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1.
Medwave ; 20(10)30-11-2020.
Artigo em Inglês, Espanhol | LILACS | ID: biblio-1145814

RESUMO

Introducción Los resultados del confinamiento obligatorio han sido perjudiciales en varios aspectos. No obstante, han surtido efecto en lograr el descenso de casos activos de COVID-19. Chile ha comenzado la desescalada y precisa conocer el mejor momento para poner fin a las restricciones. Objetivos Discutir las mejores condiciones y garantías para el fin del confinamiento obligatorio sobre la base de los casos nuevos, casos activos y positividad de exámenes de reacción en cadena de la polimerasa. Métodos Estudio basado en un modelo de tendencia con estimación de predicciones. Los datos de las variables de interés fueron sometidas a estudios de regresión lineal, con el objeto de determinar la curva que mejor explicaba los datos. Se estimó el coeficiente de determinación, la desviación estándar de y en x y el intervalo de confianza de la curva observada. Posteriormente, fue escogida la curva de tendencia en concordancia con las estimaciones de regresión. Resultados Se encontró que todas las variables dependientes tendían a disminuir con el tiempo de forma cuadrática, con excepción de la variable casos nuevos. En general, las estimaciones de coeficiente de determinación (R2) y error porcentual absoluto medio son satisfactorias, con excepción de la variable: número de exámenes de reacción en cadena de la polimerasa por día. Conclusiones Se deben tomar medidas graduales y cautelosas antes de poner fin al confinamiento obligatorio. En la actual desescalada, se deben aumentar los exámenes de reacción en cadena de la polimerasa diarios y mantener vigilancia en los indicadores de incidencia, prevalencia y positividad de dichos exámenes. La evidencia sugiere con cierto grado de confiabilidad que el confinamiento obligatorio podría levantarse de forma segura a contar del día 30 de agosto de 2020. Se deben hacer preparativos a largo plazo en contención de las futuras olas, es decir, una nueva alza de casos nuevos y activos luego del descenso.


Introduction The results of mandatory confinement have been detrimental in several respects. Nonetheless, they have resulted in reducing the number of active cases of COVID-19. Chile has begun the de-escalation and needs to know the best time to end the restrictions. Objective We discuss the best conditions and guarantees for the end of compulsory confinement. Methods This study is based on a trend model with prediction estimation. The data of the variables of interest were subjected to linear regression studies to determine the curve that best explained the data. The coefficient of determination, the standard deviation of y in x, and the confidence interval of the observed curve were estimated. The trend curve was chosen in accordance with the regression estimates. Outcomes It was found that all dependent variables tended to decrease over time in a quadratic fashion, except for the new cases variable. In general, the R2 and MAPE estimates are satisfactory, except for the variable number of PCR tests per day. Conclusions Gradual and cautious steps should be taken before ending mandatory confinement. In the current de-escalator, daily PCR tests should be increased, maintaining vigilance on indicators of incidence, prevalence, and positivity of PCR tests. Evidence suggests with some degree of confidence that mandatory confinement could be safely lifted as of August 30, 2020. Long-term preparations must be made to contain future waves of new cases.


Assuntos
Pneumonia Viral/prevenção & controle , Pneumonia Viral/epidemiologia , Quarentena/normas , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Pandemias , Betacoronavirus , Intervalos de Confiança , Modelos Lineares , Chile/epidemiologia , Incidência , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências
2.
J Infect Dis ; 217(7): 1060-1068, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29294035

RESUMO

Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building.


