Fulltext search in archive

Background: Femoroacetabular impingement syndrome (FAI) is a complex, often post-traumatically developing impairment of the hip joint. It is characterized by ambiguous symptomatology, which makes early diagnosis difficult. Aim: The study was conducted to evaluate the applicability of a triaxial gyroscopic sensor in routine practice as an additional indication criterion for operative versus conservative treatment procedures. Methods: Ninety-two patients were included in the experimental retrospective study and 62 completed the examination. All patients signed informed consent. A gyroscopic sensor was placed on the right side of the pelvis above the hip joint and patients walked approximately 15 steps. Data were also evaluated while the patients climbed stairs. A complete clinical examination of the dynamics and physiological movements in the joint was performed. The data measured by the gyroscopic sensor were processed using differential geometry methods and subsequently evaluated using spectral analysis and neural networks. Results: FAI diagnosis using gyroscopic measurement is fast and easy to implement. Our approach to processing the gyroscopic signals used to detect the stage of osteoarthritis and post-traumatic FAI could lead to more accurate detection and capture early in FAI development. Conclusions: The obtained data are easily evaluated, interpretable, and beneficial in the diagnosis of the early stages of FAI. The results of the study show that this approach can lead to more accurate and early detection of osteoarthritis and post-traumatic FAI.

Agaritine, a naturally occurring amino acid and phenylhydrazine derivative found in mushrooms of the genus Agaricus, has gained attention due to its potential impact on human health. The presence of the hydrazine moiety in the structure of agaritine plays a crucial role in its toxicological properties. It has raised concerns due to its high reactivity as chemical radicals. Therefore, research is commonly focused on the potential health risks of agaritine and its possible role as a pro-carcinogenic agent. However, some studies did not provide evidence of agaritine's toxicological effects. Therefore, further research is needed to understand agaritine's mechanisms of action and its safe consumption levels in humans. This review aims to provide an overview of current knowledge surrounding agaritine and its potential health risks.

Acetylcholinesterase (AChE) reactivators (oximes) are generally used as antidotes in case of nerve agent poisoning. Because of their affinity to AChE, they may also act as weak inhibitors of AChE. Their inhibition potency against AChE was determined by an in vitro method based on the interaction between AChE and oxime reactivator in the concentration range 10^-1 to 10^-8 M. We used eel AChE for these assays. We found that AChE inhibition strongly depends on the oxime structure. The aim of the present study is to describe the structure-activity relationship (SAR) between oxime structure and inhibition of AChE. AChE reactivators tested include both monoquaternary and bisquaternary structures with the oxime group in different positions on the pyridine ring and with changes in the connecting linker in the case of the bisquaternary compounds.We found AChE inhibition to be highest in bisquaternary oximes that have a longer linker length and have the oxime group in the ortho position. Increased AChE inhibition in monoquaternary oximes was highest when the meta position was occupied by the oxime nucleophile. In addition, different substituents in the connecting chain (in case of bisquaternary oximes) modulated their inhibition potency.

Phosphonylated butyrylcholiesterase (BChE) is a marker of exposure to organophosphorus compounds, including nerve agents and pesticides. In cases of poisoning with nerve agents, it is important not only to establish the fact of poisoning, but also to give a quantitative estimate. The most common quantitative characteristic is BChE inhibition. We developed a highly sensitive method for the quantification of BChE inhibition by Russian Vx (VR) by mass spectrometry. For model experiments we used donor human blood plasma exposed to VR at concentrations of 1‒100 ng/ml. Butyrylcholinesterase was selectively isolated from plasma by immunoprecipitation and then subjected to enzymatic hydrolysis with pepsin. The hydrolysate was analyzed by HPLC-MS/MS using MRM mode, which allowed determination of the VR-modified nonapeptide  FGESAGAAS (m/z 930 Da) at a very low level of VR (1 ng/mL). To measure the inhibition of BChE, an excess of VR is added to one sample, and the nonapeptide peak area is considered to correspond to 100% inhibition. The inhibition of BChE in samples containing different concentrations of VR are determined by ratio of the nonapeptide peak area in each specific  sample to that at 100% inhibition. BChE inhibition,% = (S₉₃₀/S₉₃₀100%)*100 It was found that the VR-modified nonapeptide peak area is linearly related to VR concentration. The BChE inhibition measured by mass spectrometry was consistent with the results of Ellman’s assay (R²≥0.98). The advantages of the proposed approach over Ellman’s assay include the possibility of quantification of inhibition at low doses of nerve agent and lack of necessity to construct calibration plots.

