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Chemical warfare nerve agents (CWNAs) are potent and irreversible inhibitors of acetylcholinesterase (AChE). Oxime reactivators are an important part of the standard treatment for CWNA exposure as they can reactivate inhibited AChE. Evaluating the oxime candidates of interest in biological samples requires analytical detection methods and oxime reactivators as a class of compounds have historically been notoriously difficult to isolate, detect and analyze in an analytical laboratory, and there are currently no sensitive or robust analytical detection methods in the literature. The goal of this study was to develop reliable and robust novel extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to detect and quantitate 2-PAM, HI-6, HLö-7, and MMB-4 in a human AChE knock-in, mouse carboxylesterase knock-out (KIKO) mouse in vivo model. This study identified an LC column that achieved retention for all four oxime compounds which is a major advancement over past oxime methods. A unique extraction and chromatographic method was developed for each oxime. The developed methods were sensitive down to 0.5 ng/mL for 2-PAM, 50 ng/mL for HI-6, and 15 ng/mL for both HLö-7 and MMB-4. These methods were validated to meet the Food and Drug Administration (FDA) bioanalytical method validation requirements under Good Laboratory Practice (GLP) conditions. The 4 methods were validated for performance by assessing linearity, sensitivity, precision, accuracy, selectivity, specificity, carryover, extraction recovery, dilution analysis, and stability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Organophosphoate (OP) chemicals are known to inhibit the enzyme acetylcholinesterase (AChE). Studying OP poisoning is difficult because common small animal research models have serum carboxylesterase, which contributes to animals' resistance to OP poisoning. Historically, guinea pigs have been used for this research; however, a novel genetically modified mouse strain (KIKO) was developed with nonfunctional serum carboxylase (Es1 KO) and an altered acetylcholinesterase (AChE) gene, which expresses the amino acid sequence of the human form of the same protein (AChE KI). KIKO mice were injected with 1xLD 50 of an OP nerve agent or vehicle control with or without atropine. After one to three minutes, animals were injected with 35 mg/kg of the currently fielded Reactivator countermeasure for OP poisoning. Postmortem brains were imaged on a Bruker RapifleX ToF/ToF instrument. Data confirmed the presence of increased acetylcholine in OP-exposed animals, regardless of treatment or atropine status. More interestingly, we detected a small amount of Reactivator within the brain of both exposed and unexposed animals; it is currently debated if reactivators can cross the blood-brain barrier. Further, we were able to simultaneously image acetylcholine, the primary affected neurotransmitter, as well as determine the location of both Reactivator and acetylcholine in the brain. This study, which utilized sensitive MALDI-MSI methods, characterized KIKO mice as a functional model for OP countermeasure development.

The addition of supplemental diets to laboratory animals, specifically rodents, is a common practice for the provision of additional nutritional support. We set out to investigate whether the use of commercially available supplemental diets during breeding affected fertility rate, litter size, pup health, and pup survival. Genetically modified female breeding mice with a C57BL/6 background were divided into 3 groups ( n = 16 per group) that received standard rodent chow alone or standard rodent chow with one of 2 commercially available supplemental diets: Love Mash (Bio-Serv) extruded pellet or Nutra-Gel (Bio-Serv) diet gel. Male and female mice began receiving the supplemental diet 1 wk before being paired with a partner of the same supplemental group. The mice were allowed to breed for 1 wk before separation from the male. The dams were continued on the diet until all pups were weaned. Overall, breeding dams supplemented with the Love Mash diet experienced significantly greater reproductive success rates and pup survivability compared with the standard diet control group. Dams supplemented with either of the 2 supplemental diets supported significantly larger litters compared with the standard diet control group. Furthermore, Love Mash supplemented diet groups produced significantly larger pups compared with the Nutra-Gel supplemented groups. This study demonstrates that supplemental diets given 1 wk before breeding and continued throughout gestation, parturition, and weaning significantly improved reproductive success, increased litter sizes, and supported pup health and survival.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro , achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 μM, a 4-fold increase from our 2018 report. The best QMP ( 1b ), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

Nerve agents have experienced a resurgence in recent times with their use against civilian targets during the attacks in Syria (2012), the poisoning of Sergei and Yulia Skripal in the United Kingdom (2018) and Alexei Navalny in Russia (2020), strongly renewing the importance of antidote development against these lethal substances. The current standard treatment against their effects relies on the use of small molecule-based oximes that can efficiently restore acetylcholinesterase (AChE) activity. Despite their efficacy in reactivating AChE, the action of drugs like 2-pralidoxime (2-PAM) is primarily limited to the peripheral nervous system (PNS) and, thus, provides no significant protection to the central nervous system (CNS). This lack of action in the CNS stems from their ionic nature that, on one end makes them very powerful reactivators and on the other renders them ineffective at crossing the Blood Brain Barrier (BBB) to reach the CNS. In this report, we describe the use of an iterative approach composed of parallel chemical and in silico syntheses, computational modeling, and a battery of detailed in vitro and in vivo assays that resulted in the identification of a promising, novel CNS-permeable oxime reactivator. Additional experiments to determine acute and chronic toxicity are ongoing., (© 2021. The Author(s).)

