Sarin exposure, mortality and cancer incidence in UK military veterans involved in human experiments at Porton Down: 52-year follow-up.

OBJECTIVES: We investigated whether UK military personnel exposed to sarin during the 'Service Volunteer Programme' at Porton Down had increased rates of mortality or cancer incidence over a 52-year follow-up. METHODS: A historical cohort study assembled from UK military records, comprising male veterans exposed to sarin during the 'Service Volunteer Programme' at Porton Down, UK (n=2975) and a comparison group of similar veterans who did not attend (n=2919). Mortality and cancer incidence data were obtained from national registries up to December 2019. Analysis was conducted using Cox regression adjusted for age, year of birth and service characteristics. RESULTS: Over a median follow-up of 52.2 years (range 2 days to 74.6 years), 1598 (53.7%) sarin-exposed veterans and 1583 (54.3%) non-exposed veterans died. Adjusted HRs for all-cause mortality were raised for any sarin exposure (HR=1.08, 95% CI 1.01 to 1.16), two or more exposures (HR=1.25, 95% CI 1.04 to 1.49) and higher doses (air >15 mg.min/m3) (HR=1.15, 95% CI 1.02 to 1.30). For cause-specific mortality, sarin exposure was associated with deaths from 'other' circulatory diseases (excludes ischaemic and cerebrovascular diseases) (HR=1.41, 95% CI 1.06 to 1.87) and alcohol-attributable deaths (HR=2.66, 95% CI 1.40 to 5.07). There was no association between sarin exposure and overall cancer incidence (HR=1.01, 95% CI 0.93 to 1.10), but cancer incidence was higher for alcohol-related neoplasms (HR=1.24, 95% CI 1.01 to 1.51). CONCLUSIONS: Sarin exposure was associated with increased rates of mortality over a 50-year follow-up. The strongest associations were observed for deaths attributable to alcohol and 'other' circulatory diseases.

Development of dispersive solid phase extraction method for the preconcentration of parathion ethyl as a simulant of nerve agent sarin from soil, plant and water samples prior to GC-MS determination.

In the presented study, an efficient and fast analytical method was developed for the determination of parathion ethyl as sarin simulant by gas chromatography-mass spectrometry (GC-MS). Dispersive solid phase extraction (DSPE) was performed to concentrate parathion ethyl from soil, plant and water samples. Reduced graphene oxide-iron (II, III) oxide (rGO-Fe3O4) nanocomposite was used as an adsorbent to collect the target analyte from the aqueous sample solutions. After the optimization of extraction/preconcentration parameters, optimum conditions for adsorbent amount, eluent type, mixing type/period, eluent volume and initial sample volume were determined as 15 mg, acetonitrile, vortex/30 s, 100 µL and 10 mL, respectively. Under the optimum conditions, analytical performance of the developed DSPE-GC-MS method was evaluated in terms of limit of detection (LOD), limit of quantitation (LOQ) and dynamic range. Dynamic range, LOD and LOQ values were figured out to be 0.94-235.15 µg/kg, 0.41 µg/kg and 1.36 µg/kg (mass based), respectively. Satisfactory percent recovery results (90.3-125% for soil, 93.5-108.7% for plant, 88.5-112.9% for tap water) were achieved for soil, plant and tap water samples which proved the accuracy and applicability of the developed method. It is predicted that the DSPE-GC-MS method can be accurately used for the detection of sarin in soil, plant and water samples taken from war territories.

Construction of logic gate computation for the assay of the nerve agent sarin based on an AChE-based dual-channel sensing system.

Nerve agents have posed a huge threat to national and human security, and their sensitive detection is crucial. Herein, based on the oxidation of Ce4+ and the aggregation-induced emission (AIE) of glutathione-protected gold nanoclusters (GSH-Au NCs), a cascade reaction was designed to prepare oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) and GSH-Au NCs crosslinked by Ce3+ (Ce3+-GSH-Au NCs). oxTMB had a broad UV-visible absorption range (500-700 nm) and was capable of quenching the fluorescence of Ce3+-GSH-Au NCs at 590 nm through the internal filtration effect (IFE). Thiocholine (TCh), the hydrolysis product of acetylthiocholine chloride (ATCl) catalyzed by acetylcholinesterase (AChE), reduced oxTMB completely, resulting in a decrease in the absorption of oxTMB and the recovery of IFE-quenched fluorescence of Ce3+-GSH-Au NCs. Nerve agent sarin (GB) hindered the production of TCh and the reduction of oxTMB by inhibiting the AChE activity, leading to the fluorescence of Ce3+-GSH-Au NCs being quenched again. The dual-output sensing system (AChE + ATCl + oxTMB + Ce3+-GSH-Au NCs) exhibited a low limit of detection to GB (2.46 nM for colorimetry and 1.18 nM for fluorimetry) and excellent selectivity toward common interferences being unable to inhibit AChE. Moreover, the intelligent logic gate constructed based on the sensing system showed promising applications in the field of smart sensing of nerve agents.

