Determining the manner in which food webs will respond to environmental changes is difficult because the relative importance of top-down vs. bottom-up forces in controlling ecosystems is still ...debated. This is especially true in the Arctic tundra where, despite relatively simple food webs, it is still unclear which forces dominate in this ecosystem. Our primary goal was to assess the extent to which a tundra food web was dominated by plant-herbivore or predator-prey interactions. Based on a 17-year (1993-2009) study of terrestrial wildlife on Bylot Island, Nunavut, Canada, we developed trophic mass balance models to address this question. Snow Geese were the dominant herbivores in this ecosystem, followed by two sympatric lemming species (brown and collared lemmings). Arctic foxes, weasels, and several species of birds of prey were the dominant predators. Results of our trophic models encompassing 19 functional groups showed that <10% of the annual primary production was consumed by herbivores in most years despite the presence of a large Snow Goose colony, but that 20-100% of the annual herbivore production was consumed by predators. The impact of herbivores on vegetation has also weakened over time, probably due to an increase in primary production. The impact of predators was highest on lemmings, intermediate on passerines, and lowest on geese and shorebirds, but it varied with lemming abundance. Predation of collared lemmings exceeded production in most years and may explain why this species remained at low density. In contrast, the predation rate on brown lemmings varied with prey density and may have contributed to the high-amplitude, periodic fluctuations in the abundance of this species. Our analysis provided little evidence that herbivores are limited by primary production on Bylot Island. In contrast, we measured strong predator-prey interactions, which supports the hypothesis that this food web is primarily controlled by top-down forces. The presence of allochthonous resources subsidizing top predators and the absence of large herbivores may partly explain the predominant role of predation in this low-productivity ecosystem.
Organophosphorus (OP) nerve agents and pesticides inhibit the enzyme acetylcholinesterase (AChE) via a covalent interaction at the active site, resulting in a buildup of excess acetylcholine in the ...synaptic cleft. Current reactivators rely exclusively on oxime moieties that can reverse this inhibition via direct nucleophilic attack of the bound OP at the enzyme active site. Absolute molar concentration is a fundamental component of the mechanism of oxime‐mediated reactivation and forms the basis of established reactivation kinetics theory and descriptive mathematics. Recently, a non‐oxime reactivator (ADOC) was discovered that operates via a general acid‐base catalysis mechanism, which calls the fundamental assumptions of established reactivation kinetics theory into question. The objective of this study is to determine if the assumption that reactivation occurs in an absolute concentration‐dependent manner holds true for ADOC. An assay was developed to elucidate the effect of reactivator and enzyme concentration relationships as a component of either inherent compound structure‐activity or external environment‐activity relationships, and this was utilized to evaluate both an oxime and non‐oxime reactivator. Initial results suggest that while chemical structure and, therefore, mechanism of reactivation play a role in the reactivator‐to‐enzyme interaction, environmental factors are suspected to also impact this relationship only for ADOC, likely via activation of an alternate reactivation mechanism. In conclusion, expanding the reactivation kinetics theory to accommodate alternate mechanisms of reactivation may pave new avenues for the development of nerve agent countermeasure therapeutics.
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 1xLD50 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.
Organophosphate (OPs) compounds make up the most common class of pesticides as well as chemical warfare nerve agents (CWNA), all of which covalently inhibit the active site of acetylcholinesterase ...(AChE), resulting in intoxication via a cholinergic crisis. While the biochemical mechanism of action resulting in acute intoxication of an exposed individual is well understood, less is known about the mechanism of OP intoxication via other exposure routes, particularly prenatal exposure. The clinical effects of non‐acute prenatal OP exposure have been previously studied primarily in cases involving diet and agricultural labor, while case studies documenting the effects on conception of acute exposure during chemical warfare attacks (Japan 1995, Syria 2013 and 2017) have also been published. Such exposure events were linked to a range of serious neurodevelopmental anomalies in children, but the underlying mechanism of action as well as the potential benefits of medical countermeasures have not been clearly elucidated. A robust human‐relevant animal model to test both is therefore essential for evaluating current and future OP countermeasures. The present study used a novel humanized mouse strain expressing human AChE and an inactivated native serum carboxylesterase to create a prenatal acute exposure and emergency medical countermeasure evaluation model. Pregnant mice were acutely exposed to an OP at a critical point in cholinergic development, generally equivalent to the first trimester in humans. Dams were challenged with either sarin or the pesticide paraoxon, followed by either a saline vehicle control or 2‐PAM and an atropine/midazolam cocktail, the currently fielded emergency treatment for OP intoxication. Mice were allowed to carry pups to term, and upon reaching maturity, cortex, cerebellum, bone marrow and gonads were collected from offspring in each exposure group. Phenotypical and histopathological evaluation of tissues and other factors in dams and offspring were evaluated. Gene expression analyses conducted for each tissue generated a detailed profile associated with each OP exposure/treatment group, providing robust criteria for model development. The resulting data demonstrated a significant beneficial impact of medical countermeasures for both sarin and paraoxon exposures, significantly reducing the amount and the scope of gene expression changes induced by OP exposure. This model provides insight into the developmental pathologies resulting from prenatal acute OP exposure as well as serves as an evaluation model for future treatments.
