DMSO: A Mixed-Competitive Inhibitor of Human Acetylcholinesterase

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DMSO: A Mixed-Competitive Inhibitor of Human Acetylcholinesterase Amit Kumar, and Taher Darreh-Shori ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00344 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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DMSO: A Mixed-Competitive Inhibitor of Human Acetylcholinesterase Amit Kumar1* and Taher Darreh-Shori1* 1

Center for Alzheimer Research, Karolinska Institutet; Department of Neurobiology, Care Sciences, and Society; Division of Translational Alzheimer Neurobiology., NOVUM, 4th Floor, 141 86 Stockholm, Sweden * Corresponding authors

Taher Darreh-Shori, E-mail [email protected] Tel +46 8 585 863 12 Fax +46 8 585 854 70

Amit Kumar, E-mail [email protected] Tel +46 8 585 838 97 Fax +46 8 585 854 70

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Abstract Dimethyl sulfoxide (DMSO) is the most common organic solvent used in biochemical and cellular assays during drug discovery programs. In spite of its wide use, the effect of DMSO on several enzyme classes, which are crucial targets of the new therapeutic agents, are still unexplored. Here, we report the detailed biochemical analysis of the effects of DMSO on the human acetylcholine-degrading enzyme, acetylcholinesterase (AChE), the primary target of current Alzheimer’s therapeutics. Our analysis showed that DMSO is a considerably potent and highly selective irreversible mixed-competitive inhibitor of human AChE with IC50 values in the lower millimolar range, corresponding to 0.88 to 2.6% DMSO (v/v). Most importantly, 1-4% (v/v) DMSO, the commonly used experimental concentrations, showed ~ 37-80% inhibition of human AChE activity. We believe that our results will assist in developing stringent protocols and help in the better interpretation of experimental outcomes during screening and biological evaluation of new drugs. Keywords: Dimethyl sulfoxide (DMSO), Acetylcholinesterase (AChE), Organic solvents, Enzyme kinetics, Drug discovery and development, Alzheimer's disease (AD)

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Introduction Alzheimer’s disease (AD) is the primary cause of dementia, affecting approximately 48 million people worldwide and this figure is projected to quadruple by 2050 with no cure or preventive treatment in sight.1 Currently, only four FDA-approved drugs are available in the market, which provide modest symptomatic relief and to some extent delay the progression of the disease.2 Three of these drugs act as cholinesterase inhibitors (ChEIs). They act mainly on acetylcholinesterase (AChE), the key enzyme responsible for hydrolyzing acetylcholine (ACh), the neurotransmitter of the cholinergic signaling system, although one of them also inhibits butyrylcholinesterase (BuChE).3 Moreover, there are still extensive pursuits on finding new more effective ChEIs as therapeutic agents mainly targeted at these enzymes. There is also increasing interest in drug repurposing and screening of the FDA approved drugs against these two enzymes. High-Throughput Screening (HTS) techniques are essential and the core component of any novel drug discovery program. HTS assays can be performed using a wide-array of methods, such as phenotypic screening, fluorometric and/or colorimetric assays, calorimetric assays and mass spectrometry.4,5 One of the most important aspects of developing any HTS assay is proper optimization of the working protocol so that it can be automated and easily adapted not merely for the soluble but also for the poorly soluble potential hits. When setting up a dose-response bioassays, a major concern is the potential exposure of the macromolecule target to the organic solvents, such as aliphatic alcohols, acetonitrile, and acetone (used as a vehicle for screening molecules), which can affect biochemical properties of macromolecules, such as its stability, conformation, tertiary and quaternary structure and activity.6-8 In this context, dimethyl sulfoxide (DMSO), due to its extensive solvent properties and 3