Introduction: Thalassemia is a healthcare challenging disease all over the world. It imparts a great burden on patients’ families and healthcare institutions. Scientists focus on new aspects to overcome these challenges and increase patient tolerance of disease complications. This study aims to quantify β-Hydroxybutyrate Dehydrogenase BHBDH activity in thalassemia patients compared to the control group and their correlation with the patient's demographic characteristics.Methods: To do so, serum was collected from patients and the control group and analyzed biochemically for targeted laboratory tests. We determined β-Hydroxybutyrate Dehydrogenase from normal human serum using biochemical molecular techniques.Results: The results showed that BHBDH activity is significantly higher in the patients compared to the control group regardless of age, sex, or marital status. The results confirmed that enzyme activity and the purification folds were (0.0214 U/ml) and (51.7) respectively for the partially purified enzyme. Furthermore, the proportional molecular weight of the incompletely isolated β-Hydroxybutyrate Dehydrogenase was (125.8±0.5 kDa) using gel filtration chromatography. The comparative molecular weight of the subunit of partially isolated β-Hydroxybutyrate Dehydrogenase was (32.1±0.5 kDa) using SDS–PAGE.Conclusion: we demonstrate that BHBDH enzymatic activity is higher than control and this could be a prognostic or diagnostic tool in thalassemia patients.

Organophosphorus nerve agents are highly toxic compounds which pose a threat worldwide. These compounds induce toxicity by covalently binding to the active site serine of acetylcholinesterase, which results in inhibition of the enzyme. Without functional acetylcholinesterase, the levels of the neurotransmitter acetylcholine in neuromuscular junctions rise quickly, causing overstimulation of the nervous system, which will culminate in death if not treated. Current treatments rely on small molecules to interact with inhibited enzyme to disrupt the covalently bound phosphorus moiety at the active site. The most effective molecules incorporate a pyridinium oxime which acts via direct nucleophilic attack on the phosphorus to achieve reactivation of the enzyme. These compounds have limited effectiveness because the charged portion of the molecule does not allow them to cross into the central nervous system where acetylcholinesterase inhibition is most harmful. The results of studies that characterized a small molecule reactivator (4-amino-2-((diethylamino)methyl)phenol [ADOC]) that does not incorporate an oxime but is capable of reactivating nerve agent-inhibited enzyme as well as or better than current treatments have been used to inform the design of additional novel compounds. This study describes the in vitro characterization of these novel compounds as reactivators of phosphonylated human acetylcholinesterase.

Human butyrylcholinesterase (BChE) is a stoichiometric bioscavenger of toxic organophosphates. It can be used as an antidote to protect acetylcholinesterase, and is a protein of choice for development of detoxification biocatalysts for clinical applications. Despite the number of different monomeric structures of recombinant human BChE obtained to date, all attempts to obtain an atomic structure of the natural glycosylated tetrameric BChE were unsuccessful. Here, we present for the first time the 3D structure of the natural tetrameric form of human butyrylcholinesterase, obtained by Cryo-EM technique at a final resolution of 8.8Å. The tetramer has a C2 symmetry, with all subunits arranged as a “propeller-like” tetramer. This is in contrast with previous “flat” model of subunits arrangement in tetramer. Cryo-EM structure shows that the two opposite BChE subunits are placed higher (or lower) the plane of the other two subunits. Despite glycan chains were obscured in the electron density due to their relative disoder, they could be modeled based on the positions of the residues anchoring these glycans. The electron density allowed to distinguish that C-terminal tails of all the subunits interact with each other and form a helix around the PRAD-peptide, supporting rigidity of the tail. The tail is situated in the center of the tetramer and is oriented nearly perpendicular to the tetramer “plane”.It was also observed that the subunits in the tetramer have different contacts with neighbouring subunits. This allows to consider the tetramer as a dimer of dimers which is additionally strengthened by the C-terminal tail interactions.

Based on many reports, an unmistakable link probably exists between diabetes mellitus and COVID-19. A major predisposing factor determining severity and mortality of COVID-19 is diabetes mellitus, diabetic patients were shown to be at higher risk for developing severe COVID-19 disease than non-diabetics; many recent studies reported a striking prevalence of DM in those diagnosed with COVID-19. Accordingly, antidiabetic drugs can possibly impact the clinical course and / or the outcome of this infection, either by alleviating diabetes-associated symptoms, minimizing its complications, or by mitigating or aggravating COVID-19 disease by effects independent from their direct antidiabetic effects. Several antidiabetic drug classes were shown to have varying effects, like blocking viral entry to cells, as well as having immunomodulatory, anti-inflammatory, antifibrotic, or cardioprotective effects; such effects could prove beneficial for COVID-19 patients. On the other hand, some antidiabetic agents may have adverse effects that aggravate patients’ condition like hypoglycemia, fluid retention, increased weight or lactic acidosis, which require special consideration in patient management. Some of the drugs were found in observational studies to either reduce mortality from COVID-19 or pose no harm, but more solid evidence from clinical trials is still lacking.