A pyrene-based chromo-fluorogenic probe for specific detection of sarin gas mimic, diethylchlorophosphate.

Nerve agents are becoming serious issues for the healthy and sustainable environment of modern civilization. Therefore, its detection and degradation are of paramount importance to the scientific community. In the present contribution, we have introduced a chromo-fluorogenic pyrene-based  probe, (E)-2-methoxy-3-(pyren-1-ylimino)-3,8a-dihydro-2H-chromen-4-ol (PMCO) to detect sarin stimulant diethylchlorophosphate (DCP) in solution and gaseous phases. On inserting DCP in PMCO solution, a visual colorimetric change from yellow to clear colourless in daylight and highly intensified blue fluorescence was observed instantly under a 365 nm portable UV lamp light. PMCO has outstanding selectivity and high sensitivity with a limit of detection of 1.32 µM in dimethyl sulfoxide (DMSO) medium and 77.5 nM in 20% H2O-DMSO. A handy strained paper strip-based experiment was demonstrated to recognize DCP in a mixture of similar toxic analytes. A dip-stick experiment was performed to identify DCP vapour, and may be used as an effective photonic tool. We also demonstrated real sample analysis utilizing different DCP-spiked water samples and validating DCP detection even in various types of soils such as sand, field, and mud. Therefore, this present study provides an effective chemosensor for instant and on-site detection of toxic nerve agents in dangerous circumstances.

Highly specific and sensitive chromo-fluorogenic detection of sarin, tabun, and mustard gas stimulants: a multianalyte recognition approach.

Nerve agents are the most notorious substances, which can be fatal to an individual because they block the activity of acetylcholinesterase. Fighting against unpredictable terrorist assaults and wars requires the simple and quick detection of chemical warfare agent vapor. In the present contribution, we have introduced a rhodamine-based chemosensor, BDHA, for the detection of nerve gas-mimicking agents diethylchlorophosphate (DCP) and diethylcyanophosphonate (DCNP) and mustard gas-mimicking agent 2-chloroethyl ethyl sulfide (CEES), both in the liquid and vapor phase. Probe BDHA provides the ability for detection by the naked eye in terms of colorimetric and fluorometric changes. It has been revealed that the interaction between nerve agents mimics and probe BDHA facilitates spirolactam ring opening due to the phosphorylation process. Thus, the highly fluorescent and colored species developed while probe BDHA is colorless and non-fluorescent due to the intramolecular spirolactam ring. Moreover, probe BDHA can effectively recognize DCP, DCNP, and CEES in the µM range despite many toxic analytes and could be identified based on the response times and quantum yield values. Inexpensive, easily carried paper strips-based test kits were developed for the quick, on-location solid and vapor phase detection of these mustard gas imitating agents (CEES) and nerve gas mimicking agents (DCP and DCNP) without needing expensive equipment or skilled personnel. More remarkably, the test strips' color and fluorescence can be rapidly restored, exposing them to triethyl amine (TEA) for cyclic use, suggesting a potential application in the real-time identification of chemical warfare agents. To accomplish the on-location application of BDHA, we have experimented with soil samples to find traces of DCP. Therefore, the chromo-fluorogenic probe BDHA is a promising, instantaneous, and on-the-spot monitoring tool for the selective detection of DCP, DCNP, and CEES in the presence of others.

Highly selective and sensitive chromogenic recognition of sarin gas mimicking diethylchlorophosphate.

The high-level toxic effects of organophosphate (OP) nerve agents severely threaten national security and public health. Generating trustworthy, accurate methods for quickly identifying these poisonous chemicals is urgently necessary. In this study, we have presented an azine-based colorimetric sensor (HBD) for the highly sensitive and selective identification of poisonous sarin gas surrogate diethylchlorophosphate (DCP). Our introduced sensor shows a purple color in contact with DCP, which is fully reversible upon the addition of triethylamine (TEA). The detection limit of our sensor for the toxic nerve agent mimic DCP is in the µM range. We have fabricated a test kit to verify the capability of HBD for on-the-spot identification of DCP to execute its practical use. To prove that HBD is an effective chemosensor, dip-stick investigation was conducted to detect DCP in the vaporous stage in the presence of different OPs, inorganic phosphates (IPs), and many other deadly analytes. A cellphone-based display method was also undertaken for on-the-spot recognition and measurement of DCP in isolated regions.

