RESUMEN
Solid organ transplant patients are vulnerable to suffering neurologic complications from a wide array of viral infections and can be sentinels in the population who are first to get serious complications from emerging infections like the recent waves of arboviruses, including West Nile virus, Chikungunya virus, Zika virus, and Dengue virus. The diverse and rapidly changing landscape of possible causes of viral encephalitis poses great challenges for traditional candidate-based infectious disease diagnostics that already fail to identify a causative pathogen in approximately 50% of encephalitis cases. We present the case of a 14-year-old girl on immunosuppression for a renal transplant who presented with acute meningoencephalitis. Traditional diagnostics failed to identify an etiology. RNA extracted from her cerebrospinal fluid was subjected to unbiased metagenomic deep sequencing, enhanced with the use of a Cas9-based technique for host depletion. This analysis identified West Nile virus (WNV). Convalescent serum serologies subsequently confirmed WNV seroconversion. These results support a clear clinical role for metagenomic deep sequencing in the setting of suspected viral encephalitis, especially in the context of the high-risk transplant patient population.
Asunto(s)
Rechazo de Injerto/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Meningoencefalitis/diagnóstico , Fiebre del Nilo Occidental/diagnóstico , Virus del Nilo Occidental/genética , Adolescente , Femenino , Tasa de Filtración Glomerular , Rechazo de Injerto/etiología , Supervivencia de Injerto , Humanos , Huésped Inmunocomprometido , Inmunosupresores/uso terapéutico , Pruebas de Función Renal , Meningoencefalitis/virología , Metagenómica , Pronóstico , Factores de Riesgo , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/patogenicidadRESUMEN
Antiretroviral therapy (ART) suppresses plasma and cerebrospinal fluid (CSF) HIV replication. Neurosymptomatic (NS) CSF escape is a rare exception in which CNS HIV replication occurs in the setting of neurologic impairment. The origins of NS escape are not fully understood. We performed a case-control study of asymptomatic (AS) escape and NS escape subjects with HIV-negative subjects as controls in which we investigated differential immunoreactivity to self-antigens in the CSF of NS escape by employing neuroanatomic CSF immunostaining and massively multiplexed self-antigen serology (PhIP-Seq). Additionally, we utilized pan-viral serology (VirScan) to deeply profile the CSF anti-viral antibody response and metagenomic next-generation sequencing (mNGS) for pathogen detection. We detected Epstein-Barr virus (EBV) DNA more frequently in the CSF of NS escape subjects than in AS escape subjects. Based on immunostaining and PhIP-Seq, there was evidence for increased immunoreactivity against self-antigens in NS escape CSF. Finally, VirScan revealed several immunodominant epitopes that map to the HIV envelope and gag proteins in the CSF of AS and NS escape subjects. Whether these additional inflammatory markers are byproducts of an HIV-driven process or whether they independently contribute to the neuropathogenesis of NS escape will require further study.
Asunto(s)
Coinfección , Infecciones por Virus de Epstein-Barr , Infecciones por VIH , Humanos , Autoinmunidad , Estudios de Casos y Controles , Herpesvirus Humano 4 , Sistema Nervioso Central , Infecciones por VIH/líquido cefalorraquídeo , AutoantígenosRESUMEN
The epidemiology of infectious causes of meningitis in sub-Saharan Africa is not well understood, and a common cause of meningitis in this region, Mycobacterium tuberculosis (TB), is notoriously hard to diagnose. Here we show that integrating cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) with a host gene expression-based machine learning classifier (MLC) enhances diagnostic accuracy for TB meningitis (TBM) and its mimics. 368 HIV-infected Ugandan adults with subacute meningitis were prospectively enrolled. Total RNA and DNA CSF mNGS libraries were sequenced to identify meningitis pathogens. In parallel, a CSF host transcriptomic MLC to distinguish between TBM and other infections was trained and then evaluated in a blinded fashion on an independent dataset. mNGS identifies an array of infectious TBM mimics (and co-infections), including emerging, treatable, and vaccine-preventable pathogens including Wesselsbron virus, Toxoplasma gondii, Streptococcus pneumoniae, Nocardia brasiliensis, measles virus and cytomegalovirus. By leveraging the specificity of mNGS and the sensitivity of an MLC created from CSF host transcriptomes, the combined assay has high sensitivity (88.9%) and specificity (86.7%) for the detection of TBM and its many mimics. Furthermore, we achieve comparable combined assay performance at sequencing depths more amenable to performing diagnostic mNGS in low resource settings.