The prophylactic antidotes are pharmaceutics which can prevent the intoxication of the human body or may improve its prognosis if administered before the exposure to a toxic substance. Pyridostigmine chloride, a reversible acetylcholinesterase(AChE) inhibitor, is used as a prophylactic antidote which may prevent the organism from organophosphorus poisoning (OP). This paper is focused on searching new AChE inhibitors that might be used in prophylactic treatment of OP intoxications. The aim of this study was to prepare 15 symmetric bisquaternary inhibitors and to evaluate their inhibition ability (IC50) via a standard in vitro method. The but-(2E)-en-1,4-diyl-1,1'-bis(4-(4-nitrobenzyl)-pyridinium) dibromide was selected, on the basis of IC50 value, the most potent AChE inhibitor.

The mechanism of toxicity from acute exposure to organophosphorus toxicants (OP) is understood.  Thousands of publications have confirmed that AChE inhibition results in muscle weakness and respiratory failure.  AChE activity returns to normal levels within one month, but symptoms can persist for a lifetime.  For example, people exposed to sarin in the 1995 Tokyo subway attack still have adverse symptoms 23 years later.  Farmers and sheep dippers exposed to OP pesticides have an elevated risk of psychiatric disorders and suicidal behavior.  Epidemiology studies show an association between OP exposure and Alzheimer’s disease and Parkinson’s disease.  We propose a mechanism to rationalize these observations independent of cholinesterase inhibition.  Mass spectrometry analysis of OP-treated proteins shows that OP make stable adducts on tyrosine and lysine.  Furthermore, we have mass spectrometry evidence that OP-lysines promote crosslinks between proteins.  The crosslinked proteins are visualized as protein aggregates on SDS gels and Western blots.  Mass spectrometry has identified γ-glutamyl-ε-lysine and aspartyl-ε-lysine isopeptide bonds between crosslinked peptides.  We propose, but have not yet proven, that isopeptide crosslinked proteins form stable, insoluble aggregates in the brain, similar to the protein aggregates found in Alzheimer’s, Parkinson’s, and prion diseases.  In summary, we propose that chronic neurotoxicity from OP exposure is initiated by OP-lysine formation followed by protein aggregation.  Our proposed mechanism could apply to a variety of compounds and lead to an understanding of neurotoxicity induced by many chemicals.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, which is still not fully understood. Crucial roles in the pathogenesis play Th2 immune response dysregulation and epidermal barrier alterations. Defects of this skin barrier have been considered in the initial step of AD development. Inflammatory skin conditions AD have a negative wide-ranging impact on a patient’s life quality. However, testing methods and/or treatment options for this disease are unsatisfactory nowadays.
To study the anti-inflammatory effect of substances on AD pathogenesis we established inflammatory models by stimulating HaCaT cells with tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). We designed two different treatment models (pre- and post-treatment) to mimic keratinocytes ̓ inflammatory conditions of AD that simulate relapse and therapy. HaCaT cells are a suitable model to follow the release of inflammatory mediators interleukin-6 (IL-6) and interleukin-8 (IL-8) in response to TNF-α and IFN-γ treatment. The AD-like models were verified by using natural flavonoid quercetin and synthetic glucocorticoid dexamethasone. Both substances should be associated with the inhibition of inflammatory cytokines production in the skin. Based on the data obtained, these models could open the way for screening of new preventive or therapeutic AD agents. The details of the experiments will be discussed in our poster contribution.

Despite its unprecedented efficacy against some cancers, anthracycline cardiotoxicity represents the main limitation of its clinical use. Its’ mechanisms are elusive, but quite recently TOP2B was addressed as a possible target in cardiomyocytes (1). This study introduces the putative TOP2B selective inhibitor XK469 (2) as a potential cardioprotective agent in the management of anthracycline cardiotoxicity. Its’ potential was compared with the cardioprotection of the only approved cardioprotective drug dexrazoxane (DEX, ICRF-187) (3). Initially, the selectivity of XK469 and the character of TOP2 inhibition were re-examined in vitro using isolated TOP2A and TOP2B isoforms and the detection of DNA-TOP2 covalent complexes. Contrary to XK469 original presentation, we found it rather non-selectively targets both enzyme isoforms and does induce only very little covalent complexes. Consequently, in vitro pilot results suggested that XK469 was protective against daunorubicin (DAU)-induced cardiotoxicity in vitro in a slightly higher concentration than DEX. Nevertheless, in vivo XK469 was not cardioprotective in both acute and chronic settings. This can be partly explained by marked differences in the pharmacokinetics of the two agents. Modified incubation protocol in neonatal cardiomyocytes reflecting the longer half-life of XK469 revealed an increased trend in its toxicity. Thus, despite the promising characteristics of XK469, the cardioprotective ability of XK469 was not confirmed.