Colorimetric Gas Detection Tubes: Limits of Detection and Evaluation Using Active Chemical Warfare Agents.

Chemical weapons continue to be an ongoing threat that necessitates the improvement of existing detection technologies where new technologies are absent. Lower limits of detection will facilitate early warning of exposure to chemical weapons and enable more rapid deployment of countermeasures. Here, we evaluate two colorimetric gas detection tubes, developed by Draeger Inc., for sarin and sulfur mustard chemical warfare agents and determine their limits of detection using active chemical agent. Being that commercial companies are only able to use chemical agent simulants during sensor development, it is imperative to determine limits of detection using active agent. The limit of detection was determined based on the absence of a reasonably perceptible color response at incrementally lower concentrations. A chemical vapor generator was constructed to produce stable and quantifiable concentrations of chemical agent vapor, with the presence of chemical agent verified and monitored by a secondary detector. The limits of detection of the colorimetric gas detection tubes were determined to be 0.0046 ± 0.0002 and 2.1 ± 0.3 mg/m3 for sarin and sulfur mustard, respectively. The response of the sarin detection tube was readily observable with little issue. The sulfur mustard detection tube exhibited a weaker response to active agent compared to the simulant that was used during development, which will affect their concept of operations in real-world detection scenarios.

Subacute sarin exposure disrupted the homeostasis of purine and pyrimidine metabolism in guinea pig striatum studied by integrated metabolomic, lipidomic and proteomic analysis.

Sarin was used as a chemical weapon due to its high neurotoxicity and mortality. Subacute sarin induced cognitive and behavioral disorder. However, the underlying mechanism is still unclear. Here we offered a multi-omic approach for the analysis of altered metabolites, lipids, and proteins to explore the neurotoxicity of subacute sarin. Guinea pigs were administered between the shoulder blades 16.8 µg/kg of sarin in a volume of 1.0 ml/kg body weight by subcutaneous injection once daily for 14 days. At the end of the final injection, guinea pigs were sacrificed, and striatum were dissected for analysis. A total of 138 different metabolites were identified in the metabolome analysis. Lipids and lipid-like molecules is the largest group (38.41%). For lipidomic analysis, a total of 216 lipids were identified. In proteomic study, over 4300 proteins were identified and quantified. By integrating these enriched components, we demonstrated that the joint pathways disturbed by subacute sarin mainly involving lipid, purine and pyrimidine metabolism in guinea pig striatum. Overall, this study highlights the powerfulness of omics platforms to deepen the understanding of nerve agents caused neurotoxicity.

Analysis of Organophosphorus-Based Nerve Agent Degradation Products by Gas Chromatography-Mass Spectrometry (GC-MS): Current Derivatization Reactions in the Analytical Chemist's Toolbox.

The field of gas chromatography-mass spectrometry (GC-MS) in the analysis of chemical warfare agents (CWAs), specifically those involving the organophosphorus-based nerve agents (OPNAs), is a continually evolving and dynamic area of research. The ever-present interest in this field within analytical chemistry is driven by the constant threat posed by these lethal CWAs, highlighted by their use during the Tokyo subway attack in 1995, their deliberate use on civilians in Syria in 2013, and their use in the poisoning of Sergei and Yulia Skripal in Great Britain in 2018 and Alexei Navalny in 2020. These events coupled with their potential for mass destruction only serve to stress the importance of developing methods for their rapid and unambiguous detection. Although the direct detection of OPNAs is possible by GC-MS, in most instances, the analytical chemist must rely on the detection of the products arising from their degradation. To this end, derivatization reactions mainly in the form of silylations and alkylations employing a vast array of reagents have played a pivotal role in the efficient detection of these products that can be used retrospectively to identify the original OPNA.

Intramuscularly administered A1 adenosine receptor agonists as delayed treatment for organophosphorus nerve agent-induced Status Epilepticus.