The identification of improved medical countermeasures against exposure to chemical warfare nerve agents (CWNAs), a class of organophosphorus compounds, is dependent on the choice of animal model used in preclinical studies. CWNAs bind to acetylcholinesterase and prevent the catalysis of acetylcholine, causing a plethora of peripheral and central physiologic manifestations, including seizure. Rodents are widely used to elucidate the effects of CWNA-induced seizure, albeit with a caveat: they express carboxylesterase activity in plasma. Carboxylesterase, an enzyme involved in the detoxification of some organophosphorus compounds, plays a scavenging role and decreases CWNA availability, thus exerting a protective effect. Furthermore, species-specific amino acid differences in acetylcholinesterase confound studies that use oximes or other compounds to restore its function after inhibition by CWNA. The creation of a human acetylcholinesterase knock-in/serum carboxylesterase knockout (C57BL/6-Ces1c tm1.1Loc AChE tm1.1Loc /J; a.k.a KIKO) mouse may facilitate better modeling of CWNA toxicity in a small rodent species. The current studies characterize the effects of exposure to soman, a highly toxic CWNA, and evaluate the efficacy of anti-seizure drugs in this newly developed KIKO mouse model. Data demonstrate that a combination of midazolam and ketamine reduces seizure duration and severity, eliminates the development of spontaneous recurrent seizures, and protects certain brain regions from neuronal damage in a genetically modified model with human relevance to organophosphorus compound toxicity. This new animal model and the results of this study and future studies using it will enhance medical countermeasures development for both defense and homeland security purposes.

Human butyrylcholinesterase (E.C. 3.1.1.8) purified from blood plasma has previously been shown to provide protection against up to five and a half times the median lethal dose of an organophosphorus nerve agent in several animal models. In this study the stoichiometric nature of the protection afforded by human butyrylcholinesterase against organophosphorus nerve agents was investigated in guinea pigs. Animals were administered human butyrylcholinesterase (26.15 mg/kg ≡ 308 nmol/kg) by the intravascular or intramuscular route. Animals were subsequently dosed with either soman or VX in accordance with a stage-wise adaptive dose design to estimate the modified median lethal dose in treated animals. Human butyrylcholinesterase (308 nmol/kg) increased the median lethal dose of soman from 154 nmol/kg to 770 nmol/kg. Comparing the molar ratio of agent molecules to enzyme active sites yielded a stoichiometric protective ratio of 2:1 for soman, likely related to the similar stereoselectivity the enzyme has compared to the toxic target, acetylcholinesterase. In contrast, human butyrylcholinesterase (308 nmol/kg) increased the median lethal dose of VX from 30 nmol/kg to 312 nmol/kg, resulting in a stoichiometric protective ratio of only 1:1, suggesting a lack of stereoselectivity for this agent., (Published by Elsevier Inc.)