The series of bisquaternary oximes with ortho-positioned chlorine moiety was designed, prepared and evaluated. The novel compounds exhibited valuable pK_a properties [1] with improved in vitro reactivation ability of sarin, cyclosarin, VX, paraoxon- and dichlorvos-inhibited human AChE exceeding the standard monoquaternary or bisquaternary reactivators (pralidoxime, methoxime, trimedoxime, obidoxime and asoxime syn. HI-6). Additionally, some chlorinated compounds presented in vitro reactivation ability of tabun-inhibited human AChE similar to the efficiency of trimedoxime. The in vitro results were further explained by molecular docking study. The in vitro non-cytotoxic properties of novel compounds were determined with miscellaneous results. However, assessment of maximum tolerated dose highlighted that the selected chlorinated reactivator is well tolerated by mice on the level similar to the clinically or experimentally used oxime reactivators [2]. The in vivo reactivation study is in progress.

Organophosphorus compounds (OP) pose a significant threat. Administration of human butyrylcholinesterase (Hu BChE) may reduce or prevent OP toxicity. Thus, we evaluated the safety and efficacy of Hu BChE in monkeys using sensitive neurobehavioral tests while concurrently characterizing absorption and elimination in the presence and absence of high-dose soman exposure to predict time course and degree of protection. Eight young adult male cynomolgus macaques were trained on two distinct automated tests of neurobehavioral functioning. Hu BChE purified under current-Good-Manufacturing Practices (CGMP) was injected intramuscularly at 13.1 mg/kg, producing an average peak plasma value (C_max) of 28 Units/ml. The apparent time to maximum concentration (T_max) approximated 12 hours and the elimination half-life approximated 80 hours, returning to pre-administration (baseline) levels by 14 days. No behavioral disruptions following Hu BChE administration were observed on either neurobehavioral test, even in monkeys injected 24 hours later with an otherwise lethal dose of soman. Thus, Hu BChE provided complete neurobehavioral protection from soman challenge. These data replicate and extend previous results that used a different route of administration (intravenous), a different species (rhesus macaque), and a different BChE product (non-CGMP material). The addition of two sensitive neurobehavioral tests coupled with the PK/PD results convincingly demonstrates the neurobehavioral safety of plasma-derived Hu BChE at therapeutic levels. Protection against an otherwise-lethal dose of soman by a pre-exposure treatment dose that is devoid of side effects establishes a foundation for additional testing using other exposure routes and treatment times, other challenge agents/routes, or other classes of organophosphate scavengers.

Currently, the best bioscavenger candidate against nerve agent intoxication is human butyrylcholinesterase (BChE). However, the effective dose cost, estimated to about 200 milligrams of pure enzyme, remains challenging despite the production and purification progresses realized these last years. A strategy for reducing dosage and cost would be to turn this scavenging protein into a nerve agent hydrolyzing enzyme, a catalytic bioscavenger. Up to now, screening of large mutant libraries has been hindered by the restricted eukaryotic expression of active BChE. Here we present the successful prokaryotic expression of an active human BChE variant designed with PROSS, a sequence- and structure-based algorithm for the soluble prokaryotic expression of difficult proteins. The protein is easily purified with two simple chromatographic steps. Despite 47 point mutations, the enzyme presents similar enzymatic parameters than the wild-type enzyme and its active site gorge structure is identical to that of the native enzyme produced in eukaryotic systems as determined by X-ray crystallography. These data validate the prokaryotic expression of human BChE which will greatly facilitate the screening of variants with nerve agent hydrolytic properties. We have initiated animal studies to assess the protein potency (immunogenicity, pharmacokinetic and bioscavenger efficiency) and will study the production of the tetramer form. On the other hand, we are currently developing high-throughput protocols for the prokaryotic expression, purification and screening of nerve agent hydrolysis.

