RESUMEN
MicroRNA miR-26a and long noncoding RNA (lncRNA) MEG3 gene have been independently reported to be tumor suppressor genes in various cancers, but neither has been previously associated with tongue squamous cell carcinoma (TSCC). We report here that miR-26a and lncRNA MEG3 gene expression were both strongly reduced in TSCC compared with levels in matched nonmalignant tissues, and combined low expression levels of both miR-26a and MEG3 emerged as an independent prognostic factor for poor clinical outcome in TSCC patients. Assays in the human TSCC cell lines SCC-15 and CAL27 showed that miR-26a targets the DNA methyltransferase 3B transcript and that its inhibition may result in the upregulation of MEG3, providing a plausible link between the observed reduction of miR-26a and MEG3 in TSCC tissue. Furthermore, the overexpression of miR-26a or MEG3 in SCC-15 and CAL27 cells inhibited cell proliferation and cell cycle progression, and promoted cell apoptosis. Considering the poor prognostic outcomes associated with reduced miR-26a and MEG3, our findings imply that these factors likely play important antitumor effects in TSCC pathogenesis. Furthermore, they represent potential prognostic biomarkers for stratification of TSCC patients.
Asunto(s)
Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , ARN Largo no Codificante/genética , Neoplasias de la Lengua/genética , Apoptosis , Western Blotting , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Ciclo Celular , Proliferación Celular , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Femenino , Estudios de Seguimiento , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Neoplasias de la Lengua/mortalidad , Neoplasias de la Lengua/patología , ADN Metiltransferasa 3BRESUMEN
Here, we describe a novel oligonucleotide array-based transcription factor (TF) interaction assay platform that can directly identify cointeracting TF complexes following binding to their regulatory DNA elements. This platform that combines immuno-coprecipitation technology with our previously reported oligonucleotide array-based TF assay (OATFA), is named targeted immuno-coprecipitation OATFA (TIC-OATFA). We illustrate use of the system to identify interaction partners of STAT1 (signal transducer and activator of transcription proteins 1) in mouse fibroblasts. Several previously known partners of STAT1, as well as new partners, were identified by TIC-OATFA, including the upstream stimulatory factors 1 and 2 (USF1, USF2), nuclear factor of activated T cells, TATA box-binding protein, nuclear factor erythroid-derived 2, nuclear factor-kappa B, and nuclear factor 1. Both USF1 and nuclear factor-kappa B are well known to interact with STAT1, but the other five TFs are previously unreported STAT1 interaction partners. We examined interactions between one new TF, USF2, and STAT1 in detail. USF2 belongs to the group of bHLH-zip transcription factors, which in a number of diseases including cancers, has enhanced activity. In summary, a novel oligonucleotide array-based assay platform was developed and used to study interactions between STAT1 and functional TF binding partners, revealing that USF2 and potentially four other new TFs are partners of STAT1 in an IFN-γ stimulated mouse fibroblast cell line.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/metabolismo , Factor de Transcripción STAT1/metabolismo , Factores de Transcripción/metabolismo , Animales , Fibroblastos/metabolismo , Células HeLa , Humanos , Inmunoprecipitación/métodos , Interferón gamma/metabolismo , Ratones , Células 3T3 NIH , Proteómica/métodos , ARN Mensajero/análisis , ARN Mensajero/genética , Factor de Transcripción STAT1/análisis , Factor de Transcripción STAT1/genética , Factores de Transcripción/análisis , Factores de Transcripción/genética , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Hepatitis B virus (HBV) encoded X protein (HBx) has been implicated in apoptotic and related pathogenic events during hepatocellular carcinoma. However, the underlying molecular mechanism through which HBx acts is largely unclear. We used tandem affinity purification under mild conditions to gain insight into the HBx interactome in HBV-producing HepG2.2.15 cells and identified 49 proteins by mass spectrometry that are potentially associated with HBx. Two of the key proteins of the caspase-independent apoptosis pathway were newly identified, apoptosis-inducing factor (AIF) and the homologous AMID (AIF-homologue mitochondrion-associated inducer of death). We confirmed the interactions of HBx with AIF and with AMID by reciprocal coimmunoprecipitation experiments, respectively. We observed the expression of HBx-reduced AIF-mediated apoptosis and HBx colocalization with AIF and AMID, principally in the cytoplasm. Furthermore, the elevated cytoplasmic levels of HBx could inhibit mitochondrion-to-nucleus translocation of AIF. Here, we present the first detailed molecular evidence that HBx can repress apoptosis via inhibition of the caspase-independent apoptosis pathway. This inhibition of apoptosis involves the repression of the mitochondrion-to-nucleus translocation of AIF, although tests with AMID were not conclusive. These findings provide important insights into the new mechanism of the apoptosis inhibition by HBV.