Use of a capture-based pathogen transcript enrichment strategy for RNA-Seq analysis of the Francisella tularensis LVS transcriptome during infection of murine macrophages.

Francisella tularensis is a zoonotic intracellular pathogen that is capable of causing potentially fatal human infections. Like all successful bacterial pathogens, F. tularensis rapidly responds to changes in its environment during infection of host cells, and upon encountering different microenvironments within those cells. This ability to appropriately respond to the challenges of infection requires rapid and global shifts in gene expression patterns. In this study, we use a novel pathogen transcript enrichment strategy and whole transcriptome sequencing (RNA-Seq) to perform a detailed characterization of the rapid and global shifts in F. tularensis LVS gene expression during infection of murine macrophages. We performed differential gene expression analysis on all bacterial genes at two key stages of infection: phagosomal escape, and cytosolic replication. By comparing the F. tularensis transcriptome at these two stages of infection to that of the bacteria grown in culture, we were able to identify sets of genes that are differentially expressed over the course of infection. This analysis revealed the temporally dynamic expression of a number of known and putative transcriptional regulators and virulence factors, providing insight into their role during infection. In addition, we identified several F. tularensis genes that are significantly up-regulated during infection but had not been previously identified as virulence factors. These unknown genes may make attractive therapeutic or vaccine targets.

Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing.

To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.

Application of CGE to virus identification.

Protein profiling is an increasingly valuable tool for the characterization of protein populations and has been used to identify microorganisms, most often using two-dimensional gel electrophoresis followed by mass spectrometry. We present a rapid method for the identification of viruses using microfluidic chip gel electrophoresis (CGE) of high-copy number proteins to generate unique protein profiles. Viral proteins are solubilized, fluorescently labeled and then analyzed using the µChemLab™ CGE system (∼10 min overall). A Bayesian classification approach is used to classify the reproducible and visually distinct protein profiles of MS2 bacteriophage, Epstein-Barr, Respiratory Syncytial, and Vaccinia viruses as well as discriminate between closely related T2 and T4 bacteriophage.

cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications.

Second-generation sequencing (SGS) has become the preferred method for RNA transcriptome profiling of organisms and single cells. However, SGS analysis of transcriptome diversity (including protein-coding transcripts and regulatory non-coding RNAs) is inefficient unless the sample of interest is first depleted of nucleic acids derived from ribosomal RNA (rRNA), which typically account for up to 95% of total intracellular RNA content. Here we describe a novel microscale hydroxyapatite chromatography (HAC) normalization method to remove eukaryotic and prokaryotic high abundant rRNA species, thereby increasing sequence coverage depth and transcript diversity across non-rRNA populations. RNA-seq analysis of Escherichia coli K-12 and human intracellular total RNA showed that HAC-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAC normalization generated rRNA-depleted cDNA libraries comparable to the well-established duplex specific nuclease (DSN) normalization and Ribo-Zero rRNA-depletion methods, thus establishing microscale HAC as an effective, cost saving, and non-destructive alternative normalization technique.

Development of an integrated microfluidic instrument for unattended water-monitoring applications.

Field-deployable detection technologies in the nation's water supplies have become a high priority in recent years. The unattended water sensor is presented which employs microfluidic chip-based gel electrophoresis for monitoring proteinaceous analytes in a small integrated sensor platform. The instrument collects samples directly from a domestic water flow. The sample is then processed in an automated microfluidic module using in-house designed fittings, microfluidic pumps and valves prior to analysis via Sandia's microChemLab module, which couples chip-based electrophoresis separations with sensitive LIF detection. The system is controlled using LabVIEW software to analyze water samples about every 12 min. The sample preparation, detection and data analysis has all been fully automated. Pressure transducers and a positive control verify correct operation of the system, remotely. A two-color LIF detector with internal standards allows corrections to migration time to account for ambient temperature changes. The initial unattended water sensor prototype is configured to detect protein biotoxins such as ricin as a first step toward a total bioanalysis capability based on protein profiling. The system has undergone significant testing at two water utilities. The design and optimization of the sample preparation train is presented with results from both laboratory and field testing.

Identification of viruses using microfluidic protein profiling and Bayesian classification.

We present a rapid method for the identification of viruses using microfluidic chip gel electrophoresis (CGE) of high-copy number proteins to generate unique protein profiles. Viral proteins are solubilized by heating at 95 degrees C in borate buffer containing detergent (5 min), then labeled with fluorescamine dye (10 s), and analyzed using the microChemLab CGE system (5 min). Analyses of closely related T2 and T4 bacteriophage demonstrate sufficient assay sensitivity and peak resolution to distinguish the two phage. CGE analyses of four additional viruses--MS2 bacteriophage, Epstein-Barr, respiratory syncytial, and vaccinia viruses--demonstrate reproducible and visually distinct protein profiles. To evaluate the suitability of the method for unique identification of viruses, we employed a Bayesian classification approach. Using a subset of 126 replicate electropherograms of the six viruses and phage for training purposes, successful classification with non-training data was 66/69 or 95% with no false positives. The classification method is based on a single attribute (elution time), although other attributes such as peak width, peak amplitude, or peak shape could be incorporated and may improve performance further. The encouraging results suggest a rapid and simple way to identify viruses without requiring specialty reagents such as PCR probes and antibodies.

Bacterial characterization using protein profiling in a microchip separations platform.

