Novel protein-based solutions for organophosphorus nerve agent detection and elimination

Organophosphorus (OP) nerve agents are some of the deadliest chemicals ever synthesized by man. These toxins, which include sarin, soman, cyclosarin, tabun, and VX, inhibit the neurotransmitter-regulating enzyme, acetylcholinesterase (AChE). This leads to continual acetylcholine muscarinic and nicotinic receptor stimulus and may eventually result in death due to prolonged muscles contraction and diaphragm incapacitation. Current treatments for OP poisoning include injections of atropine, to dampen acetylcholine stimulation, a strong-nucleophile oxime, such a 2-pralidoxime, to reactivate inhibited AChE, and diazepam for seizures. These treatments are limited, however, because they do not protect against poisoning, cannot be administered prior to exposure, and don't address the long-term side effects associated with nerve agent poisoning. Additionally, there is no broad-spectrum oxime effective against all nerve agents. A better therapeutic would be a prophylactic molecule capable of catalytically degrading the OP prior to AChE inhibition. Protein-based therapeutics are an emerging remedy for OP toxicity. It has been shown that pre-administration of excess AChE in mice can protein against 8-10 normally lethal doses of soman. Current enzyme therapeutics can be categorized as either stoichiometric or catalytic. Stoichiometric OP binders, such as AChE or the homologous butyrylcholinesterase (BChE), benefit from nanomolar (nM) dissociation constants, but suffer in their ability to recover after OP exposure, thereby requiring large enzyme doses for effective treatment. Catalytic protein therapeutics, including serum paraoxonase (PON1) or the bacterial organophosphate hydrolase (OPH) exhibit rapid rates of in vitro nerve agent hydrolysis, but are limited by high dissociation constants, making them ineffective in vivo. Human carboxylesterase 1 (hCE1) is a liver serine hydrolase in the same [alpha]/[beta] super family as AChE and BChE, which may have more favorable attributes as an OP bioscavenger. Indeed rodents express a serum carboxylesterase that affords them high levels of protection against OPs. Using structural and biochemical studies, we determined the stereopreference, rates of spontaneous reactivation, and availability of rapid oxime-assisted reactivation of hCE1 with nerve agents. Next, using structurally guided protein design, we engineered a form of the hCE1 that combines the benefits of both bioscavenger classes, exhibiting nM dissociation constants and enhanced rates of hydrolysis, up to 48,700-fold, against the nerve agents sarin, soman, and cyclosarin. Finally, novel mutants of hCE1 were developed that exhibit-increased rates of reactivation against specific agents, and can be utilized to detect and identify chemical agents

Immobilization of active human carboxylesterase 1 in biomimetic silica nanoparticles

The encapsulation of proteins in biomimetic silica has recently been shown to successfully maintain enzymes in their active state. Organophosphate (OP) compounds are employed as pesticides as well as potent chemical warfare nerve agents. Because these toxicants are life threatening, we sought to generate biomimetic silicas capable of responding to OPs. Here, we present the silica encapsulation of human drug metabolism enzyme carboxylesterase 1 (hCE1) in the presence of a range of catalysts. hCE1 was successfully encapsulated into silica particles when lysozyme or the peptide R5 were used as catalysts; in contrast, polyethyleneimine (PEI), a catalyst employed to encapuslate other enzymes, did not facilitate hCE1 entrapment. hCE1 silica particles in a column chromatography format respond to the presence of the organophosphate (OP) pesticides paraoxon and dimethyl-p-nitrophenyl phosphate in solution. These results may lead to novel approaches to detect OP pesticides or other weaponized agents that bind hCE1

Nerve Agent Hydrolysis Activity Designed into a Human Drug Metabolism Enzyme

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

Towards robust alkane oxidation catalysts: electronic variations in non-heme iron(II) complexes and their effect in catalytic alkane oxidation