Asunto(s)
Meningitis , Mycobacterium tuberculosis , Tuberculosis Meníngea , Sistema Nervioso Central , Humanos , Meningitis/microbiología , Metagenómica , Mycobacterium tuberculosis/genética , Tuberculosis Meníngea/líquido cefalorraquídeo , Tuberculosis Meníngea/diagnóstico , Tuberculosis Meníngea/genéticaRESUMEN
Cardioviruses (e.g., Theiler's murine encephalomyelitis virus [TMEV]) are members of the Picornaviridae family that cause myocarditis and encephalitis in rodents. Recently, several studies have identified human cardioviruses, including Saffold virus (SAFV) and a related virus named human TMEV-like cardiovirus (HTCV). At least eight cardiovirus genotypes are now recognized, with SAFV and most strains of HTCV belonging to genotypes 1 and 2, respectively; genotype 2 strains are the most common in the population. Although a genotype 3 cardiovirus has recently been cultured (SAFV-3), the genotype 1 and 2 cardioviruses have been difficult to propagate in vitro, hindering efforts to understand their seroprevalence and pathogenicity. Here we present the isolation and characterization of a genotype 2 human cardiovirus (HTCV-UC6). Notably, successful cultivation of HTCV-UC6 from stool required the addition of cytokine-blocking antibodies to interrupt downstream antiviral pathways. Unlike SAFV-3, HTCV-UC6 exhibited slow replication kinetics and demonstrated only a moderate cytopathic effect. Serologic assays revealed that 91% of U.S. adults carry antibodies to the genotype 2 cardioviruses, of which 80% generate neutralizing antibodies, in agreement with previous data showing that cardiovirus infection is widespread in humans. We also demonstrate an acute cardiovirus seroconversion event in a child with diarrhea and vomiting, thus reporting for the first time evidence linking cardiovirus infection to diarrheal disease in humans.
Asunto(s)
Infecciones por Cardiovirus/epidemiología , Cardiovirus/aislamiento & purificación , Cardiovirus/fisiología , Diarrea/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Cardiovirus/genética , Cardiovirus/crecimiento & desarrollo , Infecciones por Cardiovirus/virología , Línea Celular , Efecto Citopatogénico Viral , Heces/virología , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Viral/genética , Análisis de Secuencia de ADN , Estudios Seroepidemiológicos , Estados Unidos/epidemiología , Replicación Viral , Adulto JovenRESUMEN
We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.
Asunto(s)
Calcineurina/genética , Ciclosporina/farmacología , Regulación Fúngica de la Expresión Génica , Inmunofilinas/genética , Inmunosupresores/farmacología , Saccharomyces cerevisiae/genética , Tacrolimus/farmacología , Diseño de Fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genotipo , Modelos Biológicos , Mutación , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/efectos de los fármacos , Transducción de SeñalRESUMEN
Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A(2) (PLA(2)s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA(2) in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA(2) activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA(2) activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.