Assuntos
Anticorpos Antivirais/sangue , Dengue/diagnóstico , Infecção por Zika virus/diagnóstico , Anticorpos Antivirais/imunologia , Qualidade de Produtos para o Consumidor , Dengue/história , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Vírus da Dengue/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/história , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/tendências , História do Século XX , História do Século XXI , Humanos , Vigilância da População , Reação em Cadeia da Polimerase Via Transcriptase Reversa/história , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Sensibilidade e Especificidade , Zika virus/genética , Zika virus/imunologia , Zika virus/isolamento & purificação , Infecção por Zika virus/história , Infecção por Zika virus/virologia
3.
Curr Pharm Biotechnol ; 11(1): 113-6, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19929820

RESUMO

A variety of technologies can be used in the detection of contagious pathogens. In the early stage of an outbreak of a new infectious disease, rtPCR is advantageous over many other assays. The rtPCR can be developed either using low fidelity DNA polymerase or high fidelity DNA polymerase. The application of high fidelity DNA polymerase allows the shortening of assay development. In addition, the synthesized DNA template used as positive controls is suggested for shortening the time for assay development. Overall comparison of time required for assay development, specificity, and sensitivity for different types of molecular diagnostic technologies, it seems that early confirmation of viral infected patients will be diagnosed primarily with PCR or rtPCR-based assays presently and likely for the near future.


Assuntos
DNA Viral/genética , Testes Genéticos/tendências , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/diagnóstico , Influenza Humana/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Análise de Sequência de DNA/tendências , Sequência de Bases , Previsões , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Dados de Sequência Molecular
4.
Rev Sci Tech ; 28(1): 233-43, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19623743

RESUMO

Avian influenza has become a serious concern from both veterinary and public health points of view. National and international organisations, veterinary health authorities, research institutions, diagnostic laboratories and field services make enormous efforts worldwide to detect, combat and prevent this important disease. Accordingly, the standard diagnostic protocols are being supported by a wide variety of molecular detection techniques, including improved polymerase chain reaction assays, microarray-based detection and characterisation methods, very rapid sequencing, simple pen-side tests and other on-site approaches. These recently developed 'closer to the field' methods allow rapid detection of influenza viruses and the identification of pathogenicity variants. However, in order to harmonise the diagnosis worldwide, attention has to be paid to the validation and standardisation of these technologies, to avoid erroneous interpretation of assay results, and, consequently, inappropriate epidemiological measures. This review gives an overview of the current and potential future developments related to avian influenza diagnostics.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Animais , Aves , Diagnóstico Diferencial , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Nanotecnologia , Doença de Newcastle/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária
5.
Euro Surveill ; 14(15)2009 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-19371514

RESUMO

Increasing rates of Clostridium difficile infection (CDI) with an unusual, severe course have been reported in several countries; this rise has partly been ascribed to the emergence of a virulent strain, C. difficile PCR ribotype 027 (CD027). An intriguing question is whether this could be related to increasing consumption of broadspectrum antibiotics. From 1997 to 2007, the number of hospital discharges in Denmark with the diagnosis enterocolitis caused by C. difficile increased from eight to 23 per 100,000 hospital discharges. This increase was proportional to a concomitant rise in the consumption of fluoroquinolones and cephalosporins. The first outbreak of CD027 in Denmark occurred from October 2006 to August 2007 and included 13 patients, most of them elderly, admitted to three hospitals in the same region. Most of the patients had overlapping periods of admission. All patients had been treated with broadspectrum antibiotics, in particular cephalosporins and fluoroquinolones, prior to positive culture of CD027. 30 days after confirmation of diagnosis, three of the 13 patients had died. Taken together, the data support the hypothesis that the increasing use of certain broadspectrum antibiotics may be related to a possible increase of C. difficile infection, and show that the specific contribution by CD027 in its emergence needs to be determined.