Exposure to organophosphorus nerve agents (NAs) like sarin (GB) and soman (GD) can lead to sustained seizure activity, or status epilepticus (SE). Previous research has shown that activation of A1 adenosine receptors (A1ARs) can inhibit neuronal excitability, which could aid in SE termination. Two A1AR agonists, 2-Chloro-N6-cyclopentyladenosine (CCPA) and N-Bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), were effective in terminating GD-induced SE in rats when administered via intraperitoneal (IP) injection. However, IP injection is not a clinically relevant route of administration. This study evaluated the efficacy of these agonists in terminating NA-induced SE when administered via intramuscular (IM) route. Adult male rats were exposed subcutaneously (SC) to either GB (150 µg/kg) or GD (90 µg/kg) and were treated with ENBA or CCPA at 15, 30, or 60 min after seizure onset or left untreated. Up to 7 days after exposure, deeply anesthetized rats were euthanized and perfused brains were removed for histologic assessment of neuropathology (i.e., neuronal damage) in six brain regions (amygdala, cerebral cortex, piriform cortex, thalamus, dorsal hippocampus, and ventral hippocampus). A total neuropathy score (0-24) was determined for each rat by adding the scores from each of the six regions. The higher the total score the more severe the neuropathology. With the GB model and 60 min treatment delay, ENBA-treated rats experienced 78.6% seizure termination (N = 14) and reduced neuropathology (11.6 ± 2.6, N = 5), CCPA-treated rats experienced 85.7% seizure termination (N = 14) and slightly reduced neuropathology (20.7 ± 1.8, N = 6), and untreated rats experienced no seizure termination (N = 13) and severe neuropathology (22.3 ± 1.0, N = 4). With the GD model and 60 min treatment delay, ENBA-treated rats experienced 92.9% seizure termination (N = 14) and reduced neuropathology (13.96 ± 1.8, N = 9), CCPA-treated rats experienced 78.6% seizure termination (N = 14) and slightly reduced neuropathology (22.0 ± 0.9, N = 10); and untreated rats experienced 16.7% seizure termination (N = 12) and severe neuropathology (22.0 ± 1.8, N = 5). While ENBA and CCPA both demonstrate a clear ability to terminate SE when administered up to 60 min after seizure onset, ENBA offers more neuroprotection, making it a promising candidate for NA-induced SE.

Improving Quantification of tabun, sarin, soman, cyclosarin, and sulfur mustard by focusing agents: A field portable gas chromatography-mass spectrometry study.

Commercial gas chromatograph-mass spectrometers, one of which being Inficon's HAPSITE® ER, have demonstrated chemical detection and identification of nerve agents (G-series) and blistering agents (mustard gas) in the field; however most analyses relies on self-contained or external calibration that inherently drifts over time. We describe an analytical approach that uses target-based thermal desorption standards, called focusing agents, to accurately calculate concentrations of chemical warfare agents that are analyzed by gas chromatograph-mass spectrometry. Here, we provide relative response factors of focusing agents (2-chloroethyl ethyl sulfide, diisopropyl fluorophosphate, diethyl methylphosphonate, diethyl malonate, methyl salicylate, and dichlorvos) that are used to quantify concentrations of tabun, sarin, soman, cyclosarin and sulfur mustard loaded on thermal desorption tubes (Tenax® TA). Aging effects of focusing agents are evaluated by monitoring deviations in quantification as thermal desorption tubes age in storage at room temperature and relative humidity. The addition of focusing agents improves the quantification of tabun, sarin, soman, cyclosarin and sulfur mustard that is analyzed within the same day as well as a 14-day period. Among the six focusing agents studied here, diisopropyl fluorophosphate has the best performance for nerve agents (G-series) and blistering agents (mustard gas) compared to other focusing agents in this work and is recommended for field use for quantification. The use of focusing agent in the field leads to more accurate and reliable quantification of Tabun (GA), Sarin (GB), Soman (GD), Cyclosarin (GF) and Sulfur Mustard (HD) than the traditional internal standard. Future improvements on the detection of chemical, biological, radiological, nuclear, and explosive materials (CBRNE) can be safely demonstrated with standards calibrated for harmful agents.

A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds.

Organophosphorus (OP)1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE),2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.

Sampling and analyses of surfaces contaminated with chemical warfare agents by using a newly developed triple layered composite wipe.