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Transactivadores/fisiología , Factor Inductor de la Apoptosis/aislamiento & purificación , Factor Inductor de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/aislamiento & purificación , Proteínas Reguladoras de la Apoptosis/metabolismo , Cromatografía de Afinidad , Células Hep G2 , Interacciones Huésped-Patógeno , Humanos , Proteínas Mitocondriales/aislamiento & purificación , Proteínas Mitocondriales/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas/fisiología , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
UNLABELLED: MicroRNAs (miRNAs) play important roles in the posttranscriptional regulation of gene expression. Recent evidence has indicated the pathological relevance of miRNA dysregulation in hepatitis virus infection; however, the roles of microRNAs in the regulation of hepatitis B virus (HBV) expression are still largely unknown. In this study we identified that miR-373 was up-regulated in HBV-infected liver tissues and that the members of the miRs-371-372-373 (miRs-371-3) gene cluster were also significantly co-up-regulated in HBV-producing HepG2.2.15 cells. A positive in vivo association was identified between hepatic HBV DNA levels and the copy number variation of the miRs-371-3 gene cluster. The enhanced expression of miRs-372/373 stimulated the production of HBV proteins and HBV core-associated DNA in HepG2 cells transfected with 1.3×HBV. Further, nuclear factor I/B (NFIB) was identified to be a direct functional target of miRs-372/373 by in silico algorithms and this was subsequently confirmed by western blotting and luciferase reporter assays. Knockdown of NFIB by small interfering RNA (siRNA) promoted HBV expression, whereas rescue of NFIB attenuated the stimulation in the 1.3×HBV-transfected HepG2 cells. CONCLUSION: Our study revealed that miRNA (miRs-372/373) can promote HBV expression through a pathway involving the transcription factor (NFIB). This novel model provides new insights into the molecular basis in HBV and host interaction.
Asunto(s)
Virus de la Hepatitis B/fisiología , MicroARNs/fisiología , Factores de Transcripción NFI/fisiología , Regiones no Traducidas 3'/fisiología , ADN Viral/análisis , Dosificación de Gen , Células Hep G2 , Humanos , Factores de Transcripción NFI/antagonistas & inhibidoresRESUMEN
Nephrotoxicity is one of the major concerns for anticancer drug safety because most drugs are metabolized and excreted by the kidneys. Convenient tools able to perform rapid in vitro cytotoxicity analysis and identify drug side effects in kidney cells during early phases of drug discovery could be beneficial to drug development programs. Here we developed an electrical cell-substrate impedance sensing system (ECIS) capable of continuously measuring the dosage and time response of human proximal tubular epithelial (HK2) cells exposed to four drugs throughout the experimental period. These drugs induced HK2 cell apoptosis/death in a dose-dependent manner, although with very different dose-response effects. DDP (50 µM) was the most cytotoxic and induced obvious HK2 cell apoptosis rapidly after exposure. The other three drugs had much lower cytotoxicity, even at concentrations approaching 1 mM. The results obtained from our ECIS system correlated well with conventional in vitro assays such as flow cytometry and cell viability assays. Notably, the continuous and automatic measurements provided by ECIS system allow for better resolution for drugs with different temporal toxicity profiles. Furthermore, we investigated the effect of DDP's antidotes, glutathione and sodium subsulfite, on DDP-induced cytotoxicity, both of which decreased nephrotoxicity of DDP in a dose-dependent manner. Overall this study illustrates the convenience of ECIS for direct, continuous assessment of the cytotoxicity of anticancer drugs in vitro. ECIS has the potential to become a useful, non-invasive analytical method for early evaluation of drugs and antidotes of toxins.