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.
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.
Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture was purified and characterized to confirm its comparability to the native human form of the molecule. The purification process uses common manufacturing‐friendly operations to get a 95% pure, active molecule. The recombinant and native enzyme have similar enzymatic behavior but different host‐specific N‐glycan structures. Despite this, the recombinant enzyme is an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro.
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.
Dynamical histories of planetary systems, as well as the atmospheric evolution of highly irradiated planets, can be studied by characterizing the ultra-short-period planet population, which the TESS ...mission is particularly well suited to discover. Here, we report on the follow-up of a transit signal detected in the TESS sector 19 photometric time series of the M3.0 V star TOI-1685 (2MASS J04342248+4302148). We confirm the planetary nature of the transit signal, which has a period of
P
b
= 0.6691403
−0.0000021
+0.0000023
d, using precise radial velocity measurements taken with the CARMENES spectrograph. From the joint photometry and radial velocity analysis, we estimate the following parameters for TOI-1685 b: a mass of
M
b
= 3.78
−0.63
+0.63
M
⊕
, a radius of
R
b
= 1.70
−0.07
+0.07
R
⊕
, which together result in a bulk density of
ρ
b
= 4.21
−0.82
+0.95
g cm
−3
, and an equilibrium temperature of
T
eq
= 1069
−16
+16
K. TOI-1685 b is the least dense ultra-short-period planet around an M dwarf known to date. TOI-1685 b is also one of the hottest transiting super-Earth planets with accurate dynamical mass measurements, which makes it a particularly attractive target for thermal emission spectroscopy. Additionally, we report with moderate evidence an additional non-transiting planet candidate in the system, TOI-1685 c, which has an orbital period of
P
c
= 9.02
−0.12
+0.10
d.
ABSTRACT
In this paper, we present an analysis of near-infrared spectropolarimetric and velocimetric data of the young M dwarf AU Mic, collected with SPIRou at the Canada–France–Hawaii telescope from ...2019 to 2022, mostly within the SPIRou Legacy Survey. With these data, we study the large- and small-scale magnetic field of AU Mic, detected through the unpolarized and circularly polarized Zeeman signatures of spectral lines. We find that both are modulated with the stellar rotation period (4.86 d), and evolve on a time-scale of months under differential rotation and intrinsic variability. The small-scale field, estimated from the broadening of spectral lines, reaches 2.61 ± 0.05 kG. The large-scale field, inferred with Zeeman–Doppler imaging from Least-Squares Deconvolved profiles of circularly polarized and unpolarized spectral lines, is mostly poloidal and axisymmetric, with an average intensity of 550 ± 30 G. We also find that surface differential rotation, as derived from the large-scale field, is ≃30 per cent weaker than that of the Sun. We detect the radial velocity (RV) signatures of transiting planets b and c, although dwarfed by activity, and put an upper limit on that of candidate planet d, putatively causing the transit-timing variations of b and c. We also report the detection of the RV signature of a new candidate planet (e) orbiting further out with a period of 33.39 ± 0.10 d, i.e. near the 4:1 resonance with b. The RV signature of e is detected at 6.5σ while those of b and c show up at ≃4σ, yielding masses of $10.2^{+3.9}_{-2.7}$ and $14.2^{+4.8}_{-3.5}$ M⊕ for b and c, and a minimum mass of $35.2^{+6.7}_{-5.4}$ M⊕ for e.
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.
•Non-oxime reactivator for nerve agent-inhibited acetylcholinesterase is evaluated.•ADOC reactivation efficiency similar to or better than 2PAM for all agents tested.•ADOC mechanism of reactivation probed and phenol found to be essential.•ADOC did not provide in vivo efficacy against any nerve agents in guinea pigs.
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