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The efficacy of prophylactic use of memantine alone or in combination with clinically used reversible acetylcholinesterase inhibitors (pyridostigmine, donepezil, or rivastigmine) against soman toxicity and their influence on post-exposure therapy consisting of atropine and HI-6 was studied. The effectiveness was assessed by comparison of LD₅₀ values over 24 h after soman poisoning. Pyridostigmine failed to decrease the acute toxicity of soman. But memantine, donepezil and rivastigmine reduced the acute toxicity of soman, with donepezil showing the best efficacy. Combination of memantine with pyridostigmine or one of the centrally-acting reversible acetylcholinesterase inhibitors attenuated soman acute toxicity significantly. The pharmacological pretreatment influenced the efficacy of post-exposure treatment in a similar fashion; i) pyridostigmine or memantine did not affect the therapeutic effectiveness of antidotal treatment ii) centrally acting reversible acetylcholinesterase inhibitors slightly increased the efficacy of antidotal treatment, iii) combination of memantine with reversible acetylcholinesterase inhibitors increased the effectiveness of antidotal treatment more markedly. In conclusion, memantine alone slightly decrease the acute toxicity of soman and failed to increase post-exposure antidotal treatment efficacy. On the other hand, the combination of memantine with donepezil significantly decrease the acute toxicity of soman and increased post-exposure effectiveness. Both drugs, when applied together, mitigate soman toxicity and boost post-exposure treatment.

Pistacia lentiscus L. (PLL) is a wild-growing shrub from the Anacardiaceae family that occurs naturally in the Mediterranean. It has been used in traditional medicine for its many effects since the days of ancient Greece (1). PLL plant and processed products have been largely employed as oral antiseptic, anti-inflammatory, analgesic, and healing agents (2). Today, the scientific interest in these edible and not-edible parts of PLL is wide-spreading, as some studies underlined the potential benefit against inflammation and infections (3). The aim of our work is to find out more details about biological activity of essential oil and aqueous extract prepared from leaves of wild occurring PLL plants growing in North Sardinia. The cytotoxicity of not only the pure essential oil but also of the nanoemulsions with PLL oil, and aqueous extract were tested on three oral cell lines (gingival fibroblasts, periodontal ligament fibroblasts, and dysplastic oral keratinocytes). The viability of human oral cells was the most diminished by lecithin nanoemulsion, less by aqueous extract, and pure essential oil had the lowest influence on the viability – it was non-toxic up to the concentration of 100 μg PLL/ml medium. The anti-inflammatory activity was measured by cyclooxygenase and lipoxygenase inhibition. PLL essential oil showed the capacity to inhibit COX1/2 more than LOX. In conclusion, PLL essential oil is not cytotoxic for oral cell lines and show an in vitro anti-inflammatory activity.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial, complex, and chronic liver disease with increasing worldwide prevalence. Metabolic syndrome and obesity are the main risk factors associated with NAFLD. High-fat diet causes an excessive fat accumulation in the liver referred to as simple steatosis, which can further progress to more severe stages of NAFLD including non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. As NAFLD has been reported to cause changes in the liver homeostasis and metabolism of xenobiotics (1), the objective of this study was to investigate NAFLD-induced changes in the expression and/or activity of selected phase I drug-metabolizing liver enzymes. For our study, liver samples from wild type mice fed with standard or high-fat diet were used. The mRNA and protein expression as well as the specific activity of several cytochrome P450 (CYP), carbonyl reductase (CBR), and aldo-keto reductase (AKR) enzymes were determined by using real-time quantitative PCR, western blotting, and spectrophotometric methods, respectively. High-fat diet caused significant induction of mRNA as well as protein expression and specific activity of CYP1A1/2. The mRNA expression of CYP3A11 and CYP3A13 (orthologs of human CYP3A4) was decreased, while its protein expression remained unchanged. Induction was observed also for AKR1C6 and AKR1C20 (orthologs of human AKR1C1) at mRNA and protein level.

Within the alpha/beta hydrolase fold superfamily of proteins, the COesterase group (carboxylesterase type B, block C, cholinesterases…) diverged from the other groups through addition of an N-terminal disulfide bond and simultaneous increase in the mean size of the protein (1). This disulfide bond creates a large loop, which is essential for the high catalytic activity of cholinesterases through formation of the upper part of the active center gorge. In some non-catalytic members of the family, the loop may be necessary for heterologous partner recognition. The shuffling of this portion of protein occurred at the time of emergence of the fungi/metazoan lineage. Homologous proteins with this N-terminal disulfide bond are absent in plants but they are found in a limited number of bacterial genomes. In prokaryotes, the genes coding for such homologous enzymes may have been acquired by horizontal transfer. However the cysteines of the first disulfide bond are often lost in bacteria. Natural expression in bacteria of CO-esterases comprising this disulfide bond may have required compensatory mutations or expression of new chaperones. This disulfide bond may also challenge expression of the eukaryote-specific cholinesterases in E. coli. Recently , catalytically active human acetylcholinesterase and butyrylcholinesterase were successfully expressed in E. coli. The key was the use of a peptidic sequence optimized through the Protein Repair One Stop Shop process, an automated structure- and sequence-based algorithm toward expression of properly folded, soluble eukaryotic proteins with an enhanced stability (2,3). Surprisingly however, the crystal structure of the optimized butyrylcholinesterase variant expressed from bacteria revealed co-existing ‘close’ and ‘open’ states of the first disulfide bond. Whether the ‘open bond’ involves two cysteines (i.e., the bond never formed) or two half-cystines (i.e., the bond properly formed, then broke during the production/analysis process) cannot be inferred from the structural data. Yet, this observation suggests that this first bond is difficult to maintain in E. coli-expressed cholinesterases.

