Tipificación con oligonucleótidos espaciadores de Mycobacterium tuberculosis en Cuba / Spacer oligonucleotide typing of Mycobacterium tuberculosis in Cuba

Introducción: el conocimiento de los linajes de Mycobacterium tuberculosis es importante para entender el origen, evolución y propagación de la bacteria. Objetivo: determinar los patrones genéticos de M. tuberculosis circulantes en Cuba. Métodos: estudio descriptivo de corte transversal con un componente analítico en Cuba, en el período comprendido de enero de 2009 a diciembre de 2010. Se aplicó la tipificación con oligonucleótidos espaciadores (Spoligotyping) a 308 aislamientos de M. tuberculosis del período 2009-2010. La clasificación en genotipos se realizó según la base de datos internacional SpolDB4. Los resultados se analizaron además con la herramienta web en línea MIRU-VNTRplus y se compararon con los patrones genéticos de M. tuberculosis identificados en 1993-1995 en Cuba. Resultados: se definieron 79 patrones genotípicos diferentes, de los cuales 46 (62 por ciento) fueron patrones no reportados anteriormente en SpolDB4. Los 22 agrupamientos definidos incluyeron al 75,4 por ciento de los aislamientos estudiados. Se encontraron cinco familias genéticas fundamentales: Beijing (25,6 por ciento), S (19,2 por ciento), LAM (16,9 por ciento), Haarlem (16,9 por ciento) y T (5,8 por ciento). La familia S prevaleció en la región Occidental (OR=3,4; 95 por ciento IC:1,8-6,3; p<0,05), Beijing en el Centro de Cuba (OR=6,7; 95 por ciento IC:3,7-11,9; p<0,05) y LAM (OR=3,0; 95 por ciento IC:1,6-5,6; p<0.05) y Haarlem en la zona Oriental (OR=1,8; 95 por ciento IC:1,0-3,4; p<0,05). Conclusiones: se observó una gran diversidad genética entre los aislamientos de M. tuberculosis circulante en Cuba en 2009-2010. En el país, la estructura genética de la población de M. tuberculosis ha variado en el tiempo con una disminución de genotipos endémicos como Haarlem y T y un aumento significativo de S y Beijing. Estos datos aportan elementos importantes para la epidemiología y control de la TB en Cuba(AU)

Introduction: knowledge about Mycobacterium tuberculosis lineages is important to understand the origin, evolution and spread of this bacterium. Objective: determine the genetic patterns of M. tuberculosis circulating in Cuba. Methods: adescriptive cross-sectional study was conducted with an analytical component in Cuba in the period extending from January 2009 to December 2010. Spacer oligonucleotide typing (Spoligotyping) was applied to 308 M. tuberculosis isolates from the period 2009-2010. Classification into genotypes was carried out according to the international database SpolDB4. Results were additionally analyzed with the online tool MIRU-VNTRplus and compared with the M. tuberculosis genetic patterns found in Cuba in 1993-1995. Results: 79 different genotypic patterns were defined, of which 46 (62 percent) had not been previously reported in SpolDB4. The 22 clusters defined included 75.4 percent of the isolates studied. Five main genetic families were found: Beijing (25.6 percent), S (19.2 percent), LAM (16.9 percent), Haarlem (16.9 percent) and T (5.8 percent). The S family prevailed in the Western region (OR=3.4; CI 95 percent:1.8-6.3; p<0.05), Beijing in Central Cuba (OR=6.7; CI 95 percent:3.7-11.9; p<0.05), and LAM (OR=3.0; CI 95 percent:1.6-5.6; p<0.05) and Haarlem in the Eastern region (OR=1.8; CI 95 percent:1.0-3.4; p<0.05). Conclusions: great diversity was observed among the M. tuberculosis isolates circulating in Cuba in the period 2009-2010. The genetic structure of M. tuberculosis has changed in the country with the passing of time, with a reduction in endemic genotypes like Haarlem and T, and a significant increase in S and Beijing. These data contribute important information for epidemiology and TB control in Cuba(AU)

Sudden Cardiac Death in Brazil: A Community-Based Autopsy Series (2006-2010) / Morte Súbita Cardíaca no Brasil: Análise dos Casos de Ribeirão Preto (2006-2010)