A rapid microanalytical protein-based approach to bacterial characterization is presented. Chip gel electrophoresis (CGE) coupled with LIF detection was used to analyze lysates from different bacterial cell lines to obtain signature profiles of the soluble protein composition. The study includes Escherichia coli, Bacillus subtilis, and Bacillus anthracis (Delta Sterne strain) vegetative cells as well as endospores formed from the latter two species as model organisms to demonstrate the method. A unified protein preparation protocol was developed for both cell types to streamline the benchtop process and aid future automation. Cells and spores were lysed and proteins solubilized using a combination of thermal and chemical lysis methods. Reducing agents, necessary to solubilize spore proteins, were eliminated using a small-scale rapid size-exclusion chromatography step to eliminate interference with down-stream protein labeling. This approach was found to be compatible with nonspore cells (i.e., vegetative cells) as well, not adversely impacting the protein signatures. Data are presented demonstrating distinct CGE protein signatures for our model organisms, suggesting the potential for discrimination of organisms on the basis of empirical protein patterns. The goal of this work is to develop a fast and field-portable method for characterizing bacteria via their proteomes.

Autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores.

For domestic and military security, an autonomous system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol collector of approximately 80% efficiency operating for 7 min.

Microchip separations of protein biotoxins using an integrated hand-held device.

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.

Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins.

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%).

Simultaneous resolution of underivatized regioisomers and stereoisomers of arachidonate epoxides by capillary electrophoresis.

cis-Epoxyeicosatrienoic acids (EETs) and their hydrolysis products (threo-DHETs) have been proposed to be endothelial-dependent hyperpolarizing factors (EDHFs) which upregulate blood flow when tissue perfusion is impaired. Various EET regioisomers and enantiomers are formed from arachidonate by inducible cytochrome P450 epoxygenase isoforms, and tissue EET profiles may vary with diet, time, and disease. Because EET actions and metabolism may be regio- and stereospecific, convenient methods to measure profiles of EET isomers in tissues are needed. In the current studies, we describe two simple capillary electrophoretic methods for resolving EETs. The first method involves capillary electrophoresis with a mixture of neutral and anionic beta-cyclodextrins, which in one step baseline-resolves underivatized EET regioisomers and their enantiomers. Low picogram amounts of EET enantiomers were identified based on migration times and UV spectra. The method was also used to assess the antipode purity of EET standards, and to determine murine hepatic levels of EET enantiomers. The second method involves capillary electrochromatography, which also baseline-resolves underivatized EET and DHET regioisomers in one step. We conclude that in EET assays the major advantages of capillary electrophoresis over reversed-phase HPLC are improved peak efficiency, sensitivity, and resolution, plus precise coelution of deuterated and nondeuterated EETs.

Capillary electrophoresis of cytochrome P-450 epoxygenase metabolites of arachidonic acid. 1. Resolution of regioisomers.

The essential fatty acid arachidonate is oxidized by cytochrome P-450 epoxygenases to four epoxyeicosatrienoic acids (EETs): 14,15-, 11,12-, 8,9-, and 5,6-EETs. Each of the four EET regioisomers and their hydrolysis products (DHETs) has multiple paracrine and autocrine functions and may also potently dilate blood vessels and activate potassium channels. The present work describes a method to resolve EETs and DHETs by capillary electrophoresis (CE) using trimethyl-beta-cyclodextrin and CH3CN as buffer additives. While stored at 25 degrees C, most of the EET and DHET regioisomers remained intact when suspended in alkaline vehicle. However, under these same conditions, 5,6-EET rapidly broke down to a lactone and was slowly converted to 5,6-DHET. When subjected to CE, the EET and DHET regioisomers were baseline resolved (R > or = 1.3); 10 pg of an EET or a DHET regioisomer was readily detectable at 194 nm. In addition, the UV spectra were regiospecific and identical to those obtained during HPLC except that an additional, weak absorption occurred at 235 nm. Together, the high-sensitivity, high-resolution, and differential UV spectra permitted the identification and quantification of EETs in phospholipids isolated from murine liver. Thus, CE was successfully used for the trace analysis of eicosanoids.

Capillary electrophoresis of cytochrome P-450 epoxygenase metabolites of arachidonic acid. 2. Resolution of stereoisomers.

Each of the four regioisomers of epoxyeicosatrienoic acids (EETs) is a candidate for being an endothelial-dependent hyperpolarizing factor (EDHF). One regioisomer, 14,15-EET, stereospecifically blocks cyclooxygenases from converting arachidonic acid to prostaglandins and stereospecifically binds to cellular receptors. Both stereospecific actions emphasize the need to establish the tissue availability of the 14,15-EET enantiomers. The present work describes a method to quantitate picogram amounts of 14,15-EET enantiomers by capillary electrophoresis. The 14,15-EET enantiomers were baseline resolved (R = 1.3) using unsubstituted beta-cyclodextrin and 32% acetonitrile (v/v). When absorption at 194 nm was monitored using a photodiode array detector, 8 and 1 pg of underivatized 14,15-EET were readily quantitated and detected, respectively. Capillary electrophoresis accurately assessed chiral excesses up to 97:3 for either 14,15-EET enantiomer. Moreover, capillary electrophoresis with a photodiode array detector was sufficiently sensitive to detect and measure 14,15-EET enantiomers from murine liver. Thus, unlike chiral-phase high-performance liquid chromatography, capillary electrophoresis can be used to directly assess the chirality of trace amounts of underivatized eicosanoids.