Cholinesterases are divided into two classes according to differences in substrate specificity, behaviour in high substrate concentrations, inhibitor sensitivity and tissue distribution: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The both enzymes are sensitive to broad spectrum of molecules and may be inhibited by several compounds, such as organophosphate and carbamate pesticides or nerve agents. In a previous study, a phenazine-derived natural product, geranyl-phenazine-diol was shown to inhibit human AChE with IC50 value of 2.62 mM. Phenazines which are naturally produced by bacteria and archaeal Methanosarcina species are nitrogen containing tricyclic molecules with antibiotic, antitumor, and antiparasitic activities. Phenazines are used as electron acceptors-donors in wide range of fields including environmental biosensors. In this study, the inhibitory effect of a synthetic phenazine dye, methylene violet 3RAX (also known as diethyl safranine) was tested on human erythrocyte AChE and human plasma BChE and its inhibitory mechanism on both enzymes was studied in detail. AChE and BChE activities were assayed spectrophotometrically at 25 ^oC in 50 mM MOPS buffer pH 8, using 0.05-0.4 mM butyrylthiocholine or  0.025-0.4 mM acetylthiocholine as substrate, 0.125 mM DTNB and 0-80 µM dye. Kinetic analyses showed that methylene violet 3RAX acts as a hyperbolic noncompetitive inhibitor of AChE with K_i value of 1.42±0.09 µM; α=1 β=0.11. On the other hand, it caused linear competitive inhibition of BChE with K_i value of 0.46±0.02 µM; α=∞. In conclusion, methylene violet 3RAX with K_i value in the low micromolar range may be a promising lead candidate for the treatment of Alzheimer’s disease.

Current symptomatic treatment has only limited clinical efficacy and minute effect on progression of Alzheimer’s disease. The research focus has thus shifted from single targets towards multifunctional ligands targeting several pathological processes of the disease [1, 2]. A potent picomolar selective inhibitor of human butyrylcholinesterase [3] was used as the starting point to develop a new series of multifunctional ligands. A focused library of derivatives was designed and synthesized that showed both butyrylcholinesterase inhibition and good antioxidant activity comparable to natural antioxidants. The crystal structure of compound 11 in complex with butyrylcholinesterase revealed the molecular basis for its low nanomolar inhibition of butyrylcholinesterase (K_i = 1.09 ±0.12 nM). In addition, compounds 8 and 11 show metal-chelating properties as determined by the UV-Vis titrations, and reduce the redox activity of chelated Cu²⁺ ions in a Cu-ascorbate redox system. Compounds 8 and 11 decrease intracellular levels of reactive oxygen species, and are not substrates of the active efflux transport system, as determined in Caco2 cells. Compound 11 also protects neuroblastoma SH-SY5Y cells from toxic Aβ_1–42 species. These data indicate that compounds 8 and 11 are promising multifunctional lead ligands for treatment of Alzheimer’s disease.

Nerve agents (NAs) belong to family of organophosphorus inhibitors (OPIs) of acetylcholinesterase (AChE) enzyme. Although the use of highly toxic OPIs as chemical warfare agents is prohibited, they have been misused several times and thus still represents an emerging threat. For these reasons, the development of novel prophylactic agents and therapeutic intervention against NAs is still up-to-date and of high importance. Prophylactic administration of antidotes not only increases body's resistance to the effects of NAa, e.g. when operating at a contaminated environment after a terrorist attack, but also potentiates the subsequent antidotal therapy after exposure. Currently, pharmacological prophylaxis can be delivered either by protection of AChE against irreversible Inhibition, administration of commonly used antidotes in advance (i.e. oxime reactivators) or by the use of bioscavengers. The aim of the recent review is to highlight the current trends in prophylaxis and outline breakthroughs in prophylaxis based on reversible cholinesterase inhibitors like huperzine A or acridine derivatives.