A series of non-heme iron(II) bis(triflate) complexes containing linear and tripodal tetradentate ligands has been prepared. Electron withdrawing and electron donating substituents in the para position of the pyridine ligands as well as the effect of pyrazine versus pyridine and sulfur or oxygen donors instead of nitrogen donors have been investigated. The electronic effects induced by these substituents influence the strength of the ligand field. UV-vis spectroscopy and magnetic susceptibility studies have been used to quantify these effects and VT 1H and 19F NMR spectroscopy as well as X-ray diffraction have been used to elucidate structural and geometrical aspects of these complexes. The catalytic properties of the iron(II) complexes as catalysts for the oxidation of cyclohexane with hydrogen peroxide have been evaluated. In the strongly oxidising environment required to oxidise alkanes, catalyst stability determines the overall catalytic efficiency of a given catalyst, which can be related to the ligand field strength and the basicity of the ligand and its propensity to undergo oxidation

Engine Burning Through

Engine Burning Through is a collection of poems

A structural examination of agrochemical processing by human carboxylesterase 1

Human carboxylesterase 1 (hCE1) is the primary carboxylesterase expressed in the liver. This critical member of the phase I drug metabolism pathway detoxifies a wide-range of endobiotics, xenobiotics, and agrochemicals. To date, more than a dozen X-ray crystal structures have been elucidated of hCE1 in complex with a broad spectrum of ligands, including organophosphates. These structures provide valuable insights into agrochemical binding and metabolism by hCE1. For example, variable binding pockets that frame the enzyme’s catalytic triad and a long, flexible loop capping this region appear to regulate substrate affinity. Stereoisomers of organophosphates illustrate the substrate selectivity of these two pockets. In contrast, pyrethroid isomers likely impact the positioning of the oxyanion hole required to stabilize the negatively charged transition-state oxygen. Finally, it appears that rates of spontaneous hCE1 reactivation in the presence of organophosphates are significantly affected by alkoxy placement within the active site

Type‐I Interferon assessment in 45 minutes using the FilmArray ®

Low concentrations of type‐I interferon (IFN) in blood seem to be associated with more severe forms of Coronavirus disease 2019 (COVID‐19). However, following the type‐I interferon response (IR) in early stage disease is a major challenge. We evaluated detection of a molecular interferon signature on a FilmArray® system, which includes PCR assays for four interferon stimulated genes. We analyzed three types of patient populations: (i) children admitted to a pediatric emergency unit for fever and suspected infection, (ii) ICU‐admitted patients with severe COVID‐19, and (iii) healthcare workers with mild COVID‐19. The results were compared to the reference tools, that is, molecular signature assessed with Nanostring® and IFN‐α2 quantification by SIMOA® (Single MOlecule Array). A strong correlation was observed between the IR measured by the FilmArray®, Nanostring®, and SIMOA® platforms (r‐Spearman 0.996 and 0.838, respectively). The FilmArray® panel could be used in the COVID‐19 pandemic to evaluate the IR in 45‐min with 2 min hand‐on‐time at hospitalization and to monitor the IR in future clinical trials

Implementation of consensus-based perioperative care pathways to reduce clinical variation for elective surgery in an Australian private hospital: a mixed-methods pre–post study protocol

Introduction Addressing clinical variation in elective surgery is challenging. A key issue is how to gain consensus between largely autonomous clinicians. Understanding how the consensus process works to develop and implement perioperative pathways and the impact of these pathways on reducing clinical variation can provide important insights into the effectiveness of the consensus process. The primary objective of this study is to understand the implementation of an organisationally supported, consensus approach to implement perioperative care pathways in a private healthcare facility and to determine its impact.Methods A mixed-methods Effectiveness-Implementation Hybrid (type III) pre–post study will be conducted in one Australian private hospital. Five new consensus-based perioperative care pathways will be developed and implemented for specific patient cohorts: spinal surgery, radical prostatectomy, cardiac surgery, bariatric surgery and total hip and knee replacement. The individual components of these pathways will be confirmed as part of a consensus-building approach and will follow a four-stage implementation process using the Exploration, Preparation, Implementation and Sustainment framework. The process of implementation, as well as barriers and facilitators, will be evaluated through semistructured interviews and focus groups with key clinical and non-clinical staff, and participant observation. We anticipate completing 30 interviews and 15–20 meeting observations. Administrative and clinical end-points for at least 152 participants will be analysed to assess the effectiveness of the pathways.Ethics and dissemination This study received ethical approval from Macquarie University Human Research Ethics Medical Sciences Committee (Reference No: 520221219542374). The findings of this study will be disseminated through peer-reviewed publications, conference presentations and reports for key stakeholders