Assuntos
Cefalosporinas/efeitos adversos , Fluoroquinolonas/efeitos adversos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Ribotipagem/tendências , Idoso , Idoso de 80 Anos ou mais , Cefalosporinas/administração & dosagem , Dinamarca , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Enterocolite Pseudomembranosa/induzido quimicamente , Enterocolite Pseudomembranosa/genética , Feminino , Fluoroquinolonas/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
6.
Zhonghua Bing Li Xue Za Zhi ; 37(8): 512-6, 2008 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-19094461

RESUMO

OBJECTIVE: To investigate the methylation status of 5'CpG island of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) in colorectal cancer and its relationship with gene expression and clinicopathologic parameters. METHODS: Semi-quantitative reverse transcription-PCR (RT-PCR) was used to detect the expression of IGFBP-rP1 in 46 cases of colorectal cancer and their matched normal mucosa. Methylation-specific PCR (MSP) was applied to evaluate the methylation status of 5'CpG island of IGFBP-rP1. Colon cancer cell lines LoVo and SW620 were treated with demethylation agent 5-aza-2'-deoxycytidine (5-aza-dC), followed by RT-PCR and MSP detection. RESULTS: At the mRNA level, the expression of IGFBP-rP1 was higher in colorectal cancer tissue than that in the matched normal mucosa (P < 0.05). IGFBP-rP1 was methylated in 28/46 (60.9%) cases of colorectal cancer and 37/46 (80.4%) matched normal mucosa samples (P < 0.05). A negative correlation was found between IGFBP-rP1 expression and its methylation status. The expression of IGFBP-rP1 was restored in LoVo and SW620 after treatment with 5-aza-dC and MSP confirmation of its demethylation status. No relationships was found between the methylation status and clinicopathologic parameters. CONCLUSIONS: IGFBP-rP1 expression is negatively correlated with its methylation status in colorectal cancer. DNA methylation is one of the mechanisms regulating the expression of IGFBP-rP1. Hypomethylation of IGFBP-rP1 gene with its overexpression plays an important role in the initiation and development of colorectal cancer.


Assuntos
Neoplasias Colorretais/genética , Ilhas de CpG/fisiologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Decitabina , Feminino , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Transcrição Genética
7.
Biotechniques ; 44(5): 619-26, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18474036

RESUMO

Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput.


Assuntos
Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/tendências , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências
8.
J. physiol. biochem ; 64(1): 61-66, ene.-mar. 2008. tab, graf
Artigo em Inglês | IBECS | ID: ibc-61324

RESUMO

The main goal of this study was to compare the expression of Zinc-á2-glycoprotein(ZAG), a recently described adipokine, in obese and lean subjects. ZAG expressionwas determined by Real-time PCR analysis in subcutaneous abdominal adiposetissue of eighteen young men, 9 lean (BMI=23.1±0.4 kg/m2) and 9 obese (34.7±1.2kg/m2) with a similar habitual dietary intake of fat and physical activity, which wereassessed by validated methods. Our data revealed that ZAG gene was downregulated(–70%; p<0.05) in subcutaneous adipose tissue of obese compared to lean subjects.Moreover, statistically significant positive correlations between ZAG gene expressionand serum adiponectin (r=0.89; p<0.01) and a negative correlation with the plasmalevels of leptin (r=–0.82; p<0.05) and waist circumference (r=–0.64; p<0.05) werefound in obese subjects. Our data suggest that this novel adipokine could play a rolein human susceptibility to obesity related disorders and that upregulation of ZAGcould be a promising therapeutic target for metabolic syndrome treatment (AU)


No disponible


Assuntos
Humanos , Adulto , Masculino , Obesidade/metabolismo , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismo , Gordura Abdominal/fisiologia , Adipocinas/genética , Peso Corporal/fisiologia , Regulação para Baixo/fisiologia , Expressão Gênica/fisiologia , Metabolismo dos Lipídeos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Obesidade/fisiopatologia , Adipocinas/metabolismo , Obesidade/epidemiologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Fatores de Risco , Gordura Subcutânea/fisiologia , Tecido Adiposo/metabolismo , Obesidade/dietoterapia
9.
Toxicology ; 243(3): 303-10, 2008 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-18068885

