Serum butyrylcholinesterase as a marker of COVID-19 mortality: Results of the monocentric prospective observational study.

The COVID-19 pandemic represents an excessive burden on health care systems worldwide and the number of patients who require special care in the clinical setting is often hard to predict. Consequently, there is an unmet need for a reliable biomarker that could predict clinical outcomes of high-risk patients. Lower serum butyrylcholinesterase (BChE) activity was recently linked with poor outcomes of COVID-19 patients. In line with this, our monocentric observational study on hospitalized COVID-19 patients focused on changes in serum BChE activity in relation to disease progression. Blood samples from 148 adult patients of both sexes were collected during their hospital stay at the Clinics of Infectiology and Clinics of Anesthesiology and Intensive Care, Trnava University Hospital in alignment with routine blood tests. Sera were analyzed using modified Ellman's method. Patient data with information about the health status, comorbidities and other blood parameters were collected in pseudonymized form. Our results show a lower serum BChE activity together with progressive decline of BChE activity in non-survivors, while higher stable values were present in discharged or transferred patients requiring further care. Lower BChE activity was associated with higher age and lower BMI. Moreover, we observed a negative correlation of serum BChE activity with the routinely used inflammatory markers, C-reactive protein and interleukin-6. Serum BChE activity mirrored clinical outcomes of COVID-19 patients and thus serves as a novel prognostic marker in high-risk patients.

Effect-Directed Profiling of Akebia quinata and Clitoria ternatea via High-Performance Thin-Layer Chromatography, Planar Assays and High-Resolution Mass Spectrometry.

Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, ß-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.

First report on the toxicity of SARS-CoV-2, alone and in combination with polyethylene microplastics in neotropical fish.

The COVID-19 pandemic has caused unprecedented negative impacts in the modern era, including economic, social, and public health losses. On the other hand, the potential effects that the input of SARS-CoV-2 in the aquatic environment from sewage may represent on non-target organisms are not well known. In addition, it is not yet known whether the association of SARS-CoV-2 with other pollutants, such as microplastics (MPs), may further impact the aquatic biota. Thus, we aimed to evaluate the possible ecotoxicological effects of exposure of male adults Poecilia reticulata, for 15 days, to inactivated SARS-CoV-2 (0.742 pg/L; isolated SARS.CoV2/SP02.2020.HIAE.Br) and polyethylene MP (PE MPs) (7.1 × 104 particles/L), alone and in combination, from multiple biomarkers. Our data suggest that exposure to SARS-CoV-2 induced behavioral changes (in the open field test), nephrotoxic effect (inferred by the increase in creatinine), hepatotoxic effect (inferred by the increase in bilirubin production), imbalance in the homeostasis of Fe, Ca, and Mg, as well as an anticholinesterase effect in the animals [marked by the reduction of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity]. On the other hand, exposure to PE MPs induced a genotoxic effect (assessed by the comet assay), as well as an increase in enzyme activity alpha-amylase, alkaline phosphatase, and carboxylesterases. However, we did not show synergistic, antagonistic, or additive effects caused by the combined exposure of P. reticulata to SARS-CoV-2 and PE MPs. Principal component analysis (PCA) and values from the "Integrated Biomarker Response" index indicate that exposure to SARS-CoV-2 was determinant for a more prominent effect in the evaluated animals. Therefore, our study sheds light on the ecotoxicity of the new coronavirus in non-target organisms and ratifies the need for more attention to the impacts of COVID-19 on aquatic biota.

Novel Organoruthenium(II) Complex C1 Selectively Inhibits Butyrylcholinesterase without Side Effects on Neuromuscular Transmission.

