Organ-on-a-chip platforms are utilized in global bioanalytical and toxicological studies as a way to reduce materials and increase throughput as compared to in vivo based experiments. These platforms bridge the infrastructure and regulatory gaps between in vivo animal work and human systems, with models that exemplify active biological pathways. In conjunction with the advent of increased capabilities associated with next generation sequencing and mass spectrometry based ‘-omic’ technologies, organ-on-a-chip platforms provide an excellent opportunity to investigate the global changes at multiple biological levels, including the transcriptome, proteome and metabolome. When investigated concurrently, a complete profile of cellular and regulatory perturbations can be characterized following treatment with specific agonists. In this study, global effects were observed and analyzed following liver chip exposure to the chemical warfare agent, VX. Even though the primary mechanism of action of VX (i.e. acetylcholinesterase inhibition) is well characterized, recent in vivo studies suggest additional protein binding partners that are implicated in metabolism and cellular energetic pathways. In addition, secondary toxicity associated with peripheral organ systems, especially in human tissues, is not well defined. Our results demonstrate the potential of utilizing an organ-on-a-chip platform as a surrogate system to traditional in vivo studies. This is realized by specifically indicating significant dysregulation of several cellular processes in response to VX exposure including but not limited to amino acid synthesis, drug metabolism, and energetics pathways.

A common requirement in the military, law enforcement, and forensic mission space is the need to collect trace samples from surfaces using a method that not only readily captures the sample but also retains its integrity for downstream identification and characterization. Additionally, collecting samples from three-dimensional objects (e.g., shell casings) is a challenge for which there is currently no validated standardized approach. Recently, hydrogels have been shown to have the potential for surface collection of trace bacterial spores, amino acids, and DNA. To test whether these hydrogels can serve as a viable collection medium for sampling DNA from surfaces, we carried out a series of preliminary tests examining collection efficiency and suitability of hydrogel material to recover samples of diluted, dried human DNA on a smooth polycarbonate surface. The recovery of surface DNA using a commercially available hydrogel was examined, and the efficiency compared to samples collected using a standard foam collection swab. DNA collected using the hydrogel and swab methods was then examined using quantitative polymerase chain reaction (qPCR) and short tandem repeat (STR) analysis to determine whether the collection material was compatible with these downstream processes. The hydrogel material used for this study collected the experimental DNA with comparable efficiency to standard collection swabs. In addition, qPCR and STR analyses demonstrated compatibility with the hydrogel collection and extraction process. These data suggest that hydrogels have the potential to be used as sample collection materials and deserve further characterization to elucidate their utility in collection from irregularly shaped, three-dimensional surfaces/materials.

The opioid crisis has continued to progress in the United States and the rest of the world. As this crisis continues, there is a pressing need for a rapid and cost-effective method for detecting fentanyl. Recent studies have suggested that lateral flow immunoassays (LFIs) could fill this technology gap. These qualitative paper-based assays contain antibodies designed to react with fentanyl and provide positive or negative results within a matter of minutes. In this study, two different LFI configurations for the detection of fentanyl were examined (dipsticks and cassettes) for effectiveness of detection using seized drug samples and postmortem urine samples. In the current study, 44 seized drug samples (32 fentanyl-positive, 12 fentanyl-negative) and 14 postmortem urine samples (10 fentanyl-positive, 4 fentanyl-negative) were analyzed. All 32 fentanyl-containing seized drug samples and 10 postmortem fentanyl-positive urine samples displayed positive LFI results with both LFI configurations. The fentanyl dipsticks displayed a sensitivity of 100%, a specificity of 75%, and an efficiency of 93.2% for seized drug samples and a sensitivity, specificity, and efficiency of 100% for postmortem urine. Analysis of the fentanyl cassettes displayed a sensitivity, specificity, and efficiency of 100% for seized drug samples and a sensitivity of 100%, a specificity of 75%, and an efficiency of 92.9% for postmortem urine samples. These data point to the utility of LFIs as a quick and low resource-dependent option for presumptive detection of fentanyl in real-world situations.

Overdoses due to the adulteration of the illegal opioid drug supply with synthetic opioids (i.e. fentanyl and fentanyl derivatives) continue to be a serious problem in the United States. The presence of these substances in this supply not only presents a risk to users, but also first responders. Synthetic opioids have a high toxicity and this toxicity leads to a high risk of overdose or accidental exposure when they are included in the illegal opioid supply. Due to this toxicity, first responders and forensic scientists have a need for rapid, accurate, and low-cost detection methods. In this study, we examined the commercially available fentanyl-directed lateral flow immunoassay (LFI) produced by BTNX, Inc. (Markham, ON Canada). Several aspects of opioid detection performance were examined: limits of detection (LODs) and sensitivity for fentanyl and fentanyl derivatives in liquid and from dried surface sampled fentanyl, effectiveness of detection from complex illicit street drug samples, as well as the ability of these LFIs to determine fentanyl presence from in vivo urine from rabbits and human urine collected from postmortem cases. The results of this study suggest that fentanyl-directed LFIs have the potential to be used by both first responders and forensic scientists to determine the presence of synthetic opioids in the field.