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relatively low chemical reactivity,9 is the most commonly used organic solvent for stock preparation of hit compounds for biological screenings and cellular assays.10 DMSO is a polar aprotic solvent with a well-ordered structure and is easily miscible with water and other organic solvents.9 Despite its wide use, few studies have been published showing possible toxic and/or inhibitory effect of DMSO in cellular and enzyme kinetic assays. For example, a study reports that 1% DMSO can suppress cellular growth after 72 hours in MTT toxicity assays,11 whereas another shows that 0.2% DMSO can inhibit P450-mediated substrate catalysis.12 A further report indicates that DMSO can increase the intrinsic catalytic rate of the enzyme glyceraldehyde-3-phosphate dehydrogenase in a concentration-dependent manner, most likely by a favorable effect on the enzyme-substrate complex formation.13 Furthermore, there are two studies published several decades ago, showing that DMSO can inhibit AChE activity in cardiac and muscle tissue.14,15 Altogether, these notions highlight the importance of considering the bidirectional effect of DMSO on the target macromolecule while designing the assays and interpreting the results. Thus, considering that DMSO is a solvent widely used in diverse cell culture studies, it is of the utmost importance to provide a detailed biochemical study on the effect of DMSO on cholinergic enzymes, in particular, AChE. In this paper, we report the effect of DMSO on the activities of human cholinergic enzymes; AChE, BuChE and choline acetyltransferase (ChAT) using optimized highthroughput assays, which were run on 384 well-plates. In addition, detailed in vitro enzyme kinetic studies were performed in order to determine the kinetic parameters (Km, Kcat, and Vmax), inhibition constant (Ki), half-maximal inhibitory concentration (IC50) and mode of inhibition of human AChE by DMSO.

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Result and Discussion We first determined the inhibitory effect of DMSO on human red blood cell (RBC) and recombinant AChE (rAChE) activity, using a modified version of Ellman’s colorimetric assay 16,17

at a single concentration of 3.3% DMSO (v/v). The initial analysis showed about 50%

inhibition of the enzyme activity as compared to the buffered enzyme control run in parallel as a reference (100% activity). This observation was quite unexpected, as 1-5% DMSO is the most commonly used concentration during almost all biochemical and cellular assays. Interestingly, 1-5% DMSO concentration did not affect the activity of other cholinergic enzymes, i. e. ChAT and BuChE. Organic solvents are widely used during drug discovery evaluation steps as a medium for dissolving potential drug candidates for biological assays. Studies have shown that different organic solvents (e.g. DMSO, Methanol, Acetonitrile etc.) can exert a diverse range of effects, which can interfere with critical experimental parameters such as macromolecule conformation, enzyme kinetics, and substrate affinity during biochemical assays. These parameters are crucial for the determination of correct IC50 values of new therapeutic molecules and any interference can lead to false interpretation of the experimental outcome. In light of all these findings, we designed our study to address the effect of DMSO on human AChE enzymatic activity in extensive detail in order to get further insight into DMSO’s mechanism of inhibition. The dose-response curves at different DMSO % and substrate concentrations were obtained to calculate the Ki and IC50 values, as shown in Figures 1A and 3. The analysis resulted in Ki value of ~ 88.70 mM and IC50 value of ~ 258.4 mM (corresponds to 1.83% DMSO (v/v)) at an enzyme substrate concentration of 0.5 mM. Moreover, the percentage of enzyme inhibition (EI) at 0.5 mM substrate for different concentrations of DMSO were also 5

