Anti-dementia drugs: a descriptive study of the prescription pattern in Italy.

INTRODUCTION: Acetylcholinesterase inhibitors (AChEIs) and memantine are currently the only anti-dementia drugs (ADDs) approved for treating Alzheimer's disease (AD) in Italy. This nationwide study aims to characterize dementia drug utilization in a population > 65 years, during 2018-2020. METHODS: Different administrative healthcare databases were queried to collect both aggregate and individual data. RESULTS: ADD consumption remained stable throughout the study period (~ 9 DDD/1000 inhabitants per day). AChEI consumption was over 5 DDD/1000 inhabitants per day. Memantine consumption was nearly 4 DDD/1000 inhabitants per day, representing 40% of ADD consumption. The prevalence of use of memantine represented nearly half of ADD consumption, substantially unchanged over the 3 years. Comparing the AD prevalence with the prevalence of ADDs use, the gap becomes wider as age increases. In 2019, the proportion of private purchases of ADDs was 38%, mostly represented by donepezil and rivastigmine. In 2020, memantine was the only ADD with an increase in consumption (Δ% 19-20, 1.3%). DISCUSSION: To our knowledge, this study represents the first attempt to investigate the ADD prescription pattern in Italy with a Public Health approach. In 2019, the proportion of ADD private purchases point out several issues concerning the reimbursability of ADDs. From a regulatory perspective, ADDs can be reimbursed by the National Health System only to patients diagnosed with AD; therefore, the off-label use of ADDs in patients with mild cognitive impairment may partially explain this phenomenon. The study extends knowledge on the use of ADDs, providing comparisons with studies from other countries that investigate the prescription pattern of ADDs.

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.

Exposure of adult zebrafish (Danio rerio) to SARS-CoV-2 at predicted environmentally relevant concentrations: Outspreading warns about ecotoxicological risks to freshwater fish.

While the multifaceted social, economic, and public health impacts associated with the COVID-19 pandemic are known, little is known about its effects on non-target aquatic ecosystems and organisms. Thus, we aimed to evaluate the potential ecotoxicity of SARS-CoV-2 lysate protein (SARS.CoV2/SP02.2020.HIAE.Br) in adult zebrafish (Danio rerio) at predicted environmentally relevant concentrations (0.742 and 2.226 pg/L), by 30 days. Although our data did not show locomotor alterations or anxiety-like or/and anxiolytic-like behavior, we noticed that exposure to SARS-CoV-2 negatively affected habituation memory and social aggregation of animals in response to a potential aquatic predator (Geophagus brasiliensis). An increased frequency of erythrocyte nuclear abnormalities was also observed in animals exposed to SARS-CoV-2. Furthermore, our data suggest that such changes were associated with a redox imbalance [↑ROS (reactive oxygen species), ↑H2O2 (hydrogen peroxide), ↓SOD (superoxide dismutase), and ↓CAT (catalase)], cholinesterasic effect [↑AChE (acetylcholinesterase) activity], as well as the induction of an inflammatory immune response [↑NO (nitric oxide), ↑IFN-γ (interferon-gamma), and ↓IL-10 (interleukin-10)]. For some biomarkers, we noticed that the response of the animals to the treatments was not concentration-dependent. However, principal component analysis (PCA) and the "Integrated Biomarker Response" index (IBRv2) indicated a more prominent ecotoxicity of SARS-CoV-2 at 2.226 pg/L. Therefore, our study advances knowledge about the ecotoxicological potential of SARS-CoV-2 and reinforces the presumption that the COVID-19 pandemic has negative implications beyond its economic, social, and public health impacts.

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.

Identification of two early blood biomarkers ACHE and CLEC12A for improved risk stratification of critically ill COVID-19 patients.