Enzyme butyrylcholinesterase (BChE) shows increased activity in some brain regions after progression of Alzheimer's disease and is therefore one of the therapeutic targets for symptomatic treatment of this neurodegenerative disorder. The organoruthenium(II) complex [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1) was designed based on the results of our previous structure-activity studies. Inhibitory activity toward cholinesterase enzymes shows that this complex selectively, competitively, and reversibly inhibits horse serum BChE (hsBChE) with an IC50 value of 2.88 µM. When tested at supra-pharmacological concentrations (30, 60, 90, and 120 µM), C1 had no significant effect on the maximal amplitude of nerve-evoked and directly elicited single-twitch and tetanic contractions. At the highest tested concentration (120 µM), C1 had no effect on resting membrane potential, but significantly decreased the amplitude of miniature end-plate potentials (MEPP) without reducing their frequency. The same concentration of C1 had no effect on the amplitude of end-plate potentials (EPP), however it shortened the half-decay time of MEPPs and EPPs. The decrease in the amplitude of MEPPs and shortening of the half-decay time of MEPPs and EPPs suggest a possible weak inhibitory effect on muscle-type nicotinic acetylcholine receptors (nAChR). These combined results show that, when applied at supra-pharmacological concentrations up to 120 µM, C1 does not importantly affect the physiology of neuromuscular transmission and skeletal muscle contraction.

Effect of Virus Inactivation by Heating on Routine Clinical Laboratory Indicators in Serum.

BACKGROUND: Highly infectious viruses such as SARS-CoV-2, MERS-CoV, and Ebola virus represent a threat to clinical laboratory workers. We aimed to investigate how virus inactivation by heating at 60°C for 1 hour affects routine clinical laboratory indicators. METHODS: Each collected serum sample was separated into two aliquots, and various indicators were measured in first aliquot after inactivation by heating at 60°C for 1 hour and in the second after room-temperature incubation for 1 hour. RESULTS: Serological test results for 36 indicators remained mostly unaffected by heat inactivation, with a mean estimated bias of < 10%. By contrast, the results for alanine transaminase, pseudocholinesterase, creatine kinase, lactate dehydrogenase, cardiac troponin I, and myoglobin were affected by heat inactivation, with the mean esti-mated bias here being > 20%, which was further increased in the case of the results for alkaline phosphatase, lipase, and creatine kinase isoenzyme MB. Immunological serological measurements showed good agreement according to Kappa consistency checks after heat inactivation of serum. The results for alanine transaminase, pseudocholinesterase, creatine kinase, lactate dehydrogenase, cardiac troponin I, and myoglobin were significantly correlated (r > 0.95) after heat inactivation, and after correction by using a regression equation, the results for the indicators still retained a clinical reference value. CONCLUSIONS: Inactivation by heating at 60°C for 1 hour exerts no marked effect on numerous routine biochemical and immunological indicators in serum, but the detection values for certain items are significantly decreased. Our method could serve as reference strategy for routine serological diagnostics in patients with suspected or confirmed infection with highly pathogenic viruses.

Prospective, observational, single-centre cohort study with an independent control group matched for age and sex aimed at investigating the significance of cholinergic activity in patients with schizophrenia: study protocol of the CLASH-study.

INTRODUCTION: Alterations in the cholinergic metabolism may cause various clinical symptoms of schizophrenia. In addition to the 'monoamine hypothesis,' neuroinflammation is also discussed as a cause of schizophrenia. To date, there has been no evidence of alterations in the central cholinergic transmitter balance in patients with schizophrenia under clinical conditions. By contrast, studies in critically ill patients have established the measurement of acetylcholinesterase activity as a suitable surrogate parameter of central cholinergic transmitter balance/possible pathophysiological changes. Butyrylcholinesterase activity has been established as a parameter indicating possible (neuro)inflammatory processes. Both parameters can now be measured using a point-of-care approach. Therefore, the primary objective of this study is to investigate whether acetylcholinesterase and butyrylcholinesterase activity differs in patients with various forms of schizophrenia. Secondary objectives address the possible association between acetylcholinesterase and butyrylcholinesterase activity and (1) schizophrenic symptoms using the Positive and Negative Syndrome Scale, (2) the quantity of antipsychotics taken and (3) the duration of illness. METHODS AND ANALYSIS: The study is designed as a prospective, observational cohort study with one independent control group. It is being carried out at the Department of Psychiatry and Psychotherapy III, Ulm University Hospital, Germany. Patient enrolment started in October 2020, and the anticipated end of the study is in January 2022. The enrolment period was set from October 2020 to December 2021 (extension required due to SARS-CoV-2 pandemic). The sample size is calculated at 50 patients in each group. Esterase activity is measured on hospital admission (acute symptomatology) and after referral to a postacute ward over a period of three consecutive days. The matched control group will be created after reaching 50 patients with schizophrenia. This will be followed by a comprehensive statistical analysis of the data set. ETHICS AND DISSEMINATION: The study was registered prospectively in the German Clinical Trials Register (DRKS-ID: DRKS00023143,URL: https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00023143) after approval by the ethics committee of the University of Ulm, Germany Trial Code No. 280/20. TRIAL REGISTRATION NUMBER: DRKS00023143; Pre-results.