Asunto(s)
Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Impedancia Eléctrica , Células Epiteliales/efectos de los fármacos , Riñón/citología , Riñón/efectos de los fármacos , Animales , Antídotos/farmacología , Apoptosis/efectos de los fármacos , Bioensayo/instrumentación , Bioensayo/métodos , Carboplatino/efectos adversos , Carboplatino/farmacología , Línea Celular/efectos de los fármacos , Cisplatino/efectos adversos , Cisplatino/farmacología , Ciclofosfamida/efectos adversos , Ciclofosfamida/farmacología , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Fluorouracilo/efectos adversos , Fluorouracilo/farmacología , Glutatión/farmacología , Humanos , Riñón/metabolismo , Túbulos Renales/citología , Tiosulfatos/farmacologíaRESUMEN
In this paper, we describe a comprehensive general system adapted for quantitative fluorescence resonance energy transfer (FRET) measurement using signals from three channels of a fluorescence instrument. The general FRET measurement system involves two established methods, as well as two novel approaches. Unlike the previous measurements, which can be taken correctly only when the quantity of the acceptor is greater than or equal to that of the donor, one of our novel methods can overcome this obstacle and take quantitative FRET measurements when the donor is in excess of the acceptor. Hence the general FRET measurement system allowed one to determine the exact distance when the donor and acceptor were present in different quantities, and integrated the methods for quantitative FRET measurements. The uniformity of measured values and utility of each method were validated using molecular standards based on DNA oligonucleotide rulers. We also discussed and validated the use of a novel method for estimating the relative quantities of the donor and acceptor fluorophores when they were not known before an appropriate method of this system can be selected.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Oligodesoxirribonucleótidos/química , ADN , Colorantes Fluorescentes , Oligodesoxirribonucleótidos/síntesis químicaRESUMEN
MiRNAs (microRNAs) are small non-coding RNAs involved in mammalian gene expression of cellular processes including differentiation, apoptosis and cancer development. Both specific miRNAs and mRNAs have been identified during monocytic differentiation, but their interactions have not been fully characterized. Here we report that by genome-wide microarray analysis for U937 monocytic differentiation induced by TPA, a large number of miRNAs and mRNAs were differentially expressed, and by bioinformatics analysis could demonstrate that their functional pathway patterns overlap strongly. While expected negative correlation between the expression levels of miRNAs and their target mRNAs was seen, several positive correlations between miRNAs and host mRNAs were also observed, such as C13orf25/miR17, MCM7/miR93, and MGC14376/miR22. These microarray data were verified by quantitative RT-PCR, and the TPA-induced differentiation of U937 cells was confirmed by flow cytometric analysis. Our study suggests an intrinsic correlation between miRNAs and mRNAs underlying their interactions which would provide new insights for defining the mechanisms occurring during monocytic differentiation.
Asunto(s)
Diferenciación Celular/genética , Perfilación de la Expresión Génica , MicroARNs/genética , Monocitos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Carcinógenos/farmacología , Diferenciación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Monocitos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Células U937RESUMEN
In vitro fertilization (IVF) therapy is an important treatment for human infertility. However, the methods for clinical IVF have only changed slightly over decades: culture medium is held in oil-covered drops in Petri dishes and manipulation occurs by manual pipetting. Here we report a novel microwell-structured microfluidic device that integrates single oocyte trapping, fertilization and subsequent embryo culture. A microwell array was used to capture and hold individual oocytes during the flow-through process of oocyte and sperm loading, medium substitution and debris cleaning. Different microwell depths were compared by computational modeling and flow washing experiments for their effectiveness in oocyte trapping and debris removal. Fertilization was achieved in the microfluidic devices with similar fertilization rates to standard oil-covered drops in Petri dishes. Embryos could be cultured to blastocyst stages in our devices with developmental status individually monitored and tracked. The results suggest that the microfluidic device may bring several advantages to IVF practices by simplifying oocyte handling and manipulation, allowing rapid and convenient medium changing, and enabling automated tracking of any single embryo development.