The aryl hydrocarbon receptor (AhR) is well known for its detoxification/bioactivation role. However, it has been suggested to participate also in the control of inflammatory responses. Diesel exhaust particles (DEP) are known to induce inflammation in respiratory tract. Here, we used standard reference mixture of diesel exhaust particles (SRM1650b) in order to evaluate the functional role of the AhR (using wild-type and AhR-deficient A549 lung epithelial cells) in the control of inflammatory responses towards DEP-associated pollutants. We compared their effects with those of pro-inflammatory cytokines, such as IL-1β. We found that the induction of cyclooxygenase-2 (and secretion of prostaglandins), as well as expression of pro-inflammatory cytokines, were notably higher in the AhR-deficient A549 cells, when exposed to inflammatory cytokines. Both crude organic extract of SRM1650b and its polar fraction induced an increased inflammatory response in AhR KO cells, including the induction of COX-2, TNFα, CXCL8 and IL-6 mRNAs. Our further experiments have identified increased nuclear factor-κB (NF-κB) activity to contribute to the exacerbated inflammatory response in the AhR-deficient A549 cells. Thus, the interplay of AhR and NF-κB signaling may modulate responses of lung epithelial cells towards complex mixtures of organic pollutants, such as those associated with DEP.

The widespread deployment of insecticide-treated bednets (ITNs) in sub-Saharan Africa has led to a dramatic decline in malaria mortality. However, wide-spread and growing resistance of Anopheles gambiae mosquitoes to the pyrethroid class of voltage-gated Na⁺ channel modulators used on these nets jeopardizes this achievement, and has prompted the search for suitable insecticidal AChE inhibitors to replace pyrethroids. Such compounds would have three favorable characteristics:  excellent contact toxicity towards susceptible adult An. gambiae, good contact toxicity to those that bear the G119S resistance mutation of AChE, and very weak inhibition of human AChE.¹ We will review our work on the development of aromatic and heterocyclic core methyl and dimethylcarbamate AChE inhibitors,² and including both enzymatic inhibition potencies and mosquito contact toxicities.  Finally, the inhibition selectivities of particular compounds will be rationalized in the context of our recently obtained high resolution X-ray structures of G119S An. gambiae AChE.³

Protective mechanism against organophosphorus compounds (OPs) toxicity are mainly based in molecular processes frequently divided conceptually in (A) catalytic and (B) non-catalytic bio-scavengers. Modified natural proteins and small molecules have been developed for applying in therapy and protection. The catalytic ones are mainly associate to the classical concept of A-esterases (phosphotriesterases, PTEs, i.e. paraoxonase); they hydrolyze carboxylesters and OPs by a divalent cation dependent mechanism.  The non-catalytic scavengers are mainly associated to covalent binding to proteins, especially B-esterases with a serine or tyrosine residue, which hydrolyzes carboxylesters. However, if an OP is bound (organophosphorylation), its represents an enzymatic inhibition in some cases considered “the target” of toxicity or initial molecular event (IME) in their mode of action developing toxicity (adverse output pathway, AOP). The binding to proteins also represents a sequestration avoiding the OP interaction to other protein. However, there are protein binding OPs (non-catalytic bioscavengers) which can be slowly dephosphorylated, having a role as catalytic scavenger. A proportion of B-esterase activity in serum and brain shows reversible inhibition and their protective role just in situ in the target tissue of toxicity need to be investigated. Serum albumin is other example of B-esterase mainly thorough a tyrosine residue; its role in detoxication have been demonstrated and adducts applied as biomarker of exposure. Moreover, for a specific phosphoramidate family hydrolysis capacity may be enhanced by copper, probably by a mechanism not related with its B-esterase activity. Therefore, we have examples in the borderline between non-catalytic and catalytic scavengers.

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093-0650, USA Backbone conformations in hundreds of PDB deposited cholinesterase (ChE) X-ray structures show surprising similarity with typical variability of ~1Å or less among native and liganded acetylcholinesterases (AChEs; 3.1.1.7) and as low as ~2 Å between AChEs and butyrylcholinesterases (BChEs; 3.1.1.8). The largest backbone deviations are observed in their covalent conjugates with organophosphate (OP) inhibitors. Those deviations are likely to influence approach, binding and reaction efficacy of nucleophilic oxime reactivators of ChEs the only true antidotes of OP intoxicated individuals and therefore need to be considered in structure based design of improved oxime antidotes. We developed a novel, reference point based principle for overlay-independent pairwise comparison of liganded and non-liganded Cα conformations from respective PDB structures and encoded it in JAVA based computer algorithm for quick analysis. Comparisons are based on differences in distances between each Cα pair based on differences in the angle between center of mass, reference point and each of Cα in the comparison, revealing a subset of Cα in two structures that maintains their relative positions in the 3D space best and that can be used as tethering points for overlay of compared structures. Using NanoPro (Nanome Inc.) VR software, we visualized results of pairwise structure analyses creating .pdb format 3D graphs to identify interaction matrices between amino acids revealed upon ligand binding. Structure comparisons will be paralleled to OP inhibition and oxime reactivation parameters for some of analyzed ChE-OP-oxime systems to emphasize the importance for complete molecular target template characterization in the structure based antidotes design.