Long-Chain Acylcholines Link Butyrylcholinesterase to Regulation of Non-neuronal Cholinergic Signaling.

Acylcholines are comprised of an acyl chain esterified to a choline moiety; acetylcholine is the best-characterized member of this class, functioning as a neurotransmitter in the central and peripheral nervous systems as well as an inhibitor of cytokine production by macrophages and other innate immune cells. Acylcholines are metabolized by a class of cholinesterases, including acetylcholinesterase (a specific regulator of acetylcholine levels) and butyrylcholinesterase (BChE, an enigmatic enzyme whose function has not been resolved by genetic knockout models). BChE provides reserve capacity to hydrolyze acetylcholine, but its importance is arguable given acetylcholinesterase is the most catalytically efficient enzyme characterized to date. While known to be substrates of BChE in vitro, endogenous production of long-chain acylcholines is a recent discovery enabled by untargeted metabolomics. Compared to acetylcholine, long-chain acylcholines show greater stability in circulation with homeostatic levels-dictated by synthesis and clearance-suggested to impact cholinergic receptor sensitivity of acetylcholine with varying levels of antagonism. Acylcholines then provide a link between BChE and non-neuronal acetylcholine signaling, filling a gap in understanding around how imbalances between acylcholines and BChE could modulate inflammatory disease, such as the "cytokine storm" identified in severe COVID-19. Areas for further research, development, and clinical testing are outlined.

Human carboxylesterase 1A plays a predominant role in the hydrolytic activation of remdesivir in humans.

Remdesivir, an intravenous nucleotide prodrug, has been approved for treating COVID-19 in hospitalized adults and pediatric patients. Upon administration, remdesivir can be readily hydrolyzed to form its active form GS-441524, while the cleavage of the carboxylic ester into GS-704277 is the first step for remdesivir activation. This study aims to assign the key enzymes responsible for remdesivir hydrolysis in humans, as well as to investigate the kinetics of remdesivir hydrolysis in various enzyme sources. The results showed that remdesivir could be hydrolyzed to form GS-704277 in human plasma and the microsomes from human liver (HLMs), lung (HLuMs) and kidney (HKMs), while the hydrolytic rate of remdesivir in HLMs was the fastest. Chemical inhibition and reaction phenotyping assays suggested that human carboxylesterase 1 (hCES1A) played a predominant role in remdesivir hydrolysis, while cathepsin A (CTSA), acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) contributed to a lesser extent. Enzymatic kinetic analyses demonstrated that remdesivir hydrolysis in hCES1A (SHUTCM) and HLMs showed similar kinetic plots and much closed Km values to each other. Meanwhile, GS-704277 formation rates were strongly correlated with the CES1A activities in HLM samples from different individual donors. Further investigation revealed that simvastatin (a therapeutic agent for adjuvant treating COVID-19) strongly inhibited remdesivir hydrolysis in both recombinant hCES1A and HLMs. Collectively, our findings reveal that hCES1A plays a predominant role in remdesivir hydrolysis in humans, which are very helpful for predicting inter-individual variability in response to remdesivir and for guiding the rational use of this anti-COVID-19 agent in clinical settings.