Asunto(s)
Embrión de Mamíferos , Fertilización In Vitro/instrumentación , Microfluídica/instrumentación , Oocitos , Medios de Cultivo , Análisis de Elementos Finitos , HumanosRESUMEN
Two-color DNA microarray platforms are widely used for determining differential amounts of target sequences in parallel between sample pairs. However, the fluorescence (or Forster) resonance energy transfer (FRET) between two fluorophores can potentially result in the distortions of the measured fluorescence signals. Here we assessed the influence of FRET on the two-color DNA microarray platform and developed a reliable and convenient method for the correction of FRET distortion. Compared to current methods of normalization based on the statistical analysis and the hypothesis that only a small part of target sequences are differentially presented between sample pairs, our FRET correction method can recover the undistorted signals by the compensation of fluorescence emission, without considering the number of target sequences differentially presented. The correction method was validated with samples at different target ratios and with microarrays spotted in different probe concentrations. We also applied the FRET correction method to gene expression profiling arrays, and the results show that FRET was present when the content of target sequence was beyond a threshold amount and that the process incorporating our FRET correction method can improve the reliability of the gene expression profiling microarray platform in comparison with the current process without FRET correction.
Asunto(s)
ADN/análisis , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Calibración , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentaciónRESUMEN
The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.
Asunto(s)
Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Esputo/microbiología , Tuberculosis/microbiología , Humanos , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Factores de TiempoRESUMEN
Fluorescence detection using two spectrally distinct fluorophores has long been used for the determination of the relative abundance of biomolecules, but overlap between the fluorescence spectra of each fluorophore can result in nonradiative Förster resonance energy transfer (FRET) and distorting the signals detected by fluorescence channels. Thus conventional methods for quantifying the relative abundance of fluorophores by fluorescence emission will not be accurate if FRET can occur. In this paper we report the development of a quantitative fluorescence correction method incorporating FRET to measure the relative abundance of fluorophores in dual-labeling experiments. The quantitative fluorescence correction method incorporating FRET is accurate, comprehensive, and convenient for the measurement of the relative abundance of fluorophores in dual-labeling experiments and can also correct the FRET distortion and provide accurate, quantitative, and convenient measurement of the hybridization efficiencies on microarrays.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Fluorescencia , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Secuencia de Bases , Color , ADN/análisis , ADN/química , ADN/genética , Perfilación de la Expresión Génica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Propiedades de SuperficieRESUMEN
Bufalin is a natural toxin with anti-leukemic properties. It induces cell differentiation and apoptosis, as well as increasing the sensitivity of leukemia cells to other chemotherapeutic agents. We investigated the biological effects and molecular mechanisms of bufalin triggered apoptosis in HL-60 cells by gene expression profiling. The broad transcriptional response to bufalin was consistent with bufalin's action of regulating HL-60 cell proliferation and apoptosis, as well as its synergistic effect with other drugs. Further transcription factor ELISA experiments suggested that the transcription factors NFkappaB and AP-1 were activated to promote bufalin-induced HL-60 cell apoptosis. Our study provides new insights into the molecular mechanisms of bufalin, might prove to be beneficial in leukemia therapy.
Asunto(s)
Apoptosis , Bufanólidos/toxicidad , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Proliferación Celular/efectos de los fármacos , Células HL-60 , Humanos , FN-kappa B/biosíntesis , Factor de Transcripción AP-1/biosíntesisRESUMEN
We demonstrate a new method, using a universal array approach termed multiplex allele-specific PCR-based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele-specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele-specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin-coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost-effective, and easily-used, and is especially appropriate for mutation detection in clinical genetic diagnostics.