Notwithstanding clinical effectiveness evidences continue to suggest benefit from the acetylcholinesterase inhibitors (AChEIs) in alleviating Alzheimer’s disease (AD) symptoms, these drugs do not appear to delay or prevent the underlying neurodegeneration. In this context, novel prospects are offered by the strategy of developing single chemical entities able to modulate multiple targets, i.e. the multi-target-directed ligands (MTDLs). On this basis, several multifunctional AChEIs have been rationally designed with the deliberate aim of enlarging their biological profiles, beyond the ability to inhibit cholinesterases. This is because it has been recognized that a balanced simultaneous modulation of multiple targets critically intertwined in AD pathological cascade can provide a superior therapeutic and toxicological profile compared to the action of a selective AChEI.[1] Building on this founding principle, we and others have developed several series of anti-AD MTDL compounds that combine cholinesterase inhibition with anti-aggregating, anti-oxidant, and anti-neuroinflammatory properties.[2] As a further step, to explore the possibility to discover new MTDLs based on inexpensive resources, we have developed a series of MTDLs obtained by properly modifying constituents from the cashew nut shell liquid (CNSL), a waste from cashew nut processing factories.[3] Such hybrid compounds, obtained from renewable and inexpensive material, might be promising bio-based, sustainable MTDLs for AD drug discovery. Working in the field for almost 20 years, we should draw lessons from the past and try our best to chart innovative directions and hopefully address the scientific and societal challenges of neurodegenerative diseases.

According to the World Malaria Report, there were 216 million cases of malaria with 445000 causalties in 2016. Current anticholinesterase insecticides, such as carbamates and organophosphates, act via covalent modification of serine at the bottom of the active site. Traditional chemical insecticides are highly toxic to insect but similarly to mammals. The cysteine-targeting concept of new insecticides is focused on cysteine 447 located in the peripheral site of mosquito acetylcholinesterase. In mammalian enzyme, the cysteine residue is replaced by phenylalanine, whereas honeybees or bumble-bees have this cysteine residue protected. This approach has been proposed to overcome insecticide resistance and to develop promising environmental-friendly insecticides. The eight cysteine-targeted insecticides (succimides or maleinimides) were prepared via optimised synthetic route. The inhibitory activity of novel compounds and standards (paraoxon, bendiocarb and carbofuran) towards human acetylcholinesterase, human butyrylcholinesterase and mosquito acetylcholinesterase from Anopheles gambiae were determined using the modified spectrophotometric Ellman’s method. The potentiometric titration using acetylcholine as a substrate was used for validation of Ellman’s method. All data showed that the IC₅₀ values obtained from both methods were almost similar. Human butyrylcholinesterase was used as common off-target for acetylcholinesterase inhibitors, and no inhibitory effect was determined. The binding mode of the inhibitors was determined using the rapid dilution assay. Pyridinium maleimides were found with excellent efficacy towards mosquito acetylcholinesterase in contrast to the human enzyme and with significantly improved selectivity index compared to paraoxon. Despite some limitations, we believe that specific optimisation of the structure of molecule connected to maleimide moiety may lead to the development of novel promising insecticides. This work was supported by Ministry of Health of the Czech Republic (no. 16-34390A) and University of Defense (Long-term organization development plan Medical Aspect of Weapons of Mass Destruction).

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus. Among them Aflatoxin B1 (AFB1) is the most frequent type in nature and the most carcinogenic and hepatotoxic for mammals. AFB1 is also inhibitor of the enzyme acetylcholinesterase (AChE) and, therefore, a potential chemical and biological warfare agent, as well as its metabolites. In order to investigate this, we performed inedited theoretical studies on the interactions of AFB1 and its metabolites inside the catalytic and the peripheral anionic sites (CAS and PAS) of human acetylcholinesterase (HssAChE), to verify their stability, suggest the preferential ways of inhibition, and compare their behavior to each other. Molecular docking, molecular dynamics and MM-PBSA calculations for the systems HssAChE/AFB1-metabolites, on both sites were performed. All the metabolites presented negative values of interaction energies in comparison to AFB1. This suggests that they can be better inhibitors of HssAChE. Also, the energy values obtained for the CAS were lower than for the PAS for all metabolites, suggesting that they may preferentially bind in the CAS and come closer to the active site. This behavior is different from the experimentally observed for AFB1, pointing to a different way of inhibition for its metabolites.

Acetylcholinesterase (AChE) is a key enzyme of the Central Nervous System (CNS) hydrolyzing the neurotransmitter acetylcholine. By targeting AChE, OPNA and organophosphorus pesticides irreversibly inhibit the cholinergic transmission leading to a certain death if untreated. The current treatment available in the French army consists of an auto-injector containing a methanesulfonate salt of 2-PAM for AChE reactivation, an anticholinergic drug, atropine and avizafone, a prodrug of diazepam for limiting convulsions. However, this treatment displays major drawbacks in terms of CNS bioavailability, restricted spectrum action and effectiveness. The aim of this project is to develop a new class of more efficient human nerve agent-inhibited acetylcholinesterase. We designed, synthesized and evaluated a new class of bifunctional uncharged hybrid reactivators composed of a 3-hydroxypyridinaldoxime linked to a tacrine derivative. The in vitro efficacy of this reactivators has been assessed. We show that this new class of reactivators outperform HI-6 in restoring the human AChE activity inhibited by VX, sarin, tabun and paraoxon. By X-ray crystallography, we have been able to observe some of these new hybrids inside of the catalytic site of hAChE and TcAChE.

