nuevo mecanismo dependiente de mutación que no involucra genes estructurales. El gen rsmG es uno de ellos y se caracteriza por codificar una metiltransferasa que sintetiza el nucleósido m 7 G527 localizado en el loop 530 del ribosoma bacteriano, este último caracterizado como sitio preferencial al cual se une la estreptomicina. Objetivo. Partiendo de las recientes asociaciones clínicas entre las mutaciones en el gen rsmG y la resistencia a estreptomicina, este estudio se propuso la caracterización de nuevos puntos calientes de mutación en este gen que puedan causar resistencia a estreptomicina usando Escherichia coli como modelo de estudio. Materiales y métodos. Se indagó sobre el mecanismo genético y molecular por el cual se adquiere la resistencia a estreptomicina y su transición a la resistencia a altas dosis mediante mutagénesis dirigida del gen rsmG y genotipificación del gen rpsL . Resultados. Se encontró que la mutación N39A en rsmG inactiva la proteína y se reportó un nuevo conjunto de mutaciones en rpsL que confieren resistencia a altas dosis de estreptomicina. Conclusiones. Aunque los mecanismos genéticos subyacentes permanecen sin esclarecer, se concluyó que dichos patrones secuenciales de mutación podrían tener lugar en otros genes modificadores del ARN bacteriano debido a la conservación evolutiva y al papel crítico que juegan tales modificaciones en la síntesis de proteínas.

Genotipo y fenotipo de la enzima tiopurina metiltransferasa en población chilena / Phenotype and genotype of thiopurine methyltransferase in Chilean individuals

Background: Thiopurines (azathioprine and 6-mercaptopurine) are highly effective medications but with potential adverse effects. Thiopurine methyltransferase (TMPT) is the key enzyme in their pharmacokinetics and is genetically regulated. A low activity of TPMT is associated with myelotoxicity. The genotype and enzyme activity can vary by ethnicity. Aim: To study the activity and genotype of TPMT in a group of Chilean subjects. Material and Methods: In 200 healthy adult blood donors, TPMT activity was determined by high performance liquid chromatography (HPLC). Deficient, low, normal or high levels were defined when enzymatic activity was < 5, 6-24,25-55 and > 56 nmol/grHb/h, respectively. Genotyping of TPMT (*1, *2, *3A, *3B, *3C) was performed by PCR. Results: Seventy seven women (38.5%) and 123 men (61.5%), with an average age of 34.9 years were studied. Eighteen subjects (9%) had a low enzymatic activity, 178 (89%) had normal activity, 4 (2%) had high activity and no genotype deficient subjects were identified. The wild type genotype (*1) was found in 184 (92%) individuals and 16 (8%) were heterozygous for the variants: *2 (n = 2), *3A (n = 13) and *3C (n = 1). No homozygous subjects for these variants were identified. Wild type genotype had an increased enzymatic activity (40.8 ± 7.2 nmol/gHb/h) compared to heterozygous group (21.2 ± 3 nmol/ gHb/h; p < 0.001). Conclusions: Less than 10% of a Chilean population sample has a low enzymatic activity or allelic variants in the TPMT gene, supporting the use of thiopurines according to international recommendations.

Toxicity of azathioprine: why and when? analysis of the prevalence of polymorphism in Joinville, SC, Brazil / Toxicidade da azatioprina: por que e quando? análise da prevalência de polimorfismo em Joinville, SC

CONTEXT: The use of thiopurine drugs such as azathioprine and 6-mercaptopurine has become quite common in the treatment of inflammatory bowel disease, transplantation and acute leukemias. Despite their effectiveness, these drugs are capable of causing drug-induced toxicity with the risk of death by myelosuppression. It is now known that these complications occur because of genetic polymorphisms of the thiopurinemethyltransferase (TPMT) enzyme, responsible for its metabolism. OBJECTIVE: To assess the prevalence of thiopurine methyltransferase polymorphisms in the population of Joinville, SC, Brazil. METHODS: We analyzed the frequency of four main allelic variants of the TPMT gene in 199 blood donors from Joinville, from February to April 2010. RESULTS: The normal allele ("wild-type") was found in 93.9% of subjects studied. TPMT variants were detected in 12 subjects (6.03%). CONCLUSIONS: From this study, it was estimated at 6% the risk of toxicity by the administration of azathioprine and 6-mercaptopurine to patients in Joinville.

CONTEXTO: A utilização de drogas tiopurinas como a azatioprina e a 6-mercaptopurina tem se tornado bastante frequente no tratamento de doenças inflamatórias intestinais, transplantes e leucemias agudas. Apesar de sua efetividade, estas drogas são capazes de causar toxicidade droga-induzida com risco de morte através de mielossupressão. Sabe-se hoje que estas complicações ocorrem em decorrência de polimorfismos genéticos da enzima tiopurina metiltransferase (TPMT), responsável por sua metabolização. OBJETIVOS: Avaliar a prevalência do polimorfismo do gene da TPMT na população de Joinville, SC. MÉTODOS: Foi analisada a frequência das quatro principais variantes alélicas do gene da TPMT em 199 doadores de sangue da cidade de Joinville, SC, no período de fevereiro a abril de 2010. RESULTADOS: O alelo normal ("selvagem") foi encontrado em 93,9% dos indivíduos estudados. Variantes da TPMT foram detectadas em 12 sujeitos (6,03%). CONCLUSÕES: A partir do presente estudo, pode-se estimar em cerca de 6% o risco de toxicidade na administração de azatioprina e 6-mercaptopurina a pacientes em Joinville.

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85 percent of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.

Análisis de los fenotipos y genotipos de resistencia a eritromicina y clindamicina en cepas de Streptococcus pyogenes aisladas en Chile en un período de 10 años / Resistance phenotypes and genotypes of Streptococcus pyogenes clinical isolates in Chile over a 10-year period

Background: Macrolide and lincosamide resistance in Streptococcus pyogenes is due to the acquisition of mef, ermB and ermA genes, which confer different resistance phenotypes, namely M, MLSBconstitutive and MLSBinducible respectively. The last report of resistance in Chile was done in the period 1990-1998, in which resistance to macrolides was 5.4 percent, with M phenotype as the predominant one. Aim: To characterize the evolution of erythromycin and clindamycin resistance and their associated genes in S. pyogenes strains isolated from patients with invasive and non-invasive infections in the period 1996 to 2005. Material and Methods: Resistance to erythromycin and clindamycin was determined in 1,282 clinical isolates using the disk diffusion test. Resistant isolates were analyzed by polymerase chain reaction (PCR) for the presence of the above mentioned resistance genes. Results: Global resistance to erythromycin and clindamycin was 3.5 and 0.7 percent respectively. Eighty percent of the resistant strains possessed the M. phenotype. Conclusions: Resistance levels of S. pyogenes have decreased in Chile in the last years. Most resistant strains have M phenotype in contrast to many countries in which the MLSB constitutive phenotype is the predominant one.