Human acetylcholinesterase (hAChE) is responsible for degrading neurotransmitter acetylcholine at synapses of the nervous system. Organophosphate (OP) nerve agents and pesticides inactivate hAChE through chemical modifications of the catalytic serine. The current generation of oxime antidotes is not highly efficient. Insights into the molecular structures of AChEs from various species reveal possible limitations in enhancing reactivation rates, but provide only limited information, because the structures have been obtained at cryo-temperatures. Moreover, X-ray crystallography usually cannot resolve positions of hydrogen atoms involved in proton transfer processes during reactivation. Thus, we use room-temperature X-ray and neutron crystallography to obtain structures at physiological conditions and to visualize hydrogen atoms. Several X-ray structures of native and VX and POX-conjugated hAChE in complex with oxime reactivators, RS2-170B and RS-194B have been obtained. hAChE crystallized in a unit cell (a=124.3, c=129.1 Å; P3₁) amenable to neutron crystallography. For the first time we show how RS2-170B binds in the non-modified and OP-conjugated active site gorge at room temperature. RS-194B is observed with its oxime group pointing away from the catalytic Ser203 and the reactivator is pushed out to bind at the peripheral site in the VX-modified structure. Dynamics of hAChE was probed by neutron vibrational spectroscopy to look at harmonic vibrations. POX binding induces significant changes in the acyl pocket loop conformation expelling the weakly binding RS-194B from the active site gorge completely, and the loop becomes more dynamic. We hypothesize that increased dynamics of the acyl pocket loop contributes to the POX-conjugated hAChE resistance to reactivation.

Organophosphorus compounds (OPs) irreversibly inhibit cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). There is wide variety of applications of OP compounds including warfare chemicals and pesticides. Oxime-type reactivators are used to reactivate the OP inhibited AChE and BChE. Present study was aimed to evaluate the reactivation potency of two novel oximes K456 and K733 against organophosphate inhibited AChE and BChE. Efficacy was compared with K27 and pralidoxime (2-PAM). Molecular mechanism of reactivation by the oximes is predicted by In silico method. Intrinsic toxicity of novel oximes in term of IC₅₀ and 50 % reactivation of inhibited enzymes (R₅₀) were evaluated by in vitro methods using human RBC-AChE and plasma BChE. In silico study revealed lower free binding energies, but novel oximes did not bind with catalytic anionic site of enzymes. In vitro studies showed higher intrinsic toxicity by K456 and K733 than K27 and pralidoxime. R₅₀ for human RBC-AChE were K456=203.59µM±66.96; K733= 405.55µM±67.36; K27=2.68µM ±0.98 and pralidoxime 30.71µM±5.10 (mean±SEM) respectively. No substantial reactivation in BChE was noted by tested concentration of novel oximes.  The study concludes that oximes with peripheral binding/far from catalytic anionic site are ineffective reactivators. K27 with central (inside the active gorge) binding was superior to all tested oximes.

Glyphosate [N-(phosphonomethyl) glycine] is one the world's most widely used agricultural herbicide. It allows farmers to spray a planted field, generally before the crops have sprouted, killing weeds but not the crops that will grow there. GMO critics claim glyphosate is linked to autism, cancer, gluten allergies, ‘leaky gut’ syndrome and other disorders. Concerns about glyphosate’s possible health impacts increased in 2015 after the International Agency for Research on Cancer, a research arm of the World Health Organization, classified glyphosate as “probably carcinogenic”. The ecological risk assessment indicates that there is potential for effects on birds, mammals, and terrestrial and aquatic animals. A joint panel from the World Health Organization and the Food and Agriculture Organization of the United Nations issued an summary evaluation of glyphosate in May 2016, concluding it poses no cancer risks as encountered in food and does not impact our genes. Although the European Food Safety Authority declared the evidence on glyphosate’s carcinogenicity for humans to be “very limited”, there is still some doubt as to whether all the studies have been made "lege artis" or whether they have not even been falsified.

Objectives: The purpose of this research is to identify effective treatments and promote prevention of emotional burnout among special services and military personnel. It also aims to highlight best practices and potential implementation strategies by specialists from Ukraine, Kazakhstan, Poland, UK, USA, Canada, and South Korea.Methods: The primary method utilized in this research is experimentation, employing practical psychology techniques to enhance the personal competencies of special services and military personnel. Psychological observation, conversations, questionnaires, diagnostics, and statistical analysis were auxiliary methods used to tailor emotional burnout prevention strategies specific to this group.Results: As a result, the research identifies features and specifics of the work of employees of special services and military structures, presents the main ways, and methods of treatment and prevention of emotional burnout and reveals the most effective of them for employees of special services and military personnel depending on their individual and personal characteristics. The application of emotional burnout training as a preventive measure is substantiated by the best practices and prospects of its implementation.Conclusions: The authors conclude that emotional burnout is one of the main problems of the 21st century, which concerns not only those whose activity is communication with people, but also any person who cannot regulate their emotional state. The specifics of the activities of employees of special services and military units require special professional and personal qualities, the absence of which can contribute to the formation of emotional burnout.