RESUMO

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg-siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , RNA Interferente Pequeno/genética , Animais , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Humanos , Imunoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Transfecção
10.
Expert Rev Mol Diagn ; 7(3): 231-6, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17489730

RESUMO

Inappropriate and inaccurate antimicrobial therapy can lead to adverse patient outcomes and also the development of antimicrobial resistance. Peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) gives rapid reporting with highly sensitive and specific results to clinicians within 3 h after blood cultures turn positive, thereby offering targeted therapeutics where necessary. It is simple to establish compared with real-time PCR and has resulted in significant cost savings for hospitals. PNA FISH is a promising future technology for the microbiology laboratory that will impact on patient management and clinical guidelines. This article will review the clinical data supporting these new technologies.


Assuntos
Infecções Bacterianas/diagnóstico , Infecções Bacterianas/terapia , Candidíase/diagnóstico , Candidíase/terapia , Previsões , Hibridização in Situ Fluorescente/tendências , Ácidos Nucleicos Peptídicos , Infecções Bacterianas/economia , Candidíase/economia , Humanos , Hibridização in Situ Fluorescente/economia , Hibridização in Situ Fluorescente/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências
11.
Brain Res ; 1118(1): 232-8, 2006 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-16996035

RESUMO

Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.


Assuntos
Expressão Gênica/genética , Hibridização In Situ/métodos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Comunicação Celular/genética , Contagem de Células/métodos , Conexina 43/genética , Convulsivantes , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Epilepsia/genética , Epilepsia/metabolismo , Junções Comunicantes/genética , Hipocampo/anatomia & histologia , Hipocampo/metabolismo , Citometria por Imagem , Hibridização In Situ/tendências , Ácido Caínico , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências
12.
Methods Mol Biol ; 334: 221-40, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16861767

RESUMO

Since the first publication on the method of in situ polymerase chain reaction (PCR), several thousand research papers have appeared in peer-reviewed journals describing various findings based solely on the application of this method or combined with other more robust methods, including solution-based PCR, immunohistochemistry, Southern blot, etc. A few years after the advent of PCR, several investigators developed in situ PCR methods that differed considerably from each other with regard to tissue preparations, fixation, mounting of slides, reverse transcription technique, primer design, target selection, size, and amplicon size, and thermocycler designs and the use, among many other fine details. This chapter describes the detail procedures that are used in the author's laboratory. It also discusses the variations and modification that can be used for the specific needs of an investigator. This protocol should serve as a primer for the investigators, and each researcher must use his or her variation according to their needs.


Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Células Cultivadas , Primers do DNA/química , DNA Complementar/biossíntese , Dietil Pirocarbonato/química , Endopeptidase K/metabolismo , Nucleotídeos/química , RNA/química , Ribonucleases/metabolismo , Coloração e Rotulagem , Fixação de Tecidos
13.
Toxicol Sci ; 94(1): 139-52, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16772331

RESUMO

Bioactive retinoids are potent limb teratogens, upregulating apoptosis, decreasing chondrogenesis, and producing limb-reduction defects. To target the origins of these effects, we examined gene expression changes in the developing murine limb after 3 h of culture with teratogenic concentrations of vitamin A. Embryonic day 12 CD-1 limbs were cultured in the absence or presence of vitamin A (retinol acetate) at 1.25 and 62.5muM (n = 5). Total RNA was used to probe Atlas 1.2 cDNA arrays. Eighty-one genes were significantly upregulated by retinol exposure; among these were key limb development signaling molecules, extracellular matrix and adhesion proteins, oncogenes, and a large number of transcriptional regulators, including Eya2, Id3, Snail, and Hes1. To relate these expression changes to teratogenic outcome, the response of these four genes was assessed after culture with vitamin A and retinoid receptor antagonists that are able to rescue retinoid-induced malformations; expression levels were correlated with limb malformations. Lastly, pathways analysis revealed that a large number of the genes significantly affected by retinoid treatment are functionally linked through direct interactions. Several regulatory gene cascades emerged relevant to morphogenesis, cell-fate, and chondrogenesis; moreover, members of these cascades crosstalk with one other. These results indicate that retinoids act in a coordinated fashion to disrupt development at multiple levels. In sum, this work proposes several unifying mechanisms for retinoid-induced limb malformations, identifies novel retinoid targets, and highlights Eya2, Id3, Snail, and Hes1 as potential key teratogenic effectors.


