Identifying qualitative differences in PPARα signaling networks in human and rat hepatocytes and their significance for next generation chemical risk assessment methods.

In this paper, we evaluate the PPARα signaling network in rats, examining transcriptional responses in primary hepatocytes exposed to a PPARα specific ligand, GW7647. These transcriptomic studies were complemented with ChIP-seq studies of PPARα binding and transcription binding motif identification for PPARα responsive genes. We also conducted a limited study of GW7647 dosing the in intact rat to examine differences in transcriptional responses for primary hepatocytes in vitro and in the intact liver. The rat network has a much larger number of down-regulated genes and pathways than we had found in the human and the PPARα binding motifs in rat differed for upregulated and down regulated genes. Based on these results and comparison with our previous work with the human PPARα signaling network, we identified qualitative differences in the transcriptional networks controlled by PPARα activation in the two species that provide an explanation of the interspecies differences in the responses of humans and rodents to GW7647 and likely to other PPARα agonists. These studies also allow some observations on the manner in which in vitro, fit-for-purpose assays in human hepatocytes could form the basis for risk assessment without recourse to in-life studies in rodents or other test species.

A map of the PPARα transcription regulatory network for primary human hepatocytes.

Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARα) in primary human hepatocytes using the selective PPARα ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIP-seq studies at 2 and 24h to assess genomic binding of PPARα. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARα binding-either directly at PPARα response elements or via alternative mechanisms. Almost half of regulated genes had no PPARα binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARα and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERα, and HNF4α. The linkage from PPARα binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10 µM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways.

MassCode liquid arrays as a tool for multiplexed high-throughput genetic profiling.

Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR) performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers.

Multiplexed molecular assay for rapid exclusion of foot-and-mouth disease.

A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV "look-alike" diagnostic assay panel contains 5 PCR and 12 reverse transcriptase PCR (RT-PCR) signatures for a total of 17 simultaneous PCR amplifications for 7 diseases plus incorporating 4 internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV "look-alike" viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

Multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at the point of care.

We have developed a nucleic acid-based assay that is rapid, sensitive, and specific and can be used for the simultaneous detection of five common human respiratory pathogens, including influenza virus A, influenza virus B, parainfluenza virus types 1 and 3, respiratory syncytial virus (RSV), and adenovirus groups B, C, and E. Typically, diagnosis on an unextracted clinical sample can be provided in less than 3 h, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs and therefore allow implementation of infection control measures and the timely administration of antiviral therapies. We present here a summary of the assay performance in terms of sensitivity and specificity. The limits of detection are provided for each targeted respiratory pathogen, and result comparisons were performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the University of California-Davis Medical Center hospital. Overall, the use of the multiplexed reverse transcription-PCR assay reduced the rate of false-negative results by 4% and reduced the rate of false-positive results by up to 10%. The assay correctly identified 99.3% of the clinical negatives and 97% of the adenovirus, 95% of the RSV, 92% of the influenza virus B, and 77% of the influenza virus A samples without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on unextracted samples.

Toward a multiplexed serotyping immunoassay for foot-and-mouth disease virus.

Initial results demonstrating the feasibility of a multiplexed liquid array immunoassay for foot-and-mouth disease viral antigen detection and simultaneous serotype differentiation are presented. Serotype-specific antibodies from rabbit and guinea pig hyperimmunesera were isolated and prepared for use in a multiplexed, bead-based assay. The performance of all of the available antibodies as both capture and detector reagents was evaluated in the multiplexed system to establish a combination exhibiting the highest homotypic responses and lowest heterotypic reactions. The multiplexed assay was evaluated against inactivated cell culture supernatant samples of the same subtype as the virus used to raise the capture and detector antibodies. Distinct serotype differentiation was observed, except in the case of serotype SAT1. Subsequently, cell culture supernatant samples from a larger pool of viral subtypes were analyzed. Distinct serotype differentiation was obtained when analyzing cell culture supernatant samples from viral serotypes C, Asia, and SAT3, irrespective of the subtype. However, limitations of the current antibody pairs were realized in some inconclusive results obtained when analyzing samples from a broader range of O, A, and SAT2 subtypes. The results obtained in this initial study will be used to further optimize the assay using polyvalent or monoclonal antibodies and move toward the analysis of clinical samples.