Human butyrylcholinesterase (hBChE) is well-known stoichiometric scavenger in case of organophosphorus (OP) intoxication. However, its major limitation lies in binding of only one OP moiety per hBChE molecule and thus necessity of its very high dosage prior or post intoxication. This issue might be resolved by use of hBChE reactivators that could cleave irreversibly bound OP moiety from the enzyme active site and restore its scavenging function. This concept has been called pseudo-catalytic scavenger. Within our contribution, we would like to present bisquaternary heteroaromatic compounds that are butyrylcholinesterase reactivators and might act as potential pseudo-catalytic bioscavengers. Recently, we have prepared and evaluated over 20 novel compounds that displayed better hBChE reactivation activity than clinically used reactivators.

The inhibitors for acetylcholinesterase (AChE), an enzyme hydrolyzing acetylcholine in cholinergic synapses, have been used for the treatment of Alzheimer's disease (AD). Alkaloids inhibiting AChE activity are commonly found in traditional Chinese medicine (TCM), e.g. gelantamine from Lycoris radiata, berberine from Coptis chinensis, huperzine A from Huperzia serrata. Many of these alkaloids also show regulatory role on inflammation, including the suppression on neuro-inflammation. Here, we aimed to reveal the possible relationship of these alkaloids in having both anti-inflammation and anti-AChE properties, in particular the role of which in “cholinergic anti-inflammatory pathway (CAP)”. A compound database containing 1,500 alkaloids from 113 kinds of TCM was developed. By molecular docking, the database was probed for AChE-inhibitory effect. Over 200 alkaloids showing AChE binding effect were further tested by its activities in inhibition of AChE, as well as in LPS-induced inflammatory responses. Thus, the current results could provide a good foundation for further research and development of TCM alkaloids on AD treatment.

Background: The level of pro-inflammatory Cluster of Differentiation 68 (CD 68) could be beneficial for examination as a biomarker for identifying cartilage or knee tissue degradation in joint problems. Because Cluster of Differentiation 68 appears to be linked to cell damage in the injury area, its measurement may be an effective and sensitive tool for detecting the early development of knee osteoarthritis (KOA) in people at risk for knee osteoarthritis.Aim of the study: The study aimed to evaluate which type of PRP (Pure-PRP and leukocyte-PRP) are suitable for patients with KOA via assessing the levels of serum CD 68 concentration.Materials and Methods: Serum Cluster of Differentiation 68 level was computed using ELISA kits. The experimental study comprises 21 pure-platelet-rich plasma (P-PRP) injections,11 leukocytes platelet-rich plasma (L-PRP) injections, and  16 control groups. Ranged from 35- 75 years old. All patients with diabetes mellitus, autoimmune disease, and severe knee osteoarthritis were excluded from this study. The period of the study was between November 2021 to June 2022. This study assessed other factors such as age, sex, family inheritance, and body mass index (BMI). The level of CD 68 was measured in the serum before and after the injection for six weeks.Results: The level of the study showed CD 68 elevated before injection in patients with knee osteoarthritis. A significant decrease of CD 68 (P< 0.01-P<0.001) in the serum concentration after injection as compared to before injection. However, the concentration was significantly higher than the control.Conclusions: In conclusion, both P-PRP and L-PRP demonstrated anti-inflammatory properties. Patients in both groups experienced a significant decrease in CD 68 serum levels, however, the P-PRP was more effective than the L-PRP.

To bypass the resistance to conventional chemotherapy, attention is paid to the inhibition of alternative targets such as members of the DNA damage response pathway. In the present study, we performed a three-step virtual screening of potential ATR inhibitors followed by evaluation of antiproliferative and chemosensitizing properties of selected compounds in vitro on a panel of cancer cell lines. According to pharmacophore resemblance to standard ATR inhibitor VX-970, a total of 17 compounds were purchased and tested. Among those 17 compounds, two proved antiproliferative efficacy in monotherapy, whereas ten compounds were effective in cisplatin co-treatment on the panel of ten different human cell lines.