Assuntos
Membro Anterior/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Organogênese/genética , Vitamina A/análogos & derivados , Animais , Apoptose/genética , Diferenciação Celular/genética , Análise por Conglomerados , Diterpenos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Membro Anterior/embriologia , Membro Anterior/metabolismo , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Transdução de Sinais/genética , Teratogênios/toxicidade , Fatores de Tempo , Técnicas de Cultura de Tecidos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Vitamina A/toxicidade
14.
Prog Neurobiol ; 76(3): 153-68, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16168556

RESUMO

Transcriptomics and proteomics are increasingly applied to gain a mechanistic insight into neurodegenerative disorders. These techniques not only identify distinct, differentially expressed mRNAs and proteins but are also employed to dissect signaling pathways and reveal networks by using an integrated approach. In part I of this back-to-back review, technical aspects are discussed: in the transcriptomics section, which includes enrichment by laser microcapture dissection, we comment on qRT-PCR, SAGE, subtractive hybridization, differential display and microarrays, including software packages. In the proteomics section we discuss two-dimensional (2D) gel electrophoresis, liquid chromatography, methods to label and enrich specific proteins or peptides, and different types of mass spectrometers. These tools have been applied to a range of neurodegenerative disorders and are discussed and integrated in part II (Functional Genomics meets neurodegenerative disorders. Part II: application and data integration).


Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Doenças Neurodegenerativas/genética , Proteômica/métodos , Animais , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel Bidimensional/tendências , Perfilação da Expressão Gênica/tendências , Genômica/tendências , Humanos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/tendências , Proteômica/tendências , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências
15.
Adv Physiol Educ ; 29(3): 151-9, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16109794

RESUMO

In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene expression as a result of physiology, pathophysiology, or development. This method can be applied to model systems to measure responses to experimental stimuli and to gain insight into potential changes in protein level and function. Thus physiology can be correlated with molecular events to gain a better understanding of biological processes. For clinical molecular diagnostics, real-time PCR can be used to measure viral or bacterial loads or evaluate cancer status. Here, we discuss the basic concepts, chemistries, and instrumentation of real-time PCR and include present applications and future perspectives for this technology in biomedical sciences and in life science education.


Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Animais , Disciplinas das Ciências Biológicas/educação , Expressão Gênica/genética , Humanos , Análise Serial de Proteínas/métodos , Análise Serial de Proteínas/tendências
16.
J Biotechnol ; 112(3): 225-45, 2004 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-15313001

RESUMO

DNA microarray technology has been widely used to simultaneously determine the expression levels of thousands of genes. A variety of approaches have been used, both in the implementation of this technology and in the analysis of the large amount of expression data. However, several practical issues still have not been resolved in a satisfactory manner, and among the most critical is the lack of agreement in the results obtained in different array platforms. In this study, we present a comparison of several microarray platforms [Affymetrix oligonucleotide arrays, custom complementary DNA (cDNA) arrays, and custom oligo arrays printed with oligonucleotides from three different sources] as well as analysis of various methods used for microarray target preparation and the reference design. The results indicate that the pairwise correlations of expression levels between platforms are relative low overall but that the log ratios of the highly expressed genes are strongly correlated, especially between Affymetrix and cDNA arrays. The microarray measurements were compared with quantitative real-time-polymerase chain reaction (QRT-PCR) results for 23 genes, and the varying degrees of agreement for each platform were characterized. We have also developed and tested a double amplification method which allows the use of smaller amounts of starting material. The added round of amplification produced reproducible results as compared to the arrays hybridized with single round amplified targets. Finally, the reliability of using a universal RNA reference for two-channel microarrays was tested and the results suggest that comparisons of multiple experimental conditions using the same control can be accurate.