Asunto(s)
Alelos , Pérdida Auditiva/diagnóstico , Tamizaje Masivo , Análisis por Micromatrices/métodos , Reacción en Cadena de la Polimerasa/métodos , Conexina 26 , Conexinas , Humanos , Luz , Sensibilidad y EspecificidadRESUMEN
Transforming growth factor-beta (TGF-beta) signaling is known to depend on the formation of Smad2/3-Smad4 transcription regulatory complexes. However, the signaling functions of Smad2/3-Smad4 during TGF-beta-induced responses are obscure as TGF-beta also initiates a number of other signaling pathways. In this study, we systematically assessed the contribution of TGF-beta-Smad2/3-Smad4 signaling to both target gene transcription and apoptosis. Individual Smads were selectively knocked down in Hep3B cells by stable RNA interference (RNAi). We identified TGF-beta-responsive genes using genome-wide oligonucleotide microarrays and confirmed their dependency on Smad2, Smad3, or Smad4 by the combination of RNAi and microarray assay. The major finding from our microarray analysis was that of the 2,039 target genes seen to be regulated via TGF-beta induction, 190 were differentially transcriptionally controlled by Smad2-Smad4 and Smad3-Smad4 signaling and the latter control mechanism appeared to be functionally more important. We also found indirect evidence of competition between Smad2 and Smad3 for their activation when controlling the transcription of target genes. Functional analysis revealed that Smad3 and Smad4 were the predominant mediators of TGF-beta-induced apoptosis in Hep3B cells. We provide evidence that up-regulation of Bcl-2-interacting mediator of cell death (Bim), under the transcriptional control of Smad3-Smad4 signaling, is crucial to TGF-beta-induced apoptosis in Hep3B cells.
Asunto(s)
Apoptosis/fisiología , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Transcripción Genética/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas de la Membrana/metabolismo , Análisis por Micromatrices , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Proteínas Smad/genética , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismo , Transfección , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia ArribaRESUMEN
A PDMS-glass based micro-device was designed and fabricated with 12 coplanar impedance sensors integrated for electrical cell-substrate impedance sensing (ECIS). The sensitivity and frequency characteristics of the sensors were investigated both theoretically (equivalent circuit model) and experimentally for the commonly used micro-electrode dimension scale (20-80 microm). The experimental results matched well with the theoretical model analysis and revealed that, within this micro-electrode dimension scale, as the electrode width decreased or as the total electrode length decreased the sensitivity of sensor increased over the whole sensing frequency range, whilst electrode to electrode distance had no influence on sensitivity. Through our frequency characteristics analysis, the whole frequency range could be divided into four parts. New functions describing the dominant components in each frequency range were defined and validated experimentally, and could be used to explain the phenomenon of an ECIS sensing frequency window. The contribution to the impedance measurement of cells growing on the edges of the electrodes was determined for the first time. Finally, novel proposals for ECIS sensor design and ECIS measurements were presented.
Asunto(s)
Técnicas Biosensibles/métodos , Impedancia Eléctrica , Técnicas Biosensibles/instrumentación , Supervivencia Celular , Electrodos , Diseño de Equipo , Células HeLa , Humanos , Sensibilidad y EspecificidadRESUMEN
MicroRNAs (miRNAs) and mRNAs constitute an important part of gene regulatory networks, influencing diverse biological phenomena. To discover novel regulatory pathways during myeloid differentiation, we performed miRNA as well as mRNA expression profiling of in vitro-differentiating HL-60 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The main findings were up-regulation of miR-146a/b, miR-21, miR-221, miR-222, miR-155, miR-26a and down-regulation of miR-199a*, miR-181c, miR-142-3p, miR-92. After integrating the miRNA and mRNA expression data into a Transcriptome Interaction Database by Molecule Annotation System (MAS) software, a number of differently expressed mRNAs were revealed as potential targets of these miRNAs.
