Plant-Expressed Cocaine Hydrolase Variants of Butyrylcholinesterase Exhibit Altered Allosteric Effects of Cholinesterase Activity and Increased Inhibitor Sensitivity

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme’s ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic

Securing Our Economic Future

The American economy is in the midst of a wrenching crisis, one caused by the COVID-19 pandemic and aggravated further by a series of climate-driven natural disasters. While the economy has made some steps towards recovery, the pandemic has laid bare the reality that too many Americans are unable to meet many of their urgent and basic needs. At the same time, it has become painfully clear that American society is not equipped to deal with the risks emerging from our changing climate. This book is a contribution towards policy options for addressing these challenges. Although it was largely written before the pandemic crises beset our country, the analyses, diagnoses, and prescriptions contained within all shed new light on the underlying fragilities that have since been exposed. The volume is composed of nine commissioned chapters and is divided into three sections, covering the 'Economics of the American Middle Class'; the 'Geographic Disparities in Economic Opportunity'; and the 'Geopolitics of the Climate and Energy Challenge and the US Policy Response.' Part I focuses on the economic wellbeing of the American middle class and the chapters in this section evaluate the prevailing narrative of its decline. The chapters in part II investigate the large variation in income and economic opportunities across places, and include a specific policy proposal for emergency rental assistance. Part III is devoted to the global climate crisis. The chapters in this final section emphasize the mounting social and economic costs of inaction and discuss potential policy approaches for tackling the climate challenge

Reversal of succinylcholine induced apnea with an organophosphate scavenging recombinant butyrylcholinesterase.

Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD100 of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates

Alterations in the Endogenous Ascorbic Acid Content Affect Flowering Time in Arabidopsis1[W][OA]

Ascorbic acid (AA) protects plants against abiotic stress. Previous studies suggested that this antioxidant is also involved in the control of flowering. To decipher how AA influences flowering time, we studied the four AA-deficient Arabidopsis (Arabidopsis thaliana) mutants vtc1-1, vtc2-1, vtc3-1, and vtc4-1 when grown under short and long days. These mutants flowered and senesced before the wild type irrespective of the photoperiod, a response that cannot simply be attributed to slightly elevated oxidative stress in the mutants. Transcript profiling of various flowering pathway genes revealed a correlation of altered mRNA levels and flowering time. For example, circadian clock and photoperiodic pathway genes were significantly higher in the vtc mutants than in the wild type under both short and long days, a result that is consistent with the early-flowering phenotype of the mutants. In contrast, when the AA content was artificially increased, flowering was delayed, which correlated with lower mRNA levels of circadian clock and photoperiodic pathway genes compared with plants treated with water. Similar observations were made for the autonomous pathway. Genetic analyses demonstrated that various photoperiodic and autonomous pathway mutants are epistatic to the vtc1-1 mutant. In conclusion, our transcript and genetic analyses suggest that AA acts upstream of the photoperiodic and autonomous pathways

Plant-derived BChE protects mice from SC-induced apnea.

<p>A Respiration rate of mice treated with SC followed by administration of pBChE or saline was monitored. Symbols represent individual animal subjects (<i>n</i> = 3 for each group). B The Kaplan–Meier estimates of survival reflecting survival of all pBChE-treated mice as opposed to 100% mortality among control, saline-treated subjects. Comparison by the Log-rank (Mantel-Cox) test was significant (<i>P</i><0.025).</p

Plant-derived BChE facilitate recovery of guinea pigs treated with sublethal dose of SC.

<p>Guinea pigs were injected with SC (0.167 mg/kg) followed by injection with BChE (<i>n</i> = 3, red) or saline (<i>n</i> = 3, blue) while monitoring oxygen saturation (A) and heart rate (B). Reported data points represent means±SEM. Means at each time point were compared by 2-way repeated ANOVA and P values are denoted on the graphs. Statistical analyses by t-test or 1-way ANOVA yielded very similar results. C Time to return to normal heart rate. Comparison by the Log-rank (Mantel-Cox) test was significant (<i>P</i><0.025).</p

Plant-derived BChE undergoes substrate activation by its succinylcholine substrate.

<p>Blue line represents data fitted by nonlinear regression to fit the equation: with the following parameters (±SEM): V_max = 2.45±0.16 µM/min, K_M = 57±7 µM, K_ss = 2.0±0.3 mM, and b = 2.9±0.1. <i>k</i>_cat = V_max/[BChE]_T was calculated to be 516±33 min⁻¹ based on the above V_max value and [BChE] = 4.74 nM.</p