Assuntos
Análise de Falha de Equipamento/métodos , Análise de Falha de Equipamento/normas , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/normas , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/normas , RNA/normas , Benchmarking/métodos , Benchmarking/tendências , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/tendências , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/tendências , RNA/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Sensibilidade e Especificidade , Avaliação da Tecnologia Biomédica/métodos , Avaliação da Tecnologia Biomédica/tendências , Estados Unidos
17.
Rev. argent. transfus ; 30(2): 111-119, abr.-jun. 2004. ilus
Artigo em Espanhol | BINACIS | ID: bin-2895

RESUMO

Los índices de morbilidad y mortalidad en receptores de sangre aumentan drásticamente debido a la transmisión de infecciones virales por vía transfusional. Entre los virus transmitidos por sangre, el de mayor impacto sanitario y social es el virus de la inmunodeficiencia humana tipo 1 (HIV-1). A pesar de que la implementación del tamizaje de anticuerpos para HIV en los Bancos de Sangre de Argentina ha permitido reducir considerablemente la transmisión del virus por vía sanguínea, existe en la actualidad evidencia suficiente de la transmisión de HIV por unidades de sangre con serología negativa. En los Bancos de Sangre de la mayoría de los países europeos y en los Estados Unidos se utilizan, desde el año 1999, técnicas de amplificación de ácidos nucleicos (NAT) para detectar HIV y HCV, con el fin de reducir el período de ventana inmunológica. En este trabajo realizamos una actualización del uso de las técnicas moleculares como herramientas de tamizaje de HIV en la sangre destinada a transfusión y presentamos los resultados preliminares obtenidos a partir del desarrollo de una técnica molecular artesanal: transcripción reversa y reacción en cadena de la polimerasa anidada (RT-Multiplex-Nested PCR), de alta sensibilidad y de bajo costo. Los resultados obtenidos demuestran que la sensibilidad y la especificidad de esta técnica para detectar cepas de HIV regionales están dentro de los límites establecidos por las reglamentaciones internacionales. En esta etapa del trabajo se está realizando la validación de la técnica desarrollada utilizando estándares internacionales. Esta última técnica podrá ser utilizada para el control de la sangre en Córdoba, con el fin de reducir el riesgo de transmisión del virus HIV por esta vía. (AU)


Assuntos
Humanos , Transfusão de Sangue/efeitos adversos , Transfusão de Sangue/mortalidade , Transfusão de Sangue/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Técnicas de Amplificação de Ácido Nucleico/tendências , Programas de Rastreamento , Bancos de Sangue , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Sensibilidade e Especificidade , Padrões de Referência , HIV-1/imunologia , Anticorpos Anti-HIV/análise
18.
Genes Chromosomes Cancer ; 38(3): 274-80, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14506704

RESUMO

One strategy to predict clinical outcome in patients with acute myeloid leukemia (AML) is detection of minimal residual disease (MRD) after achievement of hematologic complete remission (CR). We established a real-time RT-PCR assay by use of TaqMan technology for the identification of MRD by quantification of the most frequent fusion transcripts resulting from t(9;11)(p22;q23). To achieve comparable PCR efficiencies between the different PCR assays, primers were chosen to obtain amplicons of nearly identical lengths. MLL/AF9 copy numbers were normalized to the housekeeping gene porphobilinogen deaminase (PBGD). The sensitivity of the assay, as determined at the cellular level, was comparable to that of qualitative single-round RT-PCR. Samples from eight patients with t(9;11)-positive AML were analyzed. At diagnosis and relapse, normalized copy numbers were positive and ranged from 490 to 5,558. Samples from two of seven patients collected at the time of CR became negative, whereas five cases still had positive normalized copy numbers with values between 5 and 5,286. The implications of MRD detection by MLL/AF9 fusion transcript quantification for the clinical management of t(9;11)-positive AML have to be determined in further studies.