Background: Sudden cardiac death (SCD) is a sudden unexpected event, from a cardiac cause, that occurs in less than one hour after the symptoms onset, in a person without any previous condition that would seem fatal or who was seen without any symptoms 24 hours before found dead. Although it is a relatively frequent event, there are only few reliable data in underdeveloped countries. Objective: We aimed to describe the features of SCD in Ribeirão Preto, Brazil (600,000 residents) according to Coroners’ Office autopsy reports. Methods: We retrospectively reviewed 4501 autopsy reports between 2006 and 2010, to identify cases of SCD. Specific cause of death as well as demographic information, date, location and time of the event, comorbidities and whether cardiopulmonary resuscitation (CPR) was attempted were collected. Results: We identified 899 cases of SCD (20%); the rate was 30/100000 residents per year. The vast majority of cases of SCD involved a coronary artery disease (CAD) (64%) and occurred in men (67%), between the 6th and the 7th decades of life. Most events occurred during the morning in the home setting (53.3%) and CPR was attempted in almost half of victims (49.7%). The most prevalent comorbidity was systemic hypertension (57.3%). Chagas’ disease was present in 49 cases (5.5%). Conclusion: The majority of victims of SCD were men, in their sixties and seventies and the main cause of death was CAD. Chagas’ disease, an important public health problem in Latin America, was found in about 5.5% of the cases. .

Fundamento: Morte súbita cardíaca (MSC) é um evento súbito e inesperado, de causa cardiovascular, que ocorre em menos de uma hora após o início dos sintomas, em indivíduo sem qualquer condição clínica prévia potencialmente fatal ou assintomático nas últimas 24 horas antes do óbito, em caso de morte não testemunhada. Apesar de ser um evento relativamente frequente, há poucos dados confiáveis na literatura sobre países em desenvolvimento. Objetivo: Descrever as características da MSC em Ribeirão Preto (SP 600.000 habitantes) baseando-se nos relatórios de autopsias do Serviço de Verificação de Óbitos do Interior. Métodos: Foram revisados retrospectivamente 4.501 relatórios de autopsias entre 2006 e 2010, para identificar casos de MSC. Foram coletados dados como causa específica do óbito, características demográficas e comorbidades das vítimas, data, local e hora do evento, e se foram realizadas manobras de ressuscitação cardiopulmonar (RCP). Resultados: Foram identificados 899 casos de MSC (20%; razão 30/100.000 habitantes por ano). A principal causa de MSC foi doença arterial coronariana (DAC - 64%), acometendo homens (67%) entre a sexta e a sétima década de vida. A maior parte dos eventos ocorreu durante a manhã, no domicílio (53,3%), e a RCP foi realizada em quase metade das vítimas (49,7%). A comorbidade mais prevalente foi hipertensão arterial sistêmica (57,3%). Doença de Chagas foi detectada em 49 casos (5,5%). Conclusão: A maioria dos casos de MSC ocorreu por DAC em homens entre a sexta e a sétima década de vida. Doença de Chagas, um importante problema de saúde pública na América Latina, foi detectada em 5,5% dos casos. .

Degenerate oligonucleotide primed-PCR technology establishment and its sensitivity test analysis / 法医学杂志

OBJECTIVE@#To establish a whole genome amplification testing system based on degenerate oligonucleotide primed-PCR (DOP-PCR) and to explore its reliability and sensitivity.@*METHODS@#DOP-PCR amplified production was detected by fluorescent labeled multiplex STR amplification and capillary electrophoresis detection system to determine reliability and sensitivity of DOP-PCR system.@*RESULTS@#DOP-PCR system was successfully established and the detection sensitivity reached 5 cells (30 pg) by pretreatment of DOP-PCR and then detection of STR genotyping.@*CONCLUSION@#The system established in this study is reliable and more testing sensitive for forensic trace evidence.

Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.

Identification of a Rare 3 bp BRAF Gene Deletion in a Thyroid Nodule by Mutant Enrichment with 3'-Modified Oligonucleotides Polymerase Chain Reaction

Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumor, and 36-69% of PTC cases are caused by mutations in the BRAF gene. The substitution of a valine for a glutamic acid (V600E) comprises up to 95-100% of BRAF mutations; therefore, most diagnostic methods, including allele-specific PCR and real-time PCR, are designed to detect this mutation. Nevertheless, other mutations can also comprise the genetic background of PTC. Recently, a novel and sensitive technique called mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR has been introduced. When we applied allelespecific PCR and MEMO-PCR for the detection of the BRAF V600E mutation, we found an unusual 3' bp deletion mutation (c.1799_1801delTGA) only when using MEMO-PCR. This deletion results in the introduction of a glutamic acid into the B-Raf activation segment (p.V600_K601delinsE), leading to an elevated basal kinase activity of BRAF. This is the first report of a rare 3 bp BRAF deletion in a PTC patient that could not be detected by allele-specific PCR.

Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking

Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5' single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5' ss tail. A bromoacetyl derivative with a side chain of 6 A was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8A approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5'ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.

Microarreglos: herramienta para el conocimiento de las enfermedades / Microarrays: tool for the knowledge of the diseases

En este artículo analizamos la importancia de los microarreglos como herramienta para el conocimiento de las enfermedades, la forma como se estudian, su clasificación y diferentes tipos, y la forma como se analizan los datos y las aplicaciones clínicas de los microarreglos de ADN

Transfection efficiency comparison of oligonucleotide and plasmid to the HL-60 cell line with liposomes / 华中科技大学学报（医学）（英德文版）

The transfection efficiency of oligonucleotide and plasmid to the HL-60 cell line with lipofectaminePLUS was compared through observing the transfection rate and the expression duration of exogenous gene in the target cells. The results showed that the transfection rate of oligonucleotide to the HL-60 was about 90% - 95% and it had no obvious attenuation within 84 h. However, the plasmid transfection rate was only 5% -25% and it was decreased significantly within 60 h. It was suggested that the transfection of oligonucleotide with liposomes was better than that of plasmid.

Integrones y cassettes genéticos de resistencia a antimicrobianos en cepas de Shigella flexneri / Integrons and antimicrobial resistance gene cassettes in Shigella flexneri strains

BACKGROUND: The resistance of Shigella flexneri to antimicrobial agents can be associated to the presence of integrons that may contain and express antimicrobial resistance gene cassettes. AIM: To study antimicrobial resistance and the presence of integrons and antimicrobial gene cassettes in Shigella flexneri strains. MATERIAL AND METHODS: In vitro susceptibility to 27 antimicrobials was studied in twenty four Shigella flexneri strains isolated from stools. The presence of integrons class 1, 2 and 3 and antimicrobial resistance gene cassettes was investigated by polymerase chain reaction (PCR) using specific primers for each gene. RESULTS: Most strains were resistant to one of the following antimicrobials: ampicillin, sulphonamide, trimethoprim, tetracycline, streptomycin, sulfamethoxazole-trimethoprim or chloramphenicol. Twenty nine percent were simultaneously resistant to all these antimicrobials. Integrons class 1 and 2 were found in 19 strains (79 per cent). Class 3 integrons were not found. Gene cassettes dfrA1 and ant(3")I were associated to integrons class 2 in most strains (15/20, 75 per cent). Genes cat, tetB and blarTEM were detected in 18/24 (75 per cent), 7/24 (29 per cent) and 4/24 (17 per cent) of the strains, respectively and were not associated to any of the studied integrons. Genes that codify enzymes AAC(6')Ib and APH(3')VI were not detected in any strain. CONCLUSIONS: The high frequency of integrons found in the studied strains, could partly explain the increasing antimicrobial resistance of Shigella flexneri strains, isolated in Chile.

Reverse-SSO hybridization provides an accurate and simple HLA-DR typing: a comparative study with HLA-DR serologic typing

The HLA-DR molecule is a polymorphic membrane glycoprotein and one of the key molecules causing allograft rejection and graft-versus-host disease in organ transplantation. Serologic typing using DR specific alloantisera has long been used, but several problems have limited its application. The purpose of this study was to establish an efficient reverse-SSO typing system that detects DRB1 and DRB3/B4/B5 alleles on a single membrane. A DR typing membrane was prepared by immobilizing 21 dT-tailed sequence specific oligonucleotides (SSOs) on a nylon membrane and was used in a hybridization assay with digoxigenin-labeled PCR-amplified target DNA. The positive signals were detected on X-ray film using chemiluminescence. A comparison study with serology using DNAs from 105 unrelated individuals demonstrated that the reverse-SSO typing system was superior to serologic typing in terms of accuracy (100% vs 90.5%), simplicity, range of application, rapidity, and cost of the test. These data indicate that the reverse-SSO typing system can replace serology as a routine DR test, and will be useful in time-restricted solid organ transplantation and in selection of an unrelated marrow donor prior to bone marrow transplantation.