In order to identify biomarkers for earlier prediction of COVID-19 outcome, we collected blood samples from patients with fatal outcomes (non-survivors) and with positive clinical outcomes (survivors) at ICU admission and after seven days. COVID-19 survivors and non-survivors showed significantly different transcript levels for 93 genes in whole blood already at ICU admission as revealed by RNA-Seq. These differences became even more pronounced at day 7, resulting in 290 differentially expressed genes. Many identified genes play a role in the differentiation of hematopoietic cells. For validation, we designed an RT-qPCR assay for C-type lectin domain family 12 member A (CLEC12A) and acetylcholinesterase (ACHE), two transcripts that showed highest potential to discriminate between survivors and non-survivors at both time points. Using our combined RT-qPCR assay we examined 33 samples to accurately predict patient survival with an AUROC curve of 0.931 (95% CI = 0.814-1.000) already at ICU admission. CLEC12A and ACHE showed improved prediction of patient outcomes compared to standard clinical biomarkers including CRP and PCT in combination (AUROC = 0.403, 95% CI = 0.108-0.697) or SOFA score (AUROC = 0.701 95% CI = 0.451-0.951) at day 0. Therefore, analyzing CLEC12A and ACHE gene expression from blood may provide a promising approach for early risk stratification of severely ill COVID-19 patients.

Synergistic action of organophosphates and COVID-19 on inflammation, oxidative stress, and renin-angiotensin system can amplify the risk of cardiovascular maladies.

Organophosphates (OPs) are ubiquitous environmental contaminants, widely used as pesticides in agricultural fields. In addition, they serve as flame-retardants, plasticizers, antifoaming or antiwear agents in lacquers, hydraulic fluids, and floor polishing agents. Therefore, world-wide and massive application of these compounds have increased the risk of unintentional exposure to non-targets including the human beings. OPs are neurotoxic agents as they inhibit the activity of acetylcholinesterase at synaptic cleft. Moreover, they can fuel cardiovascular issues in the form of myocardities, cardiac oedema, arrhythmia, systolic malfunction, infarction, and altered electrophysiology. Such pathological outcomes might increase the severity of cardiovascular diseases which are the leading cause of mortality in the developing world. Coronavirus disease-19 (COVID-19) is the ongoing global health emergency caused by SARS-CoV-2 infection. Similar to OPs, SARS-CoV-2 disrupts cytokine homeostasis, redox-balance, and angiotensin-II/AT1R axis to promote cardiovascular injuries. Therefore, during the current pandemic milieu, unintentional exposure to OPs through several environmental sources could escalate cardiac maladies in patients with COVID-19.

Action of the Purinergic and Cholinergic Anti-inflammatory Pathways on Oxidative Stress in Patients with Alzheimer's Disease in the Context of the COVID-19 Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of the 2019 coronavirus disease (COVID-19), has affected more than 20 million people in Brazil and caused a global health emergency. This virus has the potential to affect various parts of the body and compromise metabolic functions. The virus-mediated neural inflammation of the nervous system is due to a storm of cytokines and oxidative stress, which are the clinical features of Alzheimer's disease (AD). This neurodegenerative disease is aggravated in cases involving SARS-CoV-2 and its inflammatory biomarkers, accelerating accumulation of ß-amyloid peptide, hyperphosphorylation of tau protein, and production of reactive oxygen species, which lead to homeostasis imbalance. The cholinergic system, through neurons and the neurotransmitter acetylcholine (ACh), modulates various physiological pathways, such as the response to stress, sleep and wakefulness, sensory information, and the cognitive system. Patients with AD have low concentrations of ACh; hence, therapeutic methods are aimed at adjusting the ACh titers available to the body for maintaining functionality. Herein, we focused on acetylcholinesterase inhibitors, responsible for the degradation of ACh in the synaptic cleft, and muscarinic and nicotinic receptor agonists of the cholinergic system owing to the therapeutic potential of the cholinergic anti-inflammatory pathway in AD associated with SARS-CoV-2 infection.

Relationship between toxicity and oxidative stress of the nanoencapsulated colchicine in a model of Drosophila melanogaster.

Drug repurposing allows searching for new biological targets, especially against emerging diseases such as Covid-19. Drug colchicine (COL) presents recognized anti-inflammatory action, while the nanotechnology purpose therapies with low doses, efficacy, and decrease the drug's side-effects. This study aims to evaluate the effects of COL and colchicine nanocapsules (NCCOL) on survival, LC50, activity locomotor, and oxidative stress parameters, elucidating the toxicity profile in acute and chronic exposure in Drosophila melanogaster. Three-day-old flies were investigated into groups: Control, 0.001, 0.0025, 0.005, and 0.010 mg/mL of COL or NCCOL. The survival rate, open field test, LC50, oxidative stress markers (reactive species (RS) production, thiobarbituric acid reactive substances), antioxidant enzyme activity (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase), protein thiols, nonprotein thiols, acetylcholinesterase activity, and cell viability were measured. As a result, acute exposure to the COL decreases the number of crosses in the open field and increases CAT activity. NCCOL reduced RS levels, increased lipoperoxidation and SOD activity. Chronic exposure to the COL and NCCOL in high concentrations implied high mortality and enzymatic inhibition of the CAT and AChE, and only the COL caused locomotor damage in the open field test. Thus, NCCOL again reduced the formation of RS while COL increased. In this comparative study, NCCOL was less toxic to the antioxidant system than COL and showed notable involvement of oxidative stress as one of their toxicity mechanisms. Future studies are needed to elucidate all aspects of nanosafety related to the NCCOL.