Introduction: The motivation of donors to donate substances of human origin, such as blood and its components, bone marrow, gametes, and others, is influenced by various factors, such as sociodemographic characteristics, motivational factors, and the level of social competence.Aim: To describe and analyse the motivation of donors to donate blood and blood components in relation to sociodemographic characteristics and motivational factors at the Blood Centre of the University Hospital Ostrava.Design and Methods: Quantitative research based on an anonymous questionnaire survey, followed by inferential analysis of bivariate data. The self-designed questionnaire included 13 questions focusing on motivation to donate, donation experience, anonymity in donation, and sociodemographic data (gender, age, education, size of place of residence, monthly household income, and type of profession).Results: Six of the nine motivational factors were found to be statistically associated with sociodemographic characteristics. Age influenced attitudes towards commemorative medals (p=0,013). Gender was significant for four factors: the publication of donor lists (p=0,050), special health insurance (p=0,003), media coverage of donor and recipient stories (p=0,000), and more targeted information campaigns (p=0,013). Education was associated with declared donor preference in the event of an organ shortage (p=0,010) and with the publicity of donor lists (p=0,048). Household income was significant in relation to commemorative medals (p=0,000) and declared donor preference in cases of organ shortage (p=0,003). The results of our research supported the Technology Agency of the Czech Republic (TA CR) research study No. TL05000144, which formulated, for example, basic principles of compensation for donation in the Czech Republic and fundamental recommendations for recruitment campaigns targeting living body part donors.Conclusions: Motivating donors to donate human substances plays a key role in sustaining the health system and saving lives. Understanding individual donor characteristics and preferences is important for developing motivational strategies to encourage new and existing donors to donate. Our research findings contribute to the targeted sustainability of blood and blood component donation programmes.

Rivastigmine is a pseudo-irreversible cholinesterase inhibitor used for therapy of Alzheimer´s disease and non-Alzheimer dementia syndromes. In humans, rivastigmine can cause significant gastrointestinal side effects that can limit its clinical use. The aim of this study was to assess the impact of rivastigmine on gastric motor function by means of electrogastrography (EGG) in experimental pigs. Six experimental adult female pigs (3-month-old; mean weight 30.7±1.2 kg) were enrolled into the study twice and created two experimental groups. In group A, a single intragastric dose of 6 mg rivastigmine hydrogen tartrate was administered in the morning to fasting pigs before EGG recording. In group B, rivastigmine was administered to overnight fasting animals in a dietary bolus in the morning for 7 days (6 mg per day). On day 8, an intragastric dose of 12 mg rivastigmine was given in the morning to fasting pigs before EGG. EGG recording was accomplished by means of an EGG standalone system. Recordings from both groups were evaluated in dominant frequency and EGG power (areas of amplitudes). In total, 1,980 one-minute EGG intervals were evaluated. In group A, basal EGG power (median 1290.5; interquartile range 736.5-2330 μV²) was significantly higher in comparison with the power of intervals T6 (882; 577-1375; p=0.001) and T10 (992.5; 385-2859; p=0.032). In group B, the dominant frequency increased significantly from basal values (1.97±1.57 cycles per minute) to intervals T9 (3.26±2.16; p<0.001) and T10 (2.14±1.16; p=0.012), respectively. In group B, basal EGG power (median 1030.5; interquartile range 549-5093) was significantly higher in comparison with the power of intervals T7 (692.5; 434-1476; p=0.002) and T8 (799; 435-1463 μV²; p=0.004). In conclusion, both single as well as repeated intragastric administration of rivastigmine hydrogen tartrate caused a significant decrease of EGG power (areas of amplitudes) in experimental pigs. EGG power may serve as an indirect indicator of gastric motor competence. These findings provide a possible explanation of rivastigmine-associated dyspepsia in humans.

Preclinical studies in experimental pigs are carried out mostly under general anaesthesia. Ketamine is commonly used for introduction to anaesthesia. However, concerns exist, whether ketamine, an NMDA-receptor antagonist, influences gastric motor function. The aim of this study was to investigate porcine gastric myoelectric activity by means of electrogastrography (EGG). Seventeen female animals (mean weight 36.2±3.8 kg) were enrolled. Drugs used as an introduction to anaesthesia were: Group A (n=5): medetomidine 0.1 mg/kg i.m.; butorphanol 0.3 mg/kg i.m.; midazolam 0.3 mg/kg i.m.; Group B (n=6): azaperon 2.2 mg/kg i.m.; Group C (n=6): ketamine 20 mg/kg i.m.; azaperon 2.2 mg/kg i.m., all groups followed by i.v. propofol (repeated one-mL-boluses, 10-12 mL in total). EGG recording started 15 min. after the introduction administration and lasted 30 min. Results were evaluated as dominant frequency of gastric slow waves (DF) and EGG power (areas of amplitudes). In total, 510 one-minute EGG intervals were assessed. DFs were (mean ± standard deviation): 1.4±0.4 (Group A), 1.3±0.3 (Group B) and 0.2±0.1 cycles per min. (Group C). The difference between group C and groups A and B was statistically significant (p<0.001). Median power (IQR) was 0.13 (0.02-0.44; Group A), 0.13 (0.03-0.54; Group B) and 0.30 V^2 (0.07-1.44; Group C). The difference between groups A and C was of borderline significance (p=0.066; type 2 error beta 0.295). In conclusion, ketamine, even in a single low-dose, affected myoelectric function of the porcine stomach. Therefore, it should be avoided in gastrointestinal motility studies in experimental pigs.