Asunto(s)
Diferenciación Celular , Macrófagos/citología , MicroARNs/metabolismo , Monocitos/citología , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Linaje de la Célula , Biología Computacional , Regulación hacia Abajo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Células HL-60 , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Regulación hacia ArribaRESUMEN
There had been little progress in development of the theoretical basis of rectangular chromatography columns until Spangler made great progress by using a more exact model than Golay's. Unfortunately, there was a deficiency in his calculations, which led to a conclusion inconsistent with the previous theories. In this paper, a simpler formula with defined variables was first established to calculate the mean permeability coefficient for a rectangular GC column. A formula was also established to calculate the height equivalent to a theoretical plate (HETP) for a rectangular column based on this work and the correction of Spangler's theory. By comparing both our predictions and Spangler's predictions with Golay's, respectively, we could demonstrate that our theory is more exact. Further, one parameter (A) was found to be not monotonous. This finding leads to the conclusion that the square column has the highest performance among all the rectangular-shaped columns used for chromatography, and that a width/depth ratio of around three is desirable if the column is used for mixing reactants in lab-on-a-chip systems, instead of for chromatography. The conclusions are applicable not only for gas but also for liquid chromatography columns.
Asunto(s)
Cromatografía de Gases , Procedimientos Analíticos en Microchip , Modelos Teóricos , Cromatografía de Gases/instrumentación , Cromatografía de Gases/métodos , Dispositivos Laboratorio en un Chip , Cómputos Matemáticos , Procedimientos Analíticos en Microchip/métodos , PermeabilidadRESUMEN
A base stacking hybridization-based microarray method was developed for rapid identification of clinical isolates within 2 h. The oligonucleotide probe sequences for species or genus-level identification were targeted against ribosomal RNA. Isolates were lysed and directly hybridized to the microarray-bound capture probes without conventional DNA or RNA isolation and prior polymerase chain reaction amplification. Five bacterial species encountered frequently in the clinical setting, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae, and one genus Enterococcus, could be discriminated by the microarray-based assay. Identification by this method matched biochemical identification for 150 of 152 clinical strains. This base-stacking hybridization microarray offers a simple, fast (=2 h), and accurate identification of bacterial cultures and is a potential tool for routine clinical diagnosis.
Asunto(s)
Infecciones Bacterianas/microbiología , Técnicas de Tipificación Bacteriana , Bacterias Gramnegativas/clasificación , Cocos Grampositivos/clasificación , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Infecciones Bacterianas/diagnóstico , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/aislamiento & purificación , Cocos Grampositivos/genética , Cocos Grampositivos/aislamiento & purificación , Humanos , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Ribosómico/genética , Especificidad de la Especie , Factores de TiempoRESUMEN
Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.
Asunto(s)
Mutagénesis/genética , Análisis de Secuencia de ADN/métodos , Secuencia Rica en At/genética , Algoritmos , Animales , Secuencia de Bases , ADN Mitocondrial/genética , ADN Protozoario/química , ADN Protozoario/genética , Dictyostelium/genética , Secuencia Rica en GC/genética , Humanos , Datos de Secuencia Molecular , ARN de Transferencia de Treonina/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
MicroRNAs are small non-coding RNAs that participate in different biological processes, providing subtle combinational regulation of cellular pathways, often by regulating components of signalling pathways. Aberrant expression of miRNAs is an important factor in the development and progression of disease. The canonical myomiRs (miR-1, -133 and -206) are central to the development and health of mammalian skeletal and cardiac muscles, but new findings show they have regulatory roles in the development of other mammalian non-muscle tissues, including nerve, brain structures, adipose and some specialised immunological cells. Moreover, the deregulation of myomiR expression is associated with a variety of different cancers, where typically they have tumor suppressor functions, although examples of an oncogenic role illustrate their diverse function in different cell environments. This review examines the involvement of the related myomiRs at the crossroads between cell development/tissue regeneration/tissue inflammation responses, and cancer development.