Rare diseases defined by genetic mutations are classic targets for gene therapy. More recently, researchers expanded the use of gene therapy in non-clinical studies to infectious diseases through the delivery of vectorized antibodies to well-defined antigens. Here, we further extend the utility of gene therapy beyond the "accepted" indications to include organophosphate poisoning. There are no approved preventives for the multi-organ damage resulting from acute or chronic exposure to organophosphates. We show that a single intramuscular injection of adeno-associated virus vector produces peak expression (~0.5 mg/ml) of active human butyrylcholinesterase (hBChE) in mice serum within 3-4 weeks post-treatment. This expression is sustained for up to 140 days post-injection with no silencing. Sustained expression of hBChE provided dose-dependent protection against VX in male and female mice despite detectable antibodies to hBChE in some mice, thereby demonstrating that expression of hBChE in vivo in mouse muscle is an effective prophylactic against organophosphate poisoning., Competing Interests: J.M. Wilson is a paid advisor to and holds equity in Scout Bio and Passage Bio; he holds equity in Surmount Bio; he also has a sponsored research agreement with Ultragenyx, Biogen, Janssen, Precision Biosciences, Moderna Therapeutics, Scout Bio, Passage Bio, Amicus Therapeutics, and Surmount Bio, which are licensees of Penn technology. JMW is an inventor on patents that have been licensed to various biopharmaceutical companies and for which he may receive payments. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Nerve agents are a class of organophosphorus compounds (OPs) that blocks communication between nerves and organs. Because of their acute neurotoxicity, it is extremely difficult to rescue the victims after exposure. Numerous efforts have been devoted to search for an effective prophylactic nerve agent bioscavenger to prevent the deleterious effects of these compounds. However, low scavenging efficiency, unfavorable pharmacokinetics, and immunological problems have hampered the development of effective drugs. Here, we report the development and testing of a nanoparticle-based nerve agent bioscavenger (nanoscavenger) that showed long-term protection against OP intoxication in rodents. The nanoscavenger, which catalytically breaks down toxic OP compounds, showed a good pharmacokinetic profile and negligible immune response in a rat model of OP intoxication. In vivo administration of the nanoscavenger before or after OP exposure in animal models demonstrated protective and therapeutic efficacy. In a guinea pig model, a single prophylactic administration of the nanoscavenger effectively prevented lethality after multiple sarin exposures over a 1-week period. Our results suggest that the prophylactic administration of the nanoscavenger might be effective in preventing the toxic effects of OP exposure in humans., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Genetics likely play a role in various responses to nerve agent (NA) exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses. This study uses different mouse strains to identify if mouse strain differences in sarin exposure exist. In Experiment 1, basal levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CE) were measured in different strains of naïve mice to account for potential pharmacokinetic determinants of individual differences. In Experiment 2, median lethal dose (MLD) levels were estimated in 8 inbred mouse strains following subcutaneous (s.c.) administration of sarin. Few strain or sex differences in esterase activity levels were observed, with the exception of erythrocyte AChE activity in the C57BL/6J strain. Both sex and strain differences in toxicity were observed, with the most resistant strains being the BALB/cByJ and FVB/NJ strains and the most sensitive strain being the DBA/2J strain. These findings can be expanded to explore pathways involved in NA response, which may provide an avenue to develop therapeutics for preventing and treating the damaging effects of NA exposure., (Published by Elsevier B.V.)

Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase nonspecifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34 000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.

Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a β-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use., (© 2018 Wiley Periodicals, Inc.)

Genetics likely play a role in various responses to nerve agent exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses to environmental stimuli. Mouse strains or selected lines can be used to identify susceptibility based on background genetic features to nerve agent exposure. Additional genetic techniques can then be used to identify mechanisms underlying resistance and sensitivity, with the ultimate goal of developing more effective and targeted therapies. Here, we discuss the available literature on strain and selected line differences in cholinesterase activity levels and response to nerve agent-induced toxicity and seizures. We also discuss the available cholinesterase and toxicity literature across different non-human primate species. The available data suggest that robust genetic differences exist in cholinesterase activity, nerve agent-induced toxicity, and chemical-induced seizures. Available cholinesterase data suggest that acetylcholinesterase activity differs across strains, but are limited by the paucity of carboxylesterase data in strains and selected lines. Toxicity and seizures, two outcomes of nerve agent exposure, have not been fully evaluated for genetic differences, and thus further studies are required to understand baseline strain and selected line differences., (Published by Elsevier B.V.)

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Several structural analogs of ADOC were used to probe the reactivation mechanism of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design of future small molecule reactivators., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Heterotopic ossification (HO) of the bone is defined as a benign condition in which abnormal bone formation occurs in soft tissue. One of the most common prophylactic treatments for HO is radiation therapy (RT). This study retrospectively reviewed 20 patients younger than the age of 40 who received radiation to prevent HO in a single fraction of 7 Gray. The purpose of this study is to assess the risk of a second malignancy in these patients by recreating their treatment fields and contouring organs at risk to estimate the radiation dose absorbed by normal tissues outside the radiation treatment field. Diagnostic computed tomography (CT) scans for each patient were used to recreate treatment fields and to calculate dose to structures of interest. The distance from the field edge to each structure and its depth was recorded. Dose measurements in a water phantom were performed for the range of depths, distances, and field sizes used in the actual treatment plans. Computer-generated doses were compared to estimates based on measurement. The structure dose recorded was the higher dose generated between the 2 methods. Scatter dose was recorded to the rectum, bladder, sigmoid colon, small bowel, ovaries and utero-cervix in female patients, and prostate and gonads in male patients. In some patients, there is considerable dose received by certain organs from scatter because of their proximity to the radiation field. The average dose to the ovarian region was 4.125Gy with a range of 1.085 to 6.228Gy. The risk estimate for these patients ranged from 0.16% to 0.93%. The average total lifetime risk estimate for the bladder in all patients is 0.22% and the average total lifetime risk estimate for the remainder organs in all patients is 1.25%. In conclusions, proper shielding created from multileaf collimators (MLCs), blocks, and shields should always be used when possible., (Published by Elsevier Inc.)