The aryl hydrocarbon receptor (AhR) is a well-known cellular sensor of xenobiotics and major transcription regulator of xenobiotics-metabolizing enzymes. Recent studies have indicated that AhR is also important for physiological immunological functions of barrier organs, such as skin, gut, and lung. Nevertheless, its functions in epithelial cells of barrier organs are far less explored. Alveolar epithelial type II cells (ATII), also known as type II pneumocytes, are important regulators of functions of alveolar epithelium, which contribute to its regeneration, and production of surfactant. Surfactant lipids and proteins, which cover alveolar epithelium, both reduce surface tension and provide protection to pneumocytes. Here, we studied potential role of the AhR in the production of surfactant in a human model of ATII cells, A549 cell line. We have used AhR wild type and AhR-deficient A549 cells, in order to compare their capacity to express surfactant proteins, synthesize surfactant phospholipids, and produce surfactant layer, when cultivated at air-liquid interface (ALI). We then evaluated presence of lamellar bodies, as functional markers of differentiated ATII cells. Our results show that cells lacking AhR have an altered pattern of ATII markers, as compared with wild-type cells. These results suggest that toxicants activating and/or inhibiting AhR might potentially contribute to disruption of ATII cell functions and thus alter their role in the maintenance of lung homeostasis.

Tuberculosis (TB) is one of the top 10 causes of death worldwide from a single infectious agent. The World Health Organization (WHO) estimated 10 million new cases and 1.5 million deaths from TB in 2020 (1). Some strains of mycobacteria causing TB show numerous resistances to first-line drugs (isoniazid /INH/ and rifampicin) and to second-line drugs (fluoroquinolones, amikacin, bedaquilin, etc.). The development of new anti-TB drugs with new mechanism of action is necessary to improve TB therapy and to fight against resistant TB as well.
We screened our in-house library of small molecules for their therapeutic potential and identified compound K1297 with good anti-TB activity with minimum inhibitory concentration MIC₉₉ = 4 μM against H₃₇R_v strain (for comparison, MIC₉₉ (INH) = 0.5 μM). The main structural motif of this molecule is purine scaffold, which was modified and functionalized to elucidate the structure-activity relationships (SAR) and to identify derivatives with low toxicity and higher efficiency than the initial hit K1297. The effect of individual structural fragments on in vitro antimycobacterial activity, toxicity and selectivity of action have been evaluated. Finally, derivatives with optimized activity/toxicity ratio have been found and their pharmacokinetic profile and in vivo efficacy will be evaluated.

Nerve agents are organophosphorus compounds (OPs) with very potent toxicity due to their irreversible inhibition of the essential enzymeacetylcholinesterase (AChE; EC 3.1.1.7), which is primarily important in the control of neurotransmission in synapses. The related enzyme butyrylcholinesterase (BChE; EC 3.1.1.8) is inhibited by OPs aswell. Compounds with an oxime group act as reactivatorsof the inhibited AChE by the nucleophilic displacement of OP moiety from the enzyme’s catalytic serine and are used as a pharmacological treatment after OP poisoning. However, restoration of the AChE activity is directly related to the oxime structure and standard oximes in medical practice are not equally effective for various OPs (1). It was demonstrated that the chlorine substituent reduce the pKa of the oxime group compared to the non-substituted analogues and result in the higher formation of oximate anion which can be correlated to their increased reactivation ability (2). In this work, the synthesis and thorough evaluation of fluorinated and chlorinated oximes is presented. Firstly, their stability and oximate forming properties (pKa) were determined. Further, the evaluation comprised testing for affinity for human recombinant AChE and human purified plasmatic BChE, the oxime’s efficacy in reactivation of sarin-, cyclosarin-, VX-, tabun-inhibited AChE or BChE. Finally, the antidotal potential of the lead halogenated oxime was tested in mice exposed to sarin and cyclosarin (3).

Tuberculosis (TB) is a transmissible infectious disease caused by the intracellular bacteria, Mycobacterium tuberculosis (MtB), (1) which is currently one of the top 10 leading causes of death in low and middle-income countries (2). During 2020, 5.8 million patients were diagnosed with TB, 1.5 million of them died. In that year, 150 thousand patients were infected with drug-resistant TB strain.1 M. tuberculosis can quickly develop resistance against anti-TB regimens, and if not cured adequately, it can evolve into MDR-TB (multidrug resistant TB) and XDR-TB (extensive-drug resistant TB) (1). Therefore, there is a critical need to develop new chemotherapeutic agents with new mechanism of action to fight against a growing public health menace caused by cross-resistant TB strains (3).

We screened our in-house library of small molecules for their potential antimycobacterial properties identifying compound K1827 with excellent antimycobacterial in vitro activity against M. Kansasii (MIC₉₉ = 0.25 μM, which is more than 100 times more efficient that INH) and moderate activity against M. tuberculosis H37Rv (MIC99 = 32 - 64 μM, for comparison MIC99 of INH = 0.5 μM). This core scaffold of K1827 is pyrimidine that was functionalized to develop derivatives with higher activity against M. tuberculosis, better safety profile, and to determine the structure-activity relationships in the series. The effect of individual structural fragments on in vitro antimycobacterial activity, toxicity and selectivity of action have been evaluated and will be discussed within our contribution.

