RESUMEN
Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.
Asunto(s)
Corteza Cerebral/citología , Organoides/metabolismo , Animales , Evolución Biológica , Encéfalo/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/genética , Corteza Cerebral/metabolismo , Redes Reguladoras de Genes/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Macaca , Neurogénesis/genética , Organoides/crecimiento & desarrollo , Pan troglodytes , Células Madre Pluripotentes/citología , Análisis de la Célula Individual , Especificidad de la Especie , Transcriptoma/genéticaRESUMEN
Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.
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Neocórtex/citología , Neocórtex/crecimiento & desarrollo , Animales , Ciclo Celular , Humanos , Macaca , Ratones , Neocórtex/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Neuroglía/citología , Neuroglía/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Nicho de Células MadreRESUMEN
Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (195 degrees C) an ethylene glycol lysis buffer to perform rapid flow-through rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices.
Asunto(s)
Bacteriólisis/fisiología , Esporas Bacterianas/fisiología , Bacillus/genética , Bacillus/crecimiento & desarrollo , Bacillus/aislamiento & purificación , Bacillus/fisiología , Bacillus anthracis/genética , Bacillus anthracis/fisiología , Bacillus cereus/genética , Bacillus cereus/fisiología , Bacillus subtilis/genética , Bacillus subtilis/fisiología , Acción Capilar , División Celular , Cartilla de ADN , ADN Bacteriano/genética , Colorantes Fluorescentes , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Solubilidad , Esporas Bacterianas/genética , Esporas Bacterianas/aislamiento & purificación , TermodinámicaRESUMEN
Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2 microl of sample. We further compare the performance of labeled linear probes, quantum dot labeled probes, molecular beacons (MBs) and tentacle probes (TPs). Each probe technology was compared and contrasted with traditional hybridization methods using labeled sample. All probes demonstrated similar sensitivity and greater specificity than traditional hybridization techniques. MBs and TPs were able to bypass a wash step due to their 'on-off' signaling mechanism. TPs demonstrated reaction kinetics 37.6 times faster than MBs, resulting in the fastest assay time of 5 min. Our data further indicate TPs had the most sensitive detection limit (<1 nM) as well as the highest specificity (>1 x 10(4) improvement) among all tested probes in these experiments. By matching the enhanced extraction efficiencies of PPM with the selectivity of TPs, we have created a format for improved sandwich assays.
Asunto(s)
Hibridación de Ácido Nucleico/métodos , Sondas de Oligonucleótidos/química , Colorantes Fluorescentes/química , Cinética , Polímeros/química , Polimorfismo de Nucleótido Simple , Puntos CuánticosRESUMEN
The majority of efforts to increase specificity or sensitivity in biosensors result in trade-offs with little to no gain in overall accuracy. This is because a biosensor cannot be more accurate than the affinity interaction it is based on. Accordingly, we have developed a new class of reagents based on mathematical principles of cooperativity to enhance the accuracy of the affinity interaction. Tentacle probes (TPs) have a hairpin structure similar to molecular beacons (MBs) for enhanced specificity, but are modified by the addition of a capture probe for increased kinetics and affinity. They produce kinetic rate constants up to 200-fold faster than MB with corresponding stem strengths. Concentration-independent specificity was observed with no false positives at up to 1 mM concentrations of variant analyte. In contrast, MBs were concentration dependent and experienced false positives above 3.88 muM of variant analyte. The fast kinetics of this label-free reagent may prove important for extraction efficiency, hence sensitivity and detection time, in microfluidic assays. The concentration-independent specificity of TPs may prove extremely useful in assays where starting concentrations and purities are unknown as would be the case in bioterror or clinical point of care diagnostics.
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Técnicas Biosensibles , Colorantes Fluorescentes/química , Sondas de Oligonucleótidos/química , Reacciones Falso Positivas , Cinética , Técnicas de Diagnóstico Molecular , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , TermodinámicaRESUMEN
In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversity not captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, but not mouse, neocortex, and inhibiting HTR2A receptors in human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmitter signaling during neurogenesis in the developing human neocortex and highlight evolutionarily divergent mechanisms of physiological signaling.