Toxicity assessment of SARS-CoV-2-derived peptides in combination with a mix of pollutants on zebrafish adults: A perspective study of behavioral, biometric, mutagenic, and biochemical toxicity.

The dispersion of SARS-CoV-2 in aquatic environments via the discharge of domestic and hospital sewage has been confirmed in different locations. Thus, we aimed to evaluate the possible impacts of zebrafish (Danio rerio) exposure to SARS-CoV-2 peptide fragments (PSPD-2001, 2002, and 2003) alone and combined with a mix of emerging pollutants. Our data did not reveal the induction of behavioral, biometric, or mutagenic changes. But we noticed an organ-dependent biochemical response. While nitric oxide and malondialdehyde production in the brain, gills, and muscle did not differ between groups, superoxide dismutase activity was reduced in the "PSPD", "Mix", and "Mix+PSPD" groups. An increase in catalase activity and a reduction in DPPH radical scavenging activity were observed in the brains of animals exposed to the treatments. However, the "Mix+PSPD" group had a higher IBRv2 value, with NO levels (brain), the reduction of acetylcholinesterase activity (muscles), and the DPPH radical scavenging activity (brain and muscles), the most discriminant factors for this group. The principal component analysis (PCA) and hierarchical clustering analysis indicated a clear separation of the "Mix+PSPD" group from the others. Thus, we conclude that exposure to viral fragments, associated with the mix of pollutants, induced more significant toxicity in zebrafish adults than in others.

New Imaging Modality of COVID-19 Pneumonia Developed on the Basis of Alzheimer's Disease Research.

Viral pneumonia caused by highly infectious SARS-CoV-2 poses a higher risk to older people and those who have underlying health conditions, including Alzheimer's disease. In this work we present newly designed tacrine-based radioconjugates with physicochemical and biological properties that are crucial for the potential application as diagnostic radiopharmaceuticals. A set of ten tacrine derivatives was synthesized, labelled with gallium-68 and fully characterized in the context of their physicochemical properties. Based on these results, the final two most promising radioconjugates, [68Ga]Ga-NODAGA-Bn-NH(CH2)9Tac and [68Ga]Ga-THP-NH(CH2)9Tac, were selected for biodistribution studies. The latter compound was proven to be a good inhibitor of cholinesterases with significant affinity toward the lungs, according to the biodistribution studies. On the basis of molecular modelling combined with in vitro studies, we unraveled which structural properties of the developed tacrine derivatives are crucial for high affinity toward acetylcholinesterase, whose increased levels in lung tissues in the course of coronavirus disease indicate the onset of pneumonia. The radiopharmaceutical [68Ga]Ga-THP-NH(CH2)9Tac was ultimately selected due to its increased accuracy and improved sensitivity in PET imaging of lung tissue with high levels of acetylcholinesterase, and it may become a novel potential diagnostic modality for the determination of lung perfusion, including in inflammation after COVID-19.

Short-term exposure of the mayfly larvae (Cloeon dipterum, Ephemeroptera: Baetidae) to SARS-CoV-2-derived peptides and other emerging pollutants: A new threat for the aquatic environments.