A three-layered composite wipe was fabricated by laminating individual layers of non-woven polypropylene, activated carbon fabric (ACF) and aramid fabric for the sampling and investigation of chemical warfare agents (CWA)-contaminated urban porous and non-porous surfaces. The material of main ACF layer was characterized to ascertain its suitability to act as an efficient adsorbent for the surface wipe sampling. The performance of ACF-based composite wipe was determined by evaluating its extraction efficiency, wiping efficacy and adsorption capacity for the sampling of blister and nerve agent class of CWA-contaminated surfaces using gas chromatography-mass spectrometry (GC-MS). Parameters like amount of wipe required, solvent selection, amount of solvent, time of extraction etc. were optimized to achieve the maximum recovery of contaminating analytes required for the forensic investigations. Overall recoveries of contaminating analytes after sampling and extraction were found to be in the range of 45-85% for all types of surfaces. No breakthrough in wiping process was noticed up to contamination density (CD) 1.6 mg/cm2 for non-porous surface and 3.2 mg/cm2 for porous surfaces. ACF-based wipe was found capable to significantly reduce the vapour hazards from liquid sulphur mustard (HD) and sarin (GB). Contamination from surfaces could be preserved within the wipe up to 15 days for the extended forensic investigation purposes. Limit of detections (LOD) of contaminants was determined in the range of 0.8-6.8 ng/cm2 while limit of quantitation (LOQ) was achieved up to the range of 2.4-14.4 ng/cm2 for wipe sampling of different surfaces. Graphical abstract.

Gas chromatography-mass spectrometry with spiral large-volume injection for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum.

A sensitive method for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum was developed using gas chromatography-mass spectrometry (GCMS) with large-volume injection. The GC injection was administered using stomach-type spiral injector (LVI, AiSTI SCIENCE) enabling introduction of only target compounds from 50 µL ethyl acetate extract after purging the solvent. For GCMS analysis of sarin (GB), 670 times higher sensitivity, based on limit of detection (LOD, S/N = 3, on extracted ion chromatogram (EIC) at m/z 99), was achieved using this injection (50 µL) compared to that achieved using 1 µL split injection (ratio 20:1). Ethyl (EtGB), isopropyl (GB), n-propyl (nPrGB), isobutyl (iBuGB), pinacolyl (GD), cyclohexyl (GF) methylphosphonofluoridates, and O-ethyl N, N-dimethylphosphoramidofluoridate (GAF) were detected with low LOD (15-75 pg/mL) and sharp peak shapes (high practical plate number (defined as 5.54 x (tR/Wh)2, where tR is the retention time and Wh is the bandwidth at half-height): 1100000-2400000) in GCMS using a polar separation column, electron ionization, and quadruple mass analyzer. During the analysis of fluoridated phosphonate-spiked ethyl acetate extract of solid phase extraction (SPE, Bond Elut NEXUS) from fluoride-mediated regeneration of blank human plasma, LOD (on EIC at m/z 99 except for GAF (m/z 126)) were 25-140 pg/mL with sharp peak shapes. The reaction recoveries in fluoride-mediated regeneration of plasma, which was inhibited by GB, GD, GA, GF, VX, and Russian VX (10 ng/mL), were 49-114% except for GD (10%). The concentration levels of 0.3-1 ng/mL of nerve agents in plasma could be determined.

Mechanisms of acetylcholinesterase protection against sarin and soman by adenosine A1 receptor agonist N6-cyclopentyladenosine.

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A1 receptor agonist N6-cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development.

The Effects of Isopropyl Methylphosphono-Fluoridate (IMPF) Poisoning on Tumor Growth and Angiogenesis in BALB/C Mice.

BACKGROUND Acetylcholinesterase (AChE) and cholinergic receptors have an important role in the immune system and angiogenesis. This work evaluated the effects of isopropyl methylphosphonofluoridate (IMPF), an irreversible inhibitor of AChE, on tumor growth and selected parameters associated with tumor angiogenesis. MATERIAL AND METHODS Experiments were performed on male BALB/c mice exposed to IMPF (study group) or saline buffer (control group) and inoculated with L-1 sarcoma; the number of new blood vessels (TIA test) and the level of αvß3 integrin (131I-MAb-antiß3 assay) were analyzed at seven, 14, or 21 days after implantation of the tumor cells. RESULTS The IMPF poisoning affected tumor angiogenesis (TIA test). There was a statistically significant increase in the number of newly forming blood vessels in the group subjected to IMPF and inoculated with tumor cells. CONCLUSIONS This study showed that IMPF had a significant effect on the regulation of lymphocyte-induced angiogenesis and the modulation of angiogenic and pro-inflammatory cytokines secretion. The observed effects suggest involvement of neuronal and/or non-neuronal cholinergic signaling pathway.