Asunto(s)
Membrana Celular/química , Forma de la Célula/genética , Epistasis Genética , Fosfolipasas A2/genética , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae , Membrana Celular/genética , Membrana Celular/fisiología , Proliferación Celular , Epistasis Genética/fisiología , Redes Reguladoras de Genes/fisiología , Ligamiento Genético , Concentración de Iones de Hidrógeno , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Organismos Modificados Genéticamente , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructura , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
DNA microarrays containing virtually every gene of Saccharomyces cerevisiae were used to carry out a comprehensive investigation of the temporal program of gene expression accompanying the metabolic shift from fermentation to respiration. The expression profiles observed for genes with known metabolic functions pointed to features of the metabolic reprogramming that occur during the diauxic shift, and the expression patterns of many previously uncharacterized genes provided clues to their possible functions. The same DNA microarrays were also used to identify genes whose expression was affected by deletion of the transcriptional co-repressor TUP1 or overexpression of the transcriptional activator YAP1. These results demonstrate the feasibility and utility of this approach to genomewide exploration of gene expression patterns.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ciclo del Ácido Cítrico , Medios de Cultivo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fermentación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Genes Reguladores , Gluconeogénesis , Glucosa/metabolismo , Glioxilatos/metabolismo , Sistemas de Lectura Abierta , Consumo de Oxígeno , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Asymmetric localization of proteins plays a key role in many cellular processes, including cell polarity and cell fate determination. Using DNA microarray analysis, we identified a plasma membrane protein-encoding mRNA (IST2) that is transported to the bud tip by an actomyosin-based process. mRNA localization created a higher concentration of IST2 protein in the bud compared with that of the mother cell, and this asymmetry was maintained by a septin-mediated membrane diffusion barrier at the mother-bud neck. These results indicate that yeast creates distinct plasma membrane compartments, as has been described in neurons and epithelial cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto , Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Cadenas Pesadas de Miosina , Miosina Tipo V , ARN Mensajero/metabolismo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Actomiosina/metabolismo , Transporte Biológico , Compartimento Celular , Ciclo Celular , Proteínas de Ciclo Celular/genética , Membrana Celular/metabolismo , Difusión , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética , Mutación , Miosinas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN de Hongos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Temperatura , Factores de Transcripción/genéticaRESUMEN
Metagenomic next-generation sequencing (mNGS) experiments involving small amounts of nucleic acid input are highly susceptible to erroneous conclusions resulting from unintentional sequencing of occult contaminants, especially those derived from molecular biology reagents. Recent work suggests that, for any given microbe detected by mNGS, an inverse linear relationship between microbial sequencing reads and sample mass implicates that microbe as a contaminant. By associating sequencing read output with the mass of a spike-in control, we demonstrate that contaminant nucleic acid can be quantified in order to identify the mass contributions of each constituent. In an experiment using a high-resolution (n = 96) dilution series of HeLa RNA spanning 3-logs of RNA mass input, we identified a complex set of contaminants totaling 9.1 ± 2.0 attograms. Given the competition between contamination and the true microbiome in ultra-low biomass samples such as respiratory fluid, quantification of the contamination within a given batch of biological samples can be used to determine a minimum mass input below which sequencing results may be distorted. Rather than completely censoring contaminant taxa from downstream analyses, we propose here a statistical approach that allows separation of the true microbial components from the actual contribution due to contamination. We demonstrate this approach using a batch of n = 97 human serum samples and note that despite E. coli contamination throughout the dataset, we are able to identify a patient sample with significantly more E. coli than expected from contamination alone. Importantly, our method assumes no prior understanding of possible contaminants, does not rely on any prior collection of environmental or reagent-only sequencing samples, and does not censor potentially clinically relevant taxa, thus making it a generalized approach to any kind of metagenomic sequencing, for any purpose, clinical or otherwise.
Asunto(s)
Escherichia coli , Metagenómica , ADN Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ADNRESUMEN
The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.
Asunto(s)
Genoma Fúngico , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Carbono/metabolismo , Cartilla de ADN/genética , Fermentación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Glicerol/metabolismo , Glicoproteínas de Membrana/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Ligasa (ATP)/metabolismo , Empalme del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1. Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end. We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions. Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle. Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle. We provide evidence for dynamic remodeling of these components at telomeres.