The input of SARS-CoV-2 or its fragments into freshwater ecosystems (via domestic or hospital sewage) has raised concerns about its possible impacts on aquatic organisms. Thus, using mayfly larvae [Cloeon dipterum (L.), Ephemeroptera: Baetidae] as a model system, we aimed to evaluate the possible effects of the combined short exposure of SARS-CoV-2-derived peptides (named PSPD-2001, PSPD-2002, and PSPD-2003 - at 266.2 ng/L) with multiple emerging pollutants at ambient concentrations. After six days of exposure, we observed higher mortality of larvae exposed to SARS-CoV-2-derived peptides (alone or in combination with the pollutant mix) and a lower-body condition index than those unexposed larvae. In the "PSPD" and "Mix+PSPD" groups, the activity of superoxide dismutase, catalase, DPPH radical scavenging activity, and the total thiol levels were also lower than in the "control" group. In addition, we evidenced the induction of nitrosative stress (inferred by increased nitrite production) and reduced acetylcholinesterase activity by SARS-CoV-2-derived peptides. On the other hand, malondialdehyde levels in larvae exposed to treatments were significantly lower than in unexposed larvae. The values of the integrated biomarker response index and the principal component analysis (PCA) results confirmed the similarity between the responses of animals exposed to SARS-CoV-2-derived peptides (alone and in combination with the pollutant mix). Although viral peptides did not intensify the effects of the pollutant mix, our study sheds light on the potential ecotoxicological risk associated with the spread of the new coronavirus in aquatic environments. Therefore, we recommend exploring this topic in other organisms and experimental contexts.

Searching for AChE inhibitors from natural compounds by using machine learning and atomistic simulations.

Acetylcholinesterase (AChE) is one of the most important drug targets for Alzheimer's disease treatment. In this work, a combined approach involving machine-learning (ML) model and atomistic simulations was established to predict the ligand-binding affinity to AChE of the natural compounds from VIETHERB database. The trained ML model was first utilized to rapidly and accurately screen the natural compound database for potential AChE inhibitors. Atomistic simulations including molecular docking and steered-molecular dynamics simulations were then used to confirm the ML outcome. Good agreement between ML and atomistic simulations was observed. Twenty compounds were suggested to be able to inhibit AChE. Especially, four of them including geranylgeranyl diphosphate, 2-phosphoglyceric acid, and 2-carboxy-d-arabinitol 1-phosphate, and farnesyl diphosphate are highly potent inhibitors with sub-nanomolar affinities.

Comment on Kopanska et al. Disorders of the Cholinergic System in COVID-19 Era-A Review of the Latest Research. Int. J. Mol. Sci. 2022, 23, 672.

We read the recent review article by Marta Kopanska et al. [...].

Shedding light on the toxicity of SARS-CoV-2-derived peptide in non-target COVID-19 organisms: A study involving inbred and outbred mice.

Despite advances in research on the vaccine and therapeutic strategies of COVID-19, little attention has been paid to the possible (eco)toxicological impacts of the dispersion of SARS-CoV-2 particles in natural environments. Thus, in this study, we aimed to evaluate the behavioral and biochemical consequences of the short exposure of outbred and inbred mice (male Swiss and C57Bl/6 J mice, respectively) to PSPD-2002 (peptide fragments of the Spike protein of SARS-CoV-2) synthesized in the laboratory. Our data demonstrated that after 24 h of intraperitoneal administration of PSPD-2002 (at 580 µg/kg) the animals did not present alterations in their locomotor, anxiolytic-like, or anxiety-like behavior (in the open field test), nor antidepressant-like or depressive behavior in the forced swimming test. However, the C57Bl/6 J mice exposed to PSPD-2002 showed memory deficit in the novel object recognition task, which was associated with higher production of thiobarbituric acid reactive substances, as well as the increased suppression of acetylcholinesterase brain activity, compared to Swiss mice also exposed to peptide fragments. In Swiss mice the reduction in the activity of superoxide dismutase and catalase in the brain was not associated with increased oxidative stress biomarkers (hydrogen peroxide), suggesting that other antioxidant mechanisms may have been activated by exposure to PSPD-2002 to maintain the animals' brain redox homeostasis. Finally, the results of all biomarkers evaluated were applied into the "Integrated Biomarker Response Index" (IBRv2) and the principal component analysis (PCA), and greater sensitivity of C57Bl/6 J mice to PSPD-2002 was revealed. Therefore, our study provides pioneering evidence of mammalian exposure-induced toxicity (non-target SARS-CoV-2 infection) to PSPD-2002, as well as "sheds light" on the influence of genetic profile on susceptibility/resistance to the effects of viral peptide fragments.

Disorders of the Cholinergic System in COVID-19 Era-A Review of the Latest Research.