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Calcio/metabolismo , Células Ependimogliales/metabolismo , Neocórtex/embriología , Neurogénesis/genética , Receptor de Serotonina 5-HT2A/metabolismo , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Linaje de la Célula , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Ratones , Neocórtex/citología , Neocórtex/metabolismo , Neurogénesis/fisiología , Análisis de Secuencia de ARN , Serotonina/metabolismo , Análisis de la Célula IndividualRESUMEN
MicroRNAs (miRNAs) regulate many cellular events during brain development by interacting with hundreds of mRNA transcripts. However, miRNAs operate nonuniformly upon the transcriptional profile with an as yet unknown logic. Shortcomings in defining miRNA-mRNA networks include limited knowledge of in vivo miRNA targets and their abundance in single cells. By combining multiple complementary approaches, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation with an antibody to AGO2 (AGO2-HITS-CLIP), single-cell profiling and computational analyses using bipartite and coexpression networks, we show that miRNA-mRNA interactions operate as functional modules that often correspond to cell-type identities and undergo dynamic transitions during brain development. These networks are highly dynamic during development and over the course of evolution. One such interaction is between radial-glia-enriched ORC4 and miR-2115, a great-ape-specific miRNA, which appears to control radial glia proliferation rates during human brain development.
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Encéfalo/crecimiento & desarrollo , Redes Reguladoras de Genes , MicroARNs/metabolismo , Transcriptoma , Encéfalo/metabolismo , Proliferación Celular , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Efficient and rapid isolation of nucleic acids is of significant importance in the field of genomics for a variety of applications. Current techniques for the isolation of specific nucleic acids or genes typically involve multiple rounds of amplification of the target sequence using polymerase chain reaction. Described here is a recent development in the fabrication and modification of porous polymer monoliths for the selective concentration and extraction of nucleic acids sequences. The rigid monoliths are cast to shape and are tunable for functionalization using a variety of amine-terminated molecules including oligonucleotide capture probes. Efficient and rapid isolation of nucleic acids can be performed using polymer monoliths in microchannels in a time frame as short as 2 s. The described materials and methods offer the ability to perform concentration of nucleic acids in solution and elute purified samples in volumes as low as 3 microL without the requirement of altering salt concentration in the wash and elution buffers.
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Microfluídica/métodos , Ácidos Nucleicos/aislamiento & purificación , Polímeros/química , Microfluídica/instrumentación , Ácidos Nucleicos/química , PorosidadRESUMEN
Systematic analyses of spatiotemporal gene expression trajectories during organogenesis have been challenging because diverse cell types at different stages of maturation and differentiation coexist in the emerging tissues. We identified discrete cell types as well as temporally and spatially restricted trajectories of radial glia maturation and neurogenesis in developing human telencephalon. These lineage-specific trajectories reveal the expression of neurogenic transcription factors in early radial glia and enriched activation of mammalian target of rapamycin signaling in outer radial glia. Across cortical areas, modest transcriptional differences among radial glia cascade into robust typological distinctions among maturing neurons. Together, our results support a mixed model of topographical, typological, and temporal hierarchies governing cell-type diversity in the developing human telencephalon, including distinct excitatory lineages emerging in rostral and caudal cerebral cortex.
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Corteza Cerebral/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Neurogénesis/genética , Telencéfalo/crecimiento & desarrollo , Corteza Cerebral/anatomía & histología , Corteza Cerebral/citología , Humanos , Neuroglía/fisiología , Neuronas , Telencéfalo/anatomía & histología , Telencéfalo/citologíaRESUMEN
[This corrects the article on p. 70 in vol. 4, PMID: 27709111.].
RESUMEN
The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based integrated fluidic circuit that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to various stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.