Assuntos
Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 9/genética , Sistemas Computacionais/tendências , Frequência do Gene , Leucemia Mieloide/genética , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Doença Aguda , Adolescente , Adulto , Linhagem Celular Tumoral , Quebra Cromossômica/genética , Células HL-60 , Humanos , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Sensibilidade e Especificidade , Translocação Genética/genética
19.
Nefrología (Madr.) ; 23(2): 165-168, mar.-abr. 2003. ilus
Artigo em Espanhol | IBECS | ID: ibc-044635

RESUMO

Aunque la infección por el virus de la hepatitis C se asocia con varias enfermedadesextrahepáticas, la detección de RNA viral en las estructuras glomerulareses difícil. No obstante, algunos autores lo han encontrado, con resultados dispares.Describimos un paciente con glomerulonefritis membranoproliferativa,crioglobulinemia mixta tipo III e infección por virus de la hepatitis C con presenciade RNA VHC en suero, crioprecipitado y tejido renal mediante RT-PCR.Estos hallazgos apoyan el papel directo del VHC en el daño renal


Although hepatitis C virus infection has been documented in several extrahepaticdiseases, the deposition of HCV RNA in glomerular structures has proved to be difficultto demonstrate. We report a patient with membranoproliferative glomerulonephritis,type III circulating cryoglobulins and hepatitis C virus infection with detectionof HCV RNA in serum, cryoprecipitate and renal tissue using specific RT-PCRtechnique. These data confirm that HCV could have a direct role in renal damage


Assuntos
Masculino , Idoso , Humanos , Crioglobulinemia/etiologia , Crioglobulinemia/patologia , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C Crônica/sangue , Glomérulos Renais/patologia , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carga Viral/métodos , Viremia/complicações , Crioglobulinemia/virologia , Viremia/virologia , Glomérulos Renais/virologia , Crioglobulinemia/imunologia , RNA Viral/isolamento & purificação , Hepatite C Crônica/complicações , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Carga Viral/tendências , Hepatite C Crônica/virologia
20.
Semin Surg Oncol ; 20(4): 304-11, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11747272

RESUMO

The detection and molecular characterization of circulating tumor cells (CTCs) and micrometastases in urinary tract and prostatic tumors may have important prognostic and therapeutic implications. In the last decade, numerous groups have attempted the detection of occult tumor cells in renal, prostatic, and urothelial carcinomas using the highly sensitive reverse-transcriptase polymerase chain reaction (RT-PCR). In prostatic carcinoma (PC), tissue-specific transcripts were detected with high specificity in the blood of patients with localized and advanced disease. PCR assays for PC detection were shown to be strong predictors of poorer outcome in some reports, while a lack of prognostic significance was found in other studies. There was a vast difference in the PCR positivity rates between various groups studying PC. This discrepancy could be due to variations in PCR methodology. In urothelial and renal cell carcinoma, the amount of research on the subject is still too limited. Currently, these assays for occult tumor cells are promising but are not yet ready to use in PC and urinary tract tumors. Because of the many limitations of PCR (e.g., false positives), many groups are developing new approaches for the detection of occult tumor cells. The most attractive technique involves immunomagnetic isolation of intact CTC and micrometastases prior to downstream analysis. The tumor-rich magnetic fraction can be subjected to RT-PCR, immunocytochemistry, and in situ hybridization. This will lead to better quantification and molecular characterization of these tumor cells.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologia , Carcinoma de Células Renais/sangue , Feminino , Humanos , Neoplasias Renais/sangue , Masculino , Neoplasias da Próstata/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/tendências , Sensibilidade e Especificidade , Neoplasias Urológicas/sangue
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