The appearance of the SARS-CoV-2 virus initiated many studies on the effects of the virus on the human body. So far, its negative influence on the functioning of many morphological and physiological units, including the nervous system, has been demonstrated. Consequently, research has been conducted on the changes that SARS-CoV-2 may cause in the cholinergic system. The aim of this study is to review the latest research from the years 2020/2021 regarding disorders in the cholinergic system caused by the SARS-CoV-2 virus. As a result of the research, it was found that the presence of the COVID-19 virus disrupts the activity of the cholinergic system, for example, causing the development of myasthenia gravis or a change in acetylcholine activity. The SARS-CoV-2 spike protein has a sequence similar to neurotoxins, capable of binding nicotinic acetylcholine receptors (nAChR). This may be proof that SARS-CoV-2 can bind nAChR. Nicotine and caffeine have similar structures to antiviral drugs, capable of binding angiotensin-converting enzyme 2 (ACE 2) epitopes that are recognized by SARS-CoV-2, with the potential to inhibit the formation of the ACE 2/SARS-CoV-2 complex. The blocking is enhanced when nicotine and caffeine are used together with antiviral drugs. This is proof that nAChR agonists can be used along with antiviral drugs in COVID-19 therapy. As a result, it is possible to develop COVID-19 therapies that use these compounds to reduce cytokine production. Another promising therapy is non-invasive stimulation of the vagus nerve, which soothes the body's cytokine storm. Research on the influence of COVID-19 on the cholinergic system is an area that should continue to be developed as there is a need for further research. It can be firmly stated that COVID-19 causes a dysregulation of the cholinergic system, which leads to a need for further research, because there are many promising therapies that will prevent the SARS-CoV-2 virus from binding to the nicotinic receptor. There is a need for further research, both in vitro and in vivo. It should be noted that in the functioning of the cholinergic system and its connection with the activity of the COVID-19 virus, there might be many promising dependencies and solutions.

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.

Pesticides use and health impacts on farmers in Thailand, Vietnam, and Lao PDR: Protocol for a survey of knowledge, behaviours and blood acetyl cholinesterase concentrations.

Pesticides use in Southeast Asia has increased steadily, driven by the growth of large-scale commercial farming, as well as a desire to maximise food production in rural subsistence economies. Given that use of chemical pesticides, such as organophosphates and carbamates, has known potential health impacts, there are concerns about the safety of agricultural workers, and a need for a better evidence base to underpin regulation and worker education. This study, undertaken in 9 districts in Lao PDR, Thailand and Vietnam, will interview agricultural workers to investigate how they use pesticides, their knowledge of risks and self-protective practices, and their self-reported illness symptoms. In each district researchers will recruit and interview 120 participants engaged in vegetable farming, who have recently used pesticides, making a total of 1080 subjects divided equally between the three study countries. Workers' degree of pesticides exposure will be determined from acetyl cholinesterase concentrations in capillary blood samples collected using field test kits, and these data will be analysed together with the interview findings. Country findings will be compared and contrasted, and general patterns noted. Knowledge gained about risky behaviours, self-protective practices and degree of association with serious pesticides exposure will assist policy makers and inform health improvement programmes.

The Positive Modulation Effect of a 6-Week Consumption of an Anthocyanin-Rich Mulberry Milk on Working Memory, Cholinergic, and Monoaminergic Functions in Healthy Working-Age Adults.

Due to the increase of stress-related memory impairment accompanying with the COVID-19 pandemic and financial crisis, the prevention of cognitive decline induced by stress has gained much attention. Based on the evidence that an anthocyanin-rich mulberry milk demonstrated the cognitive enhancing effect, we hypothesized that it should be able to enhance memory in working-age volunteers who are exposed to working stress. This study is an open-label, two-arm randomized study. Both men and women volunteers at age between 18 and 60 years old were randomly assigned to consume the tested product either 1 or 2 servings daily for 6 weeks. All subjects were assessed for cortisol, acetylcholinesterase (AChE), monoamine oxidase (MAO), monoamine oxidase type A (MAO-A), and monoamine oxidase type B (MAO-B) in saliva, and their working memory was determined both at baseline and at a 6-week period. The results showed that the working memory of subjects in both groups was enhanced at the end of the study period together with the reduction of saliva cortisol. The suppression of AChE, MAO, and MAO-A was also observed in subjects who consumed the tested product 2 servings daily. Therefore, we suggest the memory enhancing effect of an anthocyanin-rich mulberry milk. The possible mechanism may occur primarily via the suppression of cortisol. In addition, the high dose of mulberry milk also suppresses AChE, MAO, and MAO-A.