Acetylcholinesterase (AChE) oxime reactivators are used as antidotes to organophosphate (OP) poisoning, while butyrylcholinesterase (BChE) reactivators are suitable for pseudocatalytic uptake of OP. OP acts as irreversible inhibitors of AChE. Due to the inhibition of AChE OPs cause impairment of cholinergic functions, which can lead to the death of the organism (1). The modification of the already known structure of pralidoxime aims primarily at overcoming the problems associated with physicochemical properties. The disadvantage of quaternary pyridinium oximes, which have a permanently positive charge, is poor penetration across the blood-brain barrier (BBB), which makes them ineffective in the central nervous system (2). Introducing a substituent that could contribute to increased lipophilicity, may thus provide better penetration into BBB. At the same time, a substituent in a suitable position relative to the oxime group on the pyridinium ring can reduce its pKa value and thus lead to easier formation of the oximate anion, which is important for the ability to reactivate inhibited cholinesterase (3). The aim of this work is to synthesize oxime reactivators derived from the structure of pralidoxime, determine their stability and pK_a values. Finally, the new derivatives will be measured in vitro for their ability to reactivate OP-inhibited cholinesterases.

Organophosphorous compounds (OPs) such as nerve agents or pesticides are irreversible inhibitors of cholinesterases, namely Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE), causing cholinergic crisis (1). While inhibition of AChE can be lethal, inhibition of BChE has no adverse effects. Therefore, BChE can be used for scavenging OPs before it reaches AChE in CNS. BChE itself has no catalytic activity, but by joint administration of BChE and reactivator we can establish pseudo-catalytic bioscavenger (2). Reactivators cleave the OP moiety from the enzyme by making a covalent bond with it. Reactivators of AChE are already widely used (eg. pralidoxime, asoxime) but there are few disadvantages. Firstly, there is no broad-spectrum reactivator. Second problem is that they have low ability to pass the blood-brain barrier (BBB) due to their double-charged structure. Moreover, when the OP-ChE complex is dealkylated (so called “aging” of ChE), it is no longer possible to reactivate. That is why nowadays the research is focusing to development of reactivators of BChE as well (3).  Promising results were obtained for the novel “K-oximes” K027, K048 and K203. The aim of this research is synthesis and in vitro evaluation of monoquaternary analogues of oxime K203 and observation of the effect of charge on physicochemical properties of compounds and on biological activity.

Introduction: “Ankylosing spondylitis (AS)” is an inflammatory disorder that affects the axial skeleton,   peripheral joints, as well as entheses, resulting in significant disability. “Tumor necrosis factor-α (TNF-α)” inhibitors are regarded to be a helpful treatment for patients with active “AS”. This study aimed to investigate the effectiveness and response rate of “etanercept” in a group of patients with “ankylosing spondylitis” in Mosul, Iraq.Methods: A prospective, “open-labeled”, non-randomized 12 weeks study was undertaken on 43 participants with “ankylosing spondylitis” in the “Rheumatology unit” of “Ibn Sina Teaching Hospital”, and the diagnosis was made using the “modified New York criteria”. Participants were assessed at the outset of the study, week 4, and week 12 after receiving etanercept 50mg subcutaneously once weekly. The “Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)” was utilized to assess disease activity, while the “Bath Ankylosing Spondylitis Function Index” was utilized to assess functional status (BASFI) at baseline, 4 weeks, and 12 weeks. BASDAI 50 was used to assess the response rate.Results: Mean patients’ age was “36.6±8.47” years; men accounted for “90.7 %” of the cases, with the mean disease length being “9.6±5.90” years. A marked decrease in BASFI and BASDAI was found four and twelve weeks after commencing treatment compared to baseline (p=0.000).  “BASDAI 50 %” response was fulfilled by 42.5 % of the participants after 4 weeks and by 65%% after 12 weeks of therapy with “etanercept”. There was a marked fall in the mean ESR and CRP after four and twelve weeks of “etanercept” therapy.Conclusion: In “AS” patients,  once weekly “etanercept” 50 mg given subcutaneously for twelve weeks was an effective therapy.