Polyaromatic hydrocarbons (PAHs) are a diverse group of environmental and dietary contaminants. PAHs are primarily metabolized through the aryl hydrocarbon receptor (AhR)-regulated enzymatic pathway. Both parent PAHs and their metabolites may exert various types of toxicity in target cells and tissues. In addition to their well-known genotoxic (and carcinogenic) effects, PAHs may alter numerous additional cellular events, including cell metabolism. In this project, we study whether strong AhR ligand benzo[k]fluoranthene (BkF) can modulate genes associated with glucose and lipid metabolism, as well as proteosynthesis, which is essential metabolic process performed at high energetic cost. We used an in vitro model of immortalized human hepatocytes, MIHA cell line. We observed a significant suppression of proteosynthesis induced by BkF in MIHA cells, as determined by the SUnSET method. We also observed a moderate deregulation of several genes associated with further evaluated metabolic processes. Based on this, we continue to evaluate possible impact of strong AhR ligands, such as BkF, on cellular metabolism, as well as functional role of this transcription factor in the complex effects of PAHs on cellular metabolism.

It is known that cholinesterases show homotropic pseudocooperative effects: their activity at millimolar substrate concentrations is higher than expected by simple saturation kinetics and they are strongly inhibited at the submolar concentrations. However, we have reported that the anionic site directed inhibitors tetramethylammonium and tetraethylamonium too, increase the activity of human butyrylcholinesterase. At that time, the same phenomenon could not be shown for the horse counterpart. Here, it was searched for other putative activators among often used compounds in cholinesterase research. Indeed, imidazole significantly  increase the activity of human enzyme, but also its atypical form and the horse enzyme. On the other hand, 2-PAM shows a certain degree of activation with both human enzymes, but inhibits the horse BChE in a classical competitive manner. To avoid substrate activation, the experiments were performed at around 50 micromolar starting substrate concentrations and were followed by its completion in the presence of different modulator(s) concentrations. Subsequently, the effect of 2-PAM on the phosphorylation by DFP was studied, since the bottom of the active site does not differ in these three enzymes. It seems that the distinctive action of activating agents on the wild type, the atypical human and horse BChE is a consequence of differences in the dynamics of the acylation loop at the active site entrance, rather then the composition of the enzyme’s peripheral anionic site.

Alzheimer´s disease (AD) is a progressive and degenerative neurological disorder resulting in memory loss and cognitive decline. The severity of AD dementia was found to correlate with the extent of the cholinergic loss and acetylcholine (ACh) depletion. In brain synapses ACh can be hydrolyzed by two cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which were found in neurons and glial cells as well as in AD neuritic plaques and tangles. AChE is the prevalent enzyme in the healthy brain, while BChE is considered to play a minor role in the regulation of synaptic ACh levels. However, in AD advanced stages, AChE activity is decreased while BChE activity is unchanged or even increased, making both ChEs stimulating targets for the treatment of AD.  Current AD therapy is based on AChE inhibitors, although they have very modest clinical effects in treating the symptoms of the disease and are unable to halt disease progression. Oxidative stress (OS) and mitochondrial dysfunction are also considered critical factors in AD pathogenesis. As a result, targeting mitochondrial oxidative stress (OS) in the prodromal phase of AD to slow or prevent the neurodegenerative process and restore neuronal function is thus viewed as a valid therapeutic approach. As part of our drug discovery program focused in oxidative stress-related diseases, and following a multi-target strategy, new mitochondriotropic antioxidants based on natural scaffolds acting as dual and bifunctional cholinesterase inhibitors have been developed. The results will be reported in this communication.

Since cancer is one of the most common medical causes of death worldwide, its prevention remains the most promising strategy for reducing its incidence and mortality. The study of the potential protective effects of natural compounds, that could be used in health protection, is therefore very important. Plants represent a source of secondary metabolites possessing such properties and curcumin isolated from plant Curcuma longa L. with many beneficial biomedical effects is one of them.
The objectives of our study were (I) to investigate the potential DNA-damaging/DNA-protective effects of curcumin using DNA topology assay; (II) to determine the possible mechanism of its protective activity using cell-free methods and (III) to evaluate its potential anti/genotoxic effect by comet assay on human lymphocytes.
Curcumin displayed DNA-protective effects against ROS induced by ferrous ions on plasmid DNA that increased in a concentration-dependent manner. Higher doses showed significant reducing power and DPPH radicals scavenging activity. Curcumin did not exhibit any chelating activity. Moreover, genotoxic effects of curcumin were observed in human lymphocytes. On the other hand, using the same concentration range, curcumin exhibited ability to protect human cells against H₂O₂ mostly in the higher concentrations. Our results represent basis in examining the effects of curcumin on biological systems, and therefore further research is needed to explain its dual effects.