The development of a butyrylcholinesterase porous pellet for innovative detection of cholinesterase inhibitors.

The aim of the presented research was the preparation of an innovative carrier with significantly improved properties for the fast and sensitive detection of cholinesterase inhibitors such as nerve agents. This innovative carrier was in the form of spherical pellets containing different amounts of Neusilin. Neusilin is a synthetic and amorphous form of magnesium aluminometasilicate with a high specific surface area, and the immobilized enzyme butyrylcholinesterase with an activity of 50nkat·g-1. Pellets were prepared by the extrusion-spheronization method and dried in a hot air oven under two conditions - at 30°C for 72h and at 60°C for 24h. Dried pellets were consequently impregnated with a solution containing butyrylcholinesterase. Impregnated pellets were evaluated for their quality parameters, enzymatic activity and inhibition. Activity and inhibition were tested according to the standard Ellman's method. It was observed that the addition of Neusilin significantly increased the hardness, intraparticular porosity, sphericity and activity of the carriers as well as intensity of the color transition. Therefore it is shown that these carriers have unquestionable advantages over common carriers of their kind. Drying temperatures have been shown to have no effect on properties of pellets except for a change in their size. Results were confirmed by statistical evaluation using ANOVA and PCA.

Blaptica dubia as sentinels for exposure to chemical warfare agents - a pilot study.

The increased interest of terrorist groups in toxic chemicals and chemical warfare agents presents a continuing threat to our societies. Early warning and detection is a key component for effective countermeasures against such deadly agents. Presently available and near term solutions have a number of major drawbacks, e.g. lack of automated, remote warning and detection of primarily low volatile chemical warfare agents. An alternative approach is the use of animals as sentinels for exposure to toxic chemicals. To overcome disadvantages of vertebrates the present pilot study was initiated to investigate the suitability of South American cockroaches (Blaptica dubia) as warning system for exposure to chemical warfare nerve and blister agents. Initial in vitro experiments with nerve agents showed an increasing inhibitory potency in the order tabun - cyclosarin - sarin - soman - VX of cockroach cholinesterase. Exposure of cockroaches to chemical warfare agents resulted in clearly visible and reproducible reactions, the onset being dependent on the agent and dose. With nerve agents the onset was related to the volatility of the agents. The blister agent lewisite induced signs largely comparable to those of nerve agents while sulfur mustard exposed animals exhibited a different sequence of events. In conclusion, this first pilot study indicates that Blaptica dubia could serve as a warning system to exposure of chemical warfare agents. A cockroach-based system will not detect or identify a particular chemical warfare agent but could trigger further actions, e.g. specific detection and increased protective status. By designing appropriate boxes with (IR) motion sensors and remote control (IR) camera automated off-site warning systems could be realized.

Exploration of Unimolecular Gas-Phase Detoxication Pathways of Sarin and Soman: A Computational Study from the Perspective of Reaction Energetics and Kinetics.

A mechanistic investigation has been carried out to explore all possible gas phase unimolecular isomerization as well as decomposition pathways of toxic organophosphorus compounds (OPCs), namely, sarin (GB) and soman (GD), which are better known as nerve agents. We have identified a total of 13 detoxication pathways for sarin, where the α-H, ß-H, and γ-H take part in the H-transfer process. However, for soman, due to the presence of ω-H, three additional detoxication pathways are obtained, where the ω-H is involved in the H-transfer process. Among all the pathways, the D3 decomposition pathway, where the phosphorus oxoacid derivative and alkene are generated via the formation of a six-membered ring in the transition state, is identified as the most feasible pathway from the perspective of both activation barrier and reaction enthalpy values. Moreover, we have studied the feasibility of the isomerization and decomposition pathways by performing the reaction kinetics in the temperature range of 300 K-1000 K using the one-dimensional Rice-Ramsperger-Kassel-Marcus (RRKM) master equation. From the RRKM calculation also, D3 pathway is confirmed as the most feasible pathway for both OPCs. The rate constant values associated with the D3 pathway within the temperature range of 600 K-700 K imply that the degradation of the OPCs is possible within this temperature range via the D3 pathway, which is in good agreement with the earlier reported experimental result. It is also observed that at higher temperature range (∼900 K), the increased rate constant values of other detoxication pathways indicate that along with D3, all other pathways become more or less equally feasible. Therefore, the entire work provides a widespread idea about the kinetic as well as thermodynamic feasibility of the explored detoxication pathways of the titled OPCs.

Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol.

We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.