Asunto(s)
Saccharomyces cerevisiae/metabolismo , Telómero/ultraestructura , Southern Blotting , Proteínas Portadoras/metabolismo , Ciclo Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Epítopos , Genotipo , Microscopía Fluorescente , Modelos Biológicos , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Telomerasa/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Encoded microparticles have become a powerful tool for a wide array of applications, including high-throughput sample tracking and massively parallel biological multiplexing. Spectral encoding, where particles are encoded with distinct luminescence spectra, provides a particularly appealing encoding strategy because of the ease of reading codes and assay flexibility. To date, spectral encoding has been limited in the number of codes that can be accurately resolved. Here, we demonstrate an automated 5-dimensional spectral encoding scheme using lanthanide nanophosphors that is capable of producing isotropic spherical microparticles with up to 1,100 unique codes, which we term MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding). We further develop a quantitative framework for evaluating global ability to distinguish codes and demonstrate that for six different sets of MRBLEs ranging from 106 to 1,101 codes in size, > 98% of MRBLEs can be assigned to a code with 99.99% confidence. These > 1,000 code sets represent the largest spectral code libraries built to date. We expect that these MRBLEs will enable a wide variety of novel multiplexed assays.
RESUMEN
Inclusion body disease (IBD) of boas and pythons is characterized by the intracytoplasmic accumulation of an antigenic 68 kDa viral protein IBDP, more recently known as the nucleoprotein (NP) of the reptarenaviruses. Blood samples of 131 captive boas and pythons (53 boa constrictors, Boa constrictor; 35 rainbow boas, Epicrates cenchria; 22 ball pythons, Python regius; 5 carpet pythons, Morelia spilota; 6 Burmese pythons, Python bivittatus; 4 Jamaican boas, Epicrates subflavus; 5 anacondas, Eunectes spp.; and 1 green tree python, Morelia viridis) were obtained from 28 collections in the USA. Diagnosis of IBD was initially made by the identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin (HE) stained blood films and isolated peripheral white blood cells (PWBC). The overall prevalence of IBD in study snakes was 25/131 or 19% (95% CI = 12.4%, 25.8%) with boa constrictors being more commonly infected (22/53 or 41.5%; 95% CI = 28.2%, 54.8%) than other species in this study. Of the 22 IBD positive boa constrictors, 87% were clinically healthy, 13% had various signs of chronic illness, and none showed signs of central nervous system disease. Using a validated monoclonal anti-NP antibody, NP was confirmed within the isolated PWBC by immunohistochemical staining and Western blots. The presence of reptarenaviruses within blood samples of 27 boa constrictors and three rainbow boas was also assessed by PCR. Among boa constrictors, very good agreements were shown between the observation of inclusion bodies (by HE stain) and the presence of NP (by immunohistochemistry, kappa = 0.92; and Western blots, kappa = 0.89), or the presence of reptarenaviruses (by PCR; kappa = 0.92).
Asunto(s)
Animales de Zoológico , Infecciones por Arenaviridae/veterinaria , Arenaviridae/aislamiento & purificación , Boidae , Animales , Infecciones por Arenaviridae/epidemiología , Infecciones por Arenaviridae/virología , Western Blotting/veterinaria , Eosina Amarillenta-(YS) , Estado de Salud , Hematoxilina , Inmunohistoquímica/veterinaria , Nucleoproteínas/aislamiento & purificación , Prevalencia , Especificidad de la Especie , Proteínas Virales/aislamiento & purificaciónRESUMEN
Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are 'DASHed' with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate a more than 99 % reduction of mitochondrial rRNA in HeLa cells, and enrichment of pathogen sequences in patient samples. We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN , Sistemas CRISPR-Cas/genética , Enfermedades Transmisibles/genética , Células HeLa , Humanos , Neoplasias/genética , ARN/genética , Edición de ARN/genética , ARN Mitocondrial , ARN Ribosómico/genéticaRESUMEN
Correction for 'Programmable microfluidic synthesis of spectrally encoded microspheres' by R. E. Gerver et al., Lab Chip, 2012, 12, 4716-4723.