RESUMEN
Repeated exposures to bioactivated cytotoxicants such as naphthalene (NA) render the target population, Clara cells, resistant to further injury through a glutathione-dependent mechanism. The current studies were designed to test the hypothesis that the mechanism for tolerance is localized in Clara cells. We used three approaches to test this hypothesis. First, using airway explants from tolerant mice maintained in culture, we sought to determine if the mechanism of Clara cell tolerance was airway-specific. Second, using inhalation as the route of exposure, we sought to determine if Clara cells at all airways levels become tolerant to repeated inhalation exposures of NA. Third, by measuring gamma-glutamylcysteine synthetase (gamma-GCS) activity and expression we determined if tolerance to inhaled NA resulted from shifts in phase-II metabolism. Our results indicate that Clara cells in explants from tolerant mice remained tolerant to NA injury in culture. When mice were exposed to repeated inhalation exposures of NA (15 ppm), we found that Clara cells at all airway levels became tolerant. Expression and activity analysis revealed that gamma-GCS, the rate-limiting enzyme in glutathione synthesis, is induced in tolerant Clara cells. Buthionine sulfoximine, a gamma-GCS inhibitor, was able to eliminate the resistance of these tolerant cells. We conclude: (1) the mechanism of NA tolerance in Clara cells is airway specific, (2) the specific mechanism allows Clara cells to become tolerant to NA vapor at levels relevant to human exposure, and (3) the mechanism of tolerance to inhaled NA is highly dependent on induction of the catalytic enzyme, gamma-GCS.
Asunto(s)
Contaminantes Ambientales/toxicidad , Células Epiteliales/efectos de los fármacos , Naftalenos/toxicidad , Administración por Inhalación , Animales , Esquema de Medicación , Tolerancia a Medicamentos , Contaminantes Ambientales/administración & dosificación , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Glutatión Transferasa/antagonistas & inhibidores , Glutatión Transferasa/biosíntesis , Inyecciones Intraperitoneales , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/metabolismo , Masculino , Ratones , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Naftalenos/administración & dosificaciónRESUMEN
Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.
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Corteza Cerebral/crecimiento & desarrollo , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , ARN Mensajero/análisis , Análisis de Secuencia de ARN/métodos , Transducción de Señal/genética , Animales , Corteza Cerebral/metabolismo , Diseño de Equipo , Humanos , Ratones , Técnicas Analíticas Microfluídicas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process. This included enzyme and reagent addition, temperature-controlled incubations, noncontact mixing of enzymes and reagents, buffer exchanges, and sample elution. Using this system, completely automated methods were developed for the purification of viral RNA and DNA from plasma and whole blood and of bacterial genomic DNA from water and whole blood. Extracted nucleic acids were detected and quantified using real-time PCR. The data indicate that automated viral DNA extraction was more efficient than sample extractions performed using a manual process, whereas automated total RNA extraction from the same samples was equivalent to controls. Additionally, we found that the process for bacterial genomic DNA extraction from either water or whole blood was equivalent to the manual extraction processes. We conclude the instrument, consumable cartridge, and reagent system enables easy, cost-effective, and robust sample preparation regardless of the experience of the operator.
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Automatización de Laboratorios/instrumentación , Automatización de Laboratorios/métodos , Ácidos Nucleicos/aislamiento & purificación , Manejo de Especímenes/instrumentación , Manejo de Especímenes/métodos , Bacterias/genética , Sangre/microbiología , Sangre/virología , Técnicas de Laboratorio Clínico/instrumentación , Técnicas de Laboratorio Clínico/métodos , Plasma/virología , Virus/genética , Microbiología del AguaRESUMEN
The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.
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Sondas de ADN/genética , Hibridación de Ácido Nucleico/métodos , Humanos , Modelos Teóricos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Efficient and rapid isolation of mRNA is important in the field of genomics as well as in the clinical and pharmaceutical arena. We have developed UV-initiated methacrylate-based porous polymer monoliths (PPM) for microfluidic trapping and concentration of eukaryotic mRNA. PPM are cast-to-shape and are tunable for functionalization using a variety of amine-terminated molecules. Efficient isolation of eukaryotic mRNA from total RNA was first mathematically modeled and then achieved using PPM in capillaries. Purification protocols using oligo dT's, locked nucleic acid substituted dT's, and tetramethylammonium chloride salts were characterized. mRNA yield and purity were compared with mRNA isolated by commercial kits with statistically equivalent yields and purities (determined by qPCR ratio of 18s rRNA and Gusb mRNA markers). Even after extracting 16 microg of mRNA from 315 microg of total RNA, the 0.4-microL volume monolith showed no signs of saturation. Elution volumes were below 20 microL with concentrations up to 1 microg/microL. In addition, the polymeric material exhibited exceptional stability in a range of conditions (pH, temperature, dryness) and was stable for a period of months. All of these characteristics make porous polymer monoliths good candidates for potential microfluidic sample preconcentrators and purifiers.