Background: The degree of blood pH affects the fundamental function of vitamin D, which is to maintain calcium and phosphorus as well as many other biochemical, neurological, and behavioural activities the difference in blood pH impacts the levels of calcium and vitamin D in the blood, and it is also in charge of ensuring that calcium levels are suitable and constant.Objective: that blood pH affects the basic function of calcium and phosphorus and vitamin D, the difference in pH affects calcium and vitamin D levels in the blood and is also responsible for keeping calcium levels appropriate and stable.Methods: 7 groups composed of 35 mature female rats were divided at random.Results: After 24 hours, none of the animals died. While stress activity increased with alkaline water, vitamin D3 and calcium levels increased in the acid water group while their concentration decreased with prednisolone, while calcium levels did not change in the alkaline water group. The animals in the two groups of vitamin D3 and acidified water with or without prednisolone moved less in the open field. Along with increased oxidative stress, vitamin D3 with acid water also inhibited cholinesterase activity. The lipid profile in the blood is impacted by the vitamin D3 group in both acidic and alkaline water.Conclusion: Prednisolone reduces serum levels of vitamin D3. And that acidic water directly affects the toxicity of vitamin D3, while alkaline water significantly reduces toxicity.

Purpose: The aim of our study was to determine and compare the activity of acetylcholinesterase (AChE) in different parts of dog brain after the exposure to nerve agent sarin with or without HI-6 oxime treatment.Material and methods: Before intoxication, beagle dogs were intravenously anaesthetized and premedicated with atropine sulphate (0.01 mg/kg). Three experimental groups were established - control, sarin (0.03 mg/kg, intramuscularly, 5 min after anaesthesia onset), and sarin + HI-6 dichloride (11.4 mg/kg, intramuscularly, 30 min after sarin poisoning). Brain (amygdaloid body, head of caudate nucleus, somatosensory cortex, Amon's horn of hippocampus, hypothalamus, brain stem ventral respiratory group, and medial nuclei of thalamus) samples were taken 4 h after sarin administration. AChE activity was detected by histochemistry using the Karnovsky-Roots method and computer image analysis.Results: Sarin poisoning decreased AChE activity in all selected brain areas. HI-6 did not affect this outcome.Conclusion: HI-6 does not reactivate brain AChE in dogs when administered 30 min after sarin poisoning.

The synaptic enzyme acetylcholinesterase (AChE) terminates transmission at cholinergic synapses by rapidly hydrolysing acetylcholine. Examination of the 3D structure of AChE¹ shows that the active site is located at the bottom of a deep and narrow gorge, lined largely by aromatic residues, with its peripheral anionic site located at the top, near the entrance to of the gorge. 3D structures of AChE have been determined for the Torpedo, Electrophorus, mouse, Drosophila and human enzymes. Overall, more than a hundred crystal structures of AChEs, and of covalent conjugates and reversible complexes with various inhibitors and substrate analogues have been determined. Although the 3D structure of the enzyme itself, and of its molecular dimer, are highly conserved, subtle structural differences are seen to occur upon the binding of certain inhibitors. These changes are well correlated with molecular dynamics data, and appear to be of functional significance. Unfortunately, upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human AChE is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. Using a novel computational strategy, we designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ~2,000-fold higher levels in E. coli compared to wild-type hAChE, and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography^2,3.

This talk will briefly discuss our newest progress in drug design, discovery and development involving cholinesterases, particularly in three major therapeutic areas. (1) On the basis of our previous design and discovery of cocaine hydrolases (CocHs) engineered from human butyrylcholinesterase (BChE), we have further developed a novel, long-acting CocH form, and demonstrated the promising clinical potential of CocHs for therapeutic treatment of cocaine overdose and addiction in clinically relevant animal models. One of the long-acting CocHs is currently in the large-scale protein drug manufacturing process development. (2) It has been demonstrated that a long-acting CocH (enzyme) is capable of both completely blocking cocaine-induced physiological effects and producing the desirable anti-obesity effects. Mice on a high-fat diet gained significantly less body weight when treated weekly with 1 mg/kg enzyme compared to control mice. (3) Most recently, we have also designed and tested a new therapeutic strategy for heroin detoxification based on a detailed analysis of the cholinesterases-involved chemical transformation and functional change of heroin in the body. It has been demonstrated in our animal models that a carefully selected cholinesterase inhibitor attenuated acute toxicity and physiological effects of heroin, whereas some other cholinesterase inhibitors may actually enhance the acute toxicity and physiological effects of heroin.