Multiplexed detection of antibodies to nonstructural proteins of foot-and-mouth disease virus.

Liquid array technology was used to develop a multiplexed assay for the detection of antibodies to viral nonstructural proteins (NSPs), raised in cattle in response to infection with foot-and-mouth disease (FMD) virus. Two assays, one based on recombinant NSPs and the other on synthetically produced peptides, were developed and compared side-by-side. Serum samples from serial bleeds of cattle, each experimentally infected with one of the seven serotypes (C, A, O, Asia, SAT1, SAT2, SAT3) of FMD virus were analyzed. A distinct pattern in the detection of NSP antibodies and a close correlation of the recombinant protein and peptide-based assays were observed. The detection of antibodies to NSPs is a method to differentiate FMD-infected and FMD-vaccinated animals, and a high-throughput assay would be an invaluable tool in the case of an outbreak of FMD in North America, when emergency vaccination may be utilized to spare vaccinated, noninfected animals from slaughter and subsequent disposal.

Fabricating optical fiber imaging sensors using ink jet printing technology: a pH sensor proof-of-concept.

We demonstrate the feasibility of using Drop-on-Demand microjet printing technology for fabricating imaging sensors by reproducibly printing an array of photo-polymerizable sensing elements, containing a pH sensitive indicator, on the surface of an optical fiber image guide. The reproducibility of the microjet printing process is excellent for microdot (i.e. micrometer-sized polymer) sensor diameter (92.2+/-2.2 microm), height (35.0+/-1.0 microm), and roundness (0.00072+/-0.00023). pH sensors were evaluated in terms of pH sensing ability (< or =2% sensor variation), response time, and hysteresis using a custom fluorescence imaging system. In addition, the microjet technique has distinct advantages over other fabrication methods, which are discussed in detail.

APDS: the autonomous pathogen detection system.

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system was developed to provide early warning to civilians in the event of a bioterrorism incident and can be used at high profile events for short-term, intensive monitoring or in major public buildings or transportation nodes for long-term monitoring. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic acid-based polymerase chain reaction (PCR) amplification and detection. Highly multiplexed antibody-based and duplex nucleic acid-based assays are combined to reduce false positives to a very low level, lower reagent costs, and significantly expand the detection capabilities of this biosensor. This article provides an overview of the current design and operation of the APDS. Certain sub-components of the ADPS are described in detail, including the aerosol collector, the automated sample preparation module that performs multiplexed immunoassays with confirmatory PCR, and the data monitoring and communications system. Data obtained from an APDS that operated continuously for 7 days in a major U.S. transportation hub is reported.

Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation.

The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.

Comparison of multiplexed techniques for detection of bacterial and viral proteins.

Immobilized antibody microarrays were compared to the Luminex flow cytometry system that utilizes suspensions of polystyrene microbeads covalently coupled with capture antibodies. The two immunoassays were performed for comparison of reproducibility, limits of detection and dynamic range. The Luminex system showed lower limits of detection and increased dynamic range among samples whereas the protein microarrays could be more amenable to miniaturization. Both technologies were capable of sensitive multiplexed detection.

Development of an automated sample preparation module for environmental monitoring of biowarfare agents.

An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.

Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents.

Liquid array-based multiplexed immunoassays designed for rapid, sensitive, specific, and simultaneous detection of multiple simulants of biological warfare agents have been developed. In both blind and standard laboratory trials, we demonstrate the simultaneous detection of four simulant agents from a single sample. The challenge agents comprise broad classes of pathogens (virus, protein toxins, bacterial spores, vegetative cells). Assay performance of each analyte was optimized, and dose-response curves and the limits of detection (LODs) for individual analytes are presented. Assay performance, including dynamic range, sensitivity, and LODs for liquid arrays and enzyme-linked immunosorbant assay were compared and are shown to be similar. Maximum assay sensitivity is obtained in approximately 1 h, and good sensitivity is achieved in as little as 30 min. Although the sample matrixes are very complex, even for highly multiplexed assays the samples do not exhibit evidence of nonspecific binding, demonstrating that the assays also have high specificity.

Autonomous detection of aerosolized Bacillus anthracis and Yersinia pestis.

We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.