Asunto(s)
Microfluídica/instrumentación , Microfluídica/métodos , Polímeros , ARN Mensajero/aislamiento & purificación , Metacrilatos , PorosidadRESUMEN
BACKGROUND: False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation. METHODS: Tentacle Probes, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan-minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection. We compared TaqMan-MGB probes to Tentacle Probes for SNP and deletion detection based on the presence or absence of a growth curve. RESULTS: With the TaqMan-MGB Y. pestis yp48 assays, false-positive results for Yersinia pseudotuberculosis occurred at every concentration tested, and with the TaqMan-MGB B. anthracis gyrA assays, false-positive results occurred in 21 of 29 boil preps of environmental samples of near neighbors. With Tentacle Probes no false-positive results occurred. CONCLUSIONS: The high specificity exhibited by Tentacle Probes may eliminate melting curve analysis for SNP and deletion mutation detection, allowing the diagnostic use of previously difficult targets.
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Bacillus anthracis/clasificación , Proteínas Bacterianas/genética , Girasa de ADN/genética , Polimorfismo de Nucleótido Simple , Eliminación de Secuencia , Yersinia pestis/clasificación , Bacillus anthracis/genética , Bacillus cereus/clasificación , Técnicas Bacteriológicas , Reacciones Falso Positivas , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Yersinia pestis/genética , Yersinia pseudotuberculosis/clasificaciónRESUMEN
The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).
Asunto(s)
Electroforesis por Microchip/instrumentación , Proteínas/aislamiento & purificación , Diseño de EquipoRESUMEN
We report the development of a hand-held instrument capable of performing two simultaneous microchip separations (gel and zone electrophoresis), and demonstrate this instrument for the detection of protein biotoxins. Two orthogonal analysis methods are chosen over a single method in order to improve the probability of positive identification of the biotoxin in an unknown mixture. Separations are performed on a single fused-silica wafer containing two separation channels. The chip is housed in a microfluidic manifold that utilizes o-ring sealed fittings to enable facile and reproducible fluidic connection to the chip. Sample is introduced by syringe injection into a septum-sealed port on the device exterior that connects to a sample loop etched onto the chip. Detection of low nanomolar concentrations of fluorescamine-labeled proteins is achieved using a miniaturized laser-induced fluorescence detection module employing two diode lasers, one per separation channel. Independently controlled miniature high-voltage power supplies enable fully programmable electrokinetic sample injection and analysis. As a demonstration of the portability of this instrument, we evaluated its performance in a laboratory field test at the Defence Science and Technology Laboratory with a series of biotoxin variants. The two separation methods cleanly distinguish between members of a biotoxin test set. Analysis of naturally occurring variants of ricin and two closely related staphylococcal enterotoxins indicates the two methods can be used to readily identify ricin in its different forms and can discriminate between two enterotoxin isoforms.
Asunto(s)
Electroforesis por Microchip/métodos , Técnicas Analíticas Microfluídicas/métodos , Toxinas Biológicas/aislamiento & purificación , Electroforesis por Microchip/instrumentación , Enterotoxinas/aislamiento & purificación , Equipo Reutilizado , Miniaturización , Ricina/aislamiento & purificación , Ricinus/química , Sensibilidad y Especificidad , Staphylococcus aureusRESUMEN
Repeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that gamma-glutamylcysteine synthetase (gamma-GCS) was induced in tolerant Clara cells by repeated exposures to NA. Treating tolerant mice with buthionine sulfoximine, a gamma-GCS inhibitor, eliminates resistance acquired by repeated exposures to NA. Broad phenotypic shifts were not present. Marker proteins of differentiation declined over the first 3 days in the development of tolerance, but returned to control levels at 14 and 21 days. Epithelial organizational structure and internal organelle composition in Clara cells from tolerant mice were similar compared to corn oil-treated controls, while subtle shifts in organelle distribution were present. We conclude that induction of gamma-GCS expression is coordinated with the development of NA tolerance, but induction of NA tolerance does not markedly alter Clara cell differentiation, epithelial organization, or organelle composition in bronchiolar epithelium.