Despite the recent epidemic of fentanyl abuse, there are few validated assays capable of rapidly detecting these compounds. In order to improve the ability to detect carfentanil at physiologically relevant concentrations, we developed a systems biology approach to discover host-based markers which are specifically amplified upon exposure in a rabbit model. For this work, two omics pipelines utilizing mass spectrometry were developed and leveraged. First, a proteomics pipeline was developed to interrogate the blood plasma for protein-based biomarkers. Due to the incredible dynamic range of the plasma protein content, a multi-dimensional fractionation technique was used to partition and more accurately investigate the circulating plasma proteome. Isobaric tandem mass tags were integrated into the workflow to make quantitative assessments across all animals for an extended time course post-exposure. In addition to the proteomics efforts, blood plasma was also processed through an untargeted metabolomics pipeline. This approach allows for the identification of >800 small molecule features. By processing and analyzing data sets in parallel, we were able to identify a unique fingerprint of protein and metabolite perturbations that manifest following exposure to carfentanil.

In 2017, 47,600 overdose deaths were reported to be associated with the abuse of opioids, including prescription painkillers (e.g. oxycodone), opiates (e.g. heroin), or synthetic opioids (e.g. fentanyl) within the United States. The recent spike in the presence of synthetic opioids in lots of heroin distributed on the street present specific and significant challenges to law enforcement. Synthetic opioids are extremely toxic substances, which can easily be inhaled. This type of exposure can lead to accidental overdoses by law enforcement and other first responders answering calls involving illicit drugs containing these substances. Due to this extreme toxicity, it is important for these individuals to have tools that can be easily deployed for accurate presumptive field tests. Currently, there are only a limited number of presumptive tests available for fentanyl detection. In this study, we addressed this technology gap by evaluating newly developed lateral flow immunoassays (LFIs) designed for the detection of fentanyl and its derivatives. These LFIs were evaluated for effectiveness in different biofluid matrices, following an in vivo exposure, cross-reactivity with fentanyl analogs, and in case samples. This study demonstrates that LFIs have the potential to be used by law enforcement for the detection of synthetic opioids.

This chapter provides an overview of each of the associated technologies and their current state of the art and describes examples of how global approaches are being applied to the development of chemical warfare agent (CWA) countermeasures. If metabolomic work has been done on the key metabolic product peaks, this information can open avenues for research to reveal more complete mechanisms of toxicity and potential biochemical points for developing effective CWA countermeasures. Genomics, transcriptomics, and gene expression profiling are all related to studying gene expression of hundreds to thousands of genes simultaneously from a single biological sample. Large-scale genome sequencing projects, such as the Human Genome Project, have revolutionized applied biology and toxicology. In addition, identifying exposure to and effects of low levels of CWA has been problematic. Genomic, proteomic, and metabolic technologies provide a new way to uncover novel biomarkers for rapid and accurate force health protection and monitorin.

Currently, all assays measuring acetylcholinesterase (AChE) activity following a suspected nerve agent exposure leverage methodologies that fail to identify the agent. This limits the overall effectiveness and ability to administer proper countermeasures. As such, there is an urgent need to identify novel, rapid, and more comprehensive approaches to establish AChE activity, including identification of the toxicant. Paper spray mass spectrometry was used to monitor the activity of acetylcholinesterase, both in-solution and on modified hydrophobic paper surface. Hydrophobic paper surfaces were prepared using vaporized trichloro(3,3,3-trifluoropropyl)silane. In both approaches, mixtures of diluted human whole blood with and without VX were mixed with a non-endogenous AChE specific substrate, 1,1-dimethyl-4-acetylthiomethylpiperidinium (MATP+). Formation of the cleaved MATP+ product was monitored over time and compared to MATP+ to determine relative AChE activity. This on-substrate assay was effective at determining AChE activity and identifying the toxicant; however, determination of AChE activity in-solution proceeded at a slower rate. The on-substrate assay serves as a pioneering example of an enzymatic reaction occurring on the surface of a paper spray ionization ticket. This work broadens the range of applications relating to paper spray ionization-based clinical diagnostic assays. Graphical Abstract ᅟ

Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions, which could be useful for the development of novel diagnostics or potential adjuvant therapeutics.