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.

Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well-known are GA, GB, GD, GF, VX, and VR. All of these compounds contain a chiral phosphorus center, with the S(P) enantiomers being significantly more toxic than the R(P) enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R(P) enantiomers. A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR has been developed. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed, stereoselectivity have been identified. A number of variants exhibited dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S(P) enantiomers. Improvements of up to 3 orders of magnitude relative to the value of the wild-type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.

Poisoning via organophosphorus (OP) nerve agents occurs when the OP binds and inhibits the enzyme acetylcholinesterase (AChE). This enzyme is responsible for the metabolism of the neurotransmitter acetylcholine (ACh) which transmits signals between nerves and several key somatic regions. When AChE is inhibited, the signal initiated by ACh is not properly terminated. Excessive levels of ACh result in a cholinergic crisis, and in severe cases can lead to death. Current treatments for OP poisoning involve the administration of atropine, which blocks ACh receptors, and oximes, which reactivate AChE after inhibition. Efforts to improve the safety, efficacy, and broad spectrum utility of these treatments are ongoing and usually require the use of appropriate animal model systems. For OP poisoning, the guinea pig (Cavia porcellus) is a commonly used animal model because guinea pigs more closely mirror primate susceptibility to OP poisoning than do other animals such as rats and mice. This is most likely because among rodents and other small mammals, guinea pigs have a very low relative concentration of serum carboxylesterase, an enzyme known to bind OPs in vitro and to act as an endogenous bioscavenger in vivo. Although guinea pigs historically have been used to test OP poisoning therapies, it has been found recently that guinea pig AChE is substantially more resistant to oxime-mediated reactivation than human AChE. To examine the molecular basis for this difference, we reverse transcribed mRNA encoding guinea pig AChE, amplified the resulting cDNA, and sequenced this product. The nucleotide and deduced amino acid sequences of guinea pig AChE were then compared to the human version. Several amino acid differences were noted, and the predicted locations of these differences were mapped onto a structural model of human AChE. To examine directly how these differences affect oxime-mediated reactivation of AChE after inhibition by OPs, human and guinea pig red blood cell ghosts were prepared and used as sources of AChE, and the relative capacity of several different oximes to reactivate each OP-inhibited AChE were determined. The differences we report between human and guinea pig AChE raise additional concerns about the suitability of the guinea pig as an appropriate small animal model to approximate human responses to OP poisoning and therapies., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA(+) background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.

Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para-aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase-based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP-binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867-876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre-incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three-dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p-aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library., (Copyright 2008 John Wiley & Sons, Ltd.)

Dif and Frz, two Myxococcus xanthus chemosensory pathways, are required in phosphatidylethanolamine (PE) chemotaxis for excitation and adaptation respectively. DifA and FrzCD, the homologues of methyl-accepting chemoreceptors in the two pathways, were examined for methylation in the context of chemotaxis and inter-pathway interactions. Evidence indicates that DifA may not undergo methylation, but signals transmitting through DifA do modulate FrzCD methylation. Results also revealed that M. xanthus possesses Dif-dependent and Dif-independent PE-sensing mechanisms. Previous studies showed that FrzCD methylation is decreased by negative chemostimuli but increased by attractants such as PE. Results here demonstrate that the Dif-dependent sensory mechanism suppresses the increase in FrzCD methylation in attractant response and elevates FrzCD methylation upon negative stimulation. In other words, FrzCD methylation is governed by opposing forces from Dif-dependent and Dif-independent sensing mechanisms. We propose that the Dif-independent but Frz-dependent PE sensing leads to increases in FrzCD methylation and subsequent adaptation, while the Dif-dependent PE signalling suppresses or diminishes the increase in FrzCD methylation to decelerate or delay adaptation. We contend that these antagonistic interactions are crucial for effective chemotaxis in this gliding bacterium to ensure that adaptation does not occur too quickly relative to the slow speed of M. xanthus movement.