The design of reactive molecules such as nerve agent antidotes is inherently challenging due to two intertwined processes imperative for their efficiency: The reversible binding of the initial non-covalent complex in a low energy conformation and the chemical reaction that proceeds via a transition state of high(er) energy. Furthermore, a structural and chemical diversity among different nerve agents and their corresponding complex with AChE complicates the design of broad-spectrum antidotes. The development of broad spectrum antidotes has proven challenging and although progress has been made, no new drugs with improved properties have been launched in several decades. Herein, we report a rational, structure-based approach for the development of broad-spectrum antidotes. Based on a hit molecule identified in a high throughput screening targeting the non-inhibited species of AChE, 18 new analogous molecules were designed and synthesized. This resulted in a set of compounds with a diversity in their potency, as desired for subsequent (quantitative) structure-activity relationship ((Q)SAR) modeling. The 18 compounds were investigated for their ability to bind to four different phosphonylated forms of AChE (i.e. human AChE inhibited by the nerve agents VX, VR, and tabun, or the substance DFP). The QSAR model was subsequently used to guide the development of a novel set of pyridinium-oxime based broad spectrum antidotes. The mechanism of reactivation of the developed antidotes has been investigated using a combination of X-ray crystallography and molecular modelling.

Freshwater planarians from Platyhelminthes, harboring a mammal-like cholinergic nervous system, have emerged as a promising in vivo model for investigating neurotoxicity. Moreover a large proportion of stem cells provide planarian an unconventional capacity of regeneration allowing for developmental disruption studies. Schmidtea mediterranea (Smed) was used as model for organophosphorus (OP) poisoning and for evaluating the efficacy of detoxifying enzymes. Acetylcholinesterase and butyrylcholinesterase from planarian (Smed-AChE and Smed-BChE) share 35% identity with their human counterpart (Hs-AChE and Hs-BChE). Structural predictions revealed strong similarities between planarian and human enzymes. Cholinesterase activities were detected in crude planarian homogenates after grinding and were inhibited after organophosphorus exposition. In situ Hybridization was further used to localize cholinesterases in planarians and showed two different patterns, Smed-AChE being mainly detected in cephalic ganglion and ventral nerve cords while Smed-BChE distribution was diffuse. Survival, behavior and regeneration were analyzed in whole planarian exposed to four OP [1]. The toxicity of OP degradation products generated by enzymatic hydrolysis with the robust phosphotriesterase enzyme SsoPox, from the archea Sulfolobus solfataricus [2], was further evaluated. OP were found to be highly toxic to planarians causing severe mortality and behavior disruption at sublethal concentrations as well as growth disruption during regeneration after cutting. Enzymatic decontamination drastically reduced toxicity and enhanced both mobility and development. These results underline that degradation products have a lower impact than initial organophosphorus substrates. A biotechnological application based on a filtration column incorporating detoxifying enzymes was developed to decontaminate wastewater with planarian as biosensor.

The ongoing coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health crisis. Flavon baicalein, a major bioactive molecule of Scutellaria baicalensis, inhibits the replication of SARS-CoV-2 which causes severe acute respiratory syndrome in humans. Animal experiments show that baicalein has the character of a broad-spectrum coronavirus drug. It is non-toxic and can inhibit SARS-CoV-2-induced damage. Baicalein may therefore be a promising therapeutic drug for the treatment of COVID-19.

Thyroid cancer is a rare type of malignancy. However, thyroid cancer constitutes more than 90 % of endocrine tumors. Metformin (N’, N’-dimethybiguanide) is the most commonly prescribed drug in the world, and the annual number of prescriptions for this drug exceeds 120 million. Metformin is the first-line oral treatment for patients with type II diabetes. Metformin has recently been investigated for potential anti-cancer activity in patients with thyroid cancer by stimulating the Adenosine Mono-Phosphate-Activated Protein Kinase (AMPK) pathway in some types of tumors. In general, the anti-cancer mechanism of metformin acts directly by blocking mitochondrial oxidative phosphorylation through down-regulation of mitochondrial complex I and mitochondrial glycerophosphate dehydrogenase. This leads to a state of metabolic stress that in turn stimulates the AMPK pathway due to ATP reduction, and leads to inhibition of the mechanical (mammalian) target of the rapamycin (mTOR) pathway, which subsequently inhibits cancer cell proliferation and stimulates apoptosis and autophagy with cell cycle perturbation. Metformin also acts in an independent manner, in addition to its indirect actions that target insulin resistance. In this review, we reviewed 21 studies on the use of metformin in thyroid cancer, which showed that administration of metformin in diabetic patients is associated with a reduced incidence of thyroid cancer. On the other hand, the use of metformin enhances the response to anticancer drugs in thyroid cancer. Overall, we need further prospective studies to elucidate the synergistic mechanism of metformin when it is used to treat thyroid cancer as adjuvant therapy with anticancer drugs.