To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184).

Abstract : This technical report is the third of three reports from the U.S. Army Edgewood Chemical Biological Center (ECBC) In-House Laboratory Independent Research (ILIR) funded project Molecular Toxicology of TICs in Human Embryonic Stem Cells . One of the goals of the study was to identify metabolites from the secretome of human embryonic stem cells (hESC) exposed to the toxic industrial chemicals (TICs) methyl parathion (MP) and methyl paraoxon (MPO). The non-targeted liquid chromatography followed by mass spectrometry (LC-MS) approach used in the Phase I work (Sekowski et al., ECBC-TR-1177) revealed hundreds of human metabolites and multiple metabolic pathways altered as a result of exposure to both chemicals. This was followed by Phase II work (Madren-Whalley et al., ECBC-TR-1178) in which twenty metabolites were down-selected for further structural confirmation. The purpose of this Phase III study was to confirm the identity of the down-selected subset of putative metabolites by comparing them with commercially available standards using liquid chromatography followed by tandem mass spectrometry (LC-MS-MS). Commercially available standards were available for nine metabolites. Of these nine, the identity of five metabolites was confirmed in this work.

: Current literature suggests diverse toxicological consequences for methyl parathion (MP) and its active metabolite, methyl paraoxon (MPO) exposure. These studies, however, have been based on either human epidemiological studies, in vivo animal studies, or in vitro studies using immortal cell lines. Currently, there remains no definitive connection to the molecular events that occur during MP and MPO exposure in normal human cells. Furthermore, chemicals that have certain known effects in adults can have dramatically different toxic effects during embryonic and prenatal development. Since undifferentiated human embryonic stem cells (hESC) maintain the ability to differentiate into any somatic cell in the body, they provide a unique window into the influence of toxicants on the entire early human development. The purpose of this study was to perform initial nontargeted metabolomic analysis on hESC exposed to MP and MPO to identify human metabolites and metabolic pathways which are perturbed following exposure to these chemicals. From this study, two main conclusions were reached: (1) hESC are not able to metabolize MP into MPO, and (2) MPO is much more disruptive to key developmental metabolic pathways at physiologically relevant concentrations than is MP.

We investigated the use of functionalized photonic block copolymer films for the detection of glucose. Polystyrene-b-poly(2-vinyl pyridine) (PS-b-P2VP) block copolymers were chemically functionalized with 2-(bromomethyl)phenylboronic acid and cast into films that reflect a visible color when exposed to aqueous media. The 2-(bromomethyl)phenylboronic acid functionality can reversibly bind to glucose. When exposed to high concentrations of glucose the polymer responded with a red shift in color. Low concentration exposure of glucose caused the polymer films to blue shift in color. The BCP films also exhibited a selective response to fructose, mannose or galactose, giving a different response depending on which sugar is present. The color of the polymer was tuned to blue, green, yellow or orange by varying the film's crosslink density. The color change can be visually observed without the use of equipment such as a spectrometer.

: The most widely used transient form of stored energy in biological systems is the complex, organic molecule, adenosine triphosphate (ATP). However, the much smaller, simpler pyrophosphate (PPi) molecule could potentially serve as an inexpensive, high energy bond, ATP mimic. Pyruvate Phosphate Dikinase (PPDK), an enzyme found primarily in a small number of primitive protists reversibly transfers phosphate (Pi) from phosphoenolpyruvate (PEP) and PPi to AMP producing ATP. Alpha-Hemolysin pore-functionalized, lipid bilayers facilitated movement of PEP and PPi through 1-2 nm pores with concomitant Luciferase coupled ATP dependent generation of light. This is an important first step towards developing self-renewing power for nano/micro systems.

W1-1 Differential gene expression profiles of acute and chronic injury and recovery in rat liver steatosis-fibrosis/cirrhosis modelHeekyoung CHUNG (KST)W1-2 SNPs in CYP450s: a preliminary pharmacogenomic study in a Chinese populationWEN Si-Yuan1, CHEN Kun2, XU Li1, LI Jian2, WANG Rui2, WANG Sheng-Qi1(1. Beijing Institute of Radiation Medicine, 2. Beijing 301 General Hospital, Beijing 100039, China)Abstract:Pharmacogenomics was established on the fact that certain genetic polymorphisms may cause significantly different responses among individuals exposed to a particular drug. Single nucleotide polymorphism (SNP) is the most common form of genetic polymorphism in human genome. The association study of SNPs with phenotypic traits is of great importance in genomic study.