RESUMEN
Multilayer soft lithography (MSL) provides a convenient and low-cost method for fabricating poly(dimethyl siloxane) (PDMS) microfluidic devices with on-chip valves for automated and precise control of fluid flow. MSL casting molds for flow channels typically incorporate small patches of rounded positive photoresist at valve locations to achieve the rounded cross-sectional profile required for these valves to function properly. Despite the importance of these rounded features for device performance, a comprehensive characterization of how the rounding process affects feature dimensions and closing pressures has been lacking. Here, we measure valve dimensions both before and after rounding and closing pressures for 120 different valve widths and lengths at post-rounding heights between 15 and 84 µm, for a total of 1200 different geometries spanning a wide range of useful sizes. We find that valve height and width after rounding depend strongly on valve aspect ratios, with these effects becoming more pronounced for taller and narrower features. Based on the measured data, we provide a simple fitted model and an online tool for estimating the pre-rounding dimensions needed to achieve desired post-rounding dimensions. We also find that valve closing pressures are well explained by modelling valve membranes in a manner analogous to a suspension bridge, shedding new light on device physics and providing a practical model for estimating closing pressures during device design.
Asunto(s)
Técnicas Analíticas Microfluídicas , Dimetilpolisiloxanos/química , Procesos Fotoquímicos , PresiónRESUMEN
Spectrally encoded fluorescent beads are an attractive platform for assay miniaturization and multiplexing in the biological sciences. Here, we synthesize hydrophilic PEG-acrylate polymer beads encoded with lanthanide nanophosphors using a fully automated microfluidic synthesis device. These beads are encoded by including varying amounts of two lanthanide nanophosphors relative to a third reference nanophosphor to generate 24 distinct ratios. These codes differ by less than 3% from their target values and can be distinguished from each other with an error rate of <0.1%. The encoded bead synthesis strategy we have used is readily extensible to larger numbers of codes, potentially up to millions, providing a new platform technology for assay multiplexing.
Asunto(s)
Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Técnicas Analíticas Microfluídicas/instrumentación , Microesferas , Nanoestructuras/química , Nanotecnología/instrumentación , Resinas Acrílicas/química , Técnicas de Química Sintética , Estudios de Factibilidad , Interacciones Hidrofóbicas e Hidrofílicas , Elementos de la Serie de los Lantanoides/química , Polietilenglicoles/químicaRESUMEN
Genome projects are providing vast amounts of sequence data. This raw material makes possible a completely new era of experimental approaches. Among these, DNA array technology, which allows one to assay thousands of unique nucleic acid samples simultaneously, will be important in genomic research, and the results of this research are likely to affect virtually every field of biology. DNA array technology is still in its infancy, but many have demonstrated its power by using it for such diverse applications as global monitoring of gene expression, mutation detection, and genetic mapping.
Asunto(s)
Genoma , Análisis de Secuencia por Matrices de Oligonucleótidos , Bases de Datos Factuales , Internet , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia/métodosRESUMEN
Specific protein-DNA complexes formed on a Xenopus 5S RNA gene were isolated and characterized using a novel technique. A DNA template reversibly immobilized on paramagnetic beads was used to capture, affinity purify, and concentrate protein--DNA complexes formed in a whole cell extract. The complexes were then released from the beads in a soluble and transcriptionally active form via restriction enzyme digestion of the DNA. A band-shift gel was used to separate and obtain the DNase I footprints of five individual complexes. Three of the complexes resulted from the independent binding of two proteins, TFIIIA and an unidentified protein binding to a large region just downstream of the 3' end of the gene. Two more slowly migrating complexes contained an additional large central protected region covering most of the gene. The most slowly migrating complex displayed protein interactions over the 5' flanking sequences. The formation of two of these complexes was shown to be dependent on TFIIIC activity. The correlation between transcriptional activity and the formation of these complexes suggests that the observed protein--DNA interactions are important for transcription of 5S RNA genes.