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calculated from the dose-response curves (Table 1). DMSO showed a concentrationdependent inhibition of activity with 16% DMSO showing ~ 98% inhibition of human rAChE enzyme. In addition, we also determined the effect of other commonly used solvents, i. e. methanol, acetone, and acetonitrile on human rAChE activity. In our analysis, both acetone and acetonitrile showed a reasonably similar inhibitory effect on AChE as DMSO, producing ~ 13-54% inhibition at 1-4% concentrations and ~74-80% inhibition at 16% concentration (Table 1). Furthermore, similar to DMSO, we also determined the Ki values of methanol, acetone, and acetonitrile from the dose-response curves at different solvent % and substrate concentrations (Figure 1). Out of all the tested solvents, methanol showed the highest Ki value (~ 4200 mM) i. e. the weakest inhibition, whereas DMSO showed the lowest Ki value (~ 88.3 mM) and hence the strongest inhibition of AChE (Figure 1; Table 1). Acetone and acetonitrile showed Ki values of 177 and 360 mM, respectively (Figure 1). Methanol showed almost no inhibition at the commonly used concentrations (1-4%) and only ~ 25% inhibition at the highest tested concentration of 16% (Table 1), making it the best choice for testing new compounds against AChE, and/or experimental models with cholinergic/cholinoceptive phenotypes. In order to provide additional guiding clues, we also determined the IC50 value of DMSO for AChE at various substrate concentrations. Importantly, DMSO exhibited IC50 values from lower to higher millimolar range (corresponding to ~ 0.88% to 2.6% DMSO (v/v)) at the different substrate concentrations, thus further indicating its high potency as an AChE inhibitor in lower DMSO concentrations (Figure 3; Table 2).

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Finally, the kinetic parameters (Kcat, Km, and Vmax) and mode of inhibition were determined by a nonlinear regression fit of the substrate-velocity curves data with GraphPad Prism 7,18 but also for illustration purposes by Lineweaver-Burk (double reciprocal) graph 19 (Figures 1A and 2, respectively). In the Lineweaver-Burk plot, when the reciprocal of a rate (1/v) is plotted as a function of the reciprocal of substrate concentration (1/S) for various inhibitor concentrations a straight line is produced. The intercepts of X- and Y-axis represent -1/Km and 1/Vmax, respectively and the slope is Km/Vmax..20 The double reciprocal plot showed a significant decrease in Vmax value and an increase in Km value with increasing DMSO concentrations, specifying mixed-competitive model inhibition of human rAChE by DMSO (Figure 2; Table 3). Moreover, Vmax, Km, and Kcat values for each DMSO concentration (Table 3) were calculated from the substrate-velocity curves (Figure 1A). The kinetic parameters of the enzyme were also significantly affected (Table 3) when DMSO concentration was increased from 0 to 4%, both Vmax and Kcat values significantly decreased from 1.16 to 0.51 mM/hr and 6.3 x 103 to 2.8 x 103 s-1, respectively. On the other hand, Km value increased from 0.17 to 0.84 mM at 4% DMSO concentration. It was not possible to calculate reliable Km, Vmax, and Kcat values at 16% DMSO concentration since the enzyme was ~ 98% inhibited (Table 1). Additionally, the Ki value for the inhibition of red blood cell (RBC) AChE activity by DMSO was estimated using the nonlinear substrate-velocity inhibition curves at different DMSO and substrate concentrations (Figure 4A). DMSO showed a Ki value of 104.70 mM for RBC AChE, almost comparable to Ki value of 88.70 mM estimated for human rAChE. Also, Vmax and Km values for 0, 1, 4, and 16% DMSO concentrations (Figure 4B) were calculated from the substrate-velocity curves (Figure 4A). Importantly, both Vmax and Km values for RBC AChE showed a similar pattern (significant decrease in Vmax values and 7

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increase in Km values with increasing DMSO %; Figure 4B) as observed for human rAChE (Table 3) and indicating mixed-competitive model inhibition of RBC AChE by DMSO. A mixed-competitive ligand is expected to be able to bind to both free enzyme and the enzymesubstrate complex.21,22 Hence, we may conclude that DMSO is a considerably potent and mixed-competitive inhibitor of both human rAChE and RBC AChE. This, in turn, suggests that DMSO should also inhibit other isoforms of AChE, such as the synaptic splice variants, since all AChE isoforms have a common catalytic domain. The neuronal AChE is the primary target of current AD treatment. The findings presented here hence may be vital for the optimization of assays for the discovery of new AChE inhibitors. Another important aspect of the findings is concerned with the interpretation of results derived from experimental cell culture studies. The cholinergic signaling system is undoubtedly the most widely spread neuronal and non-neuronal signaling system in the body and has regulatory functions in diverse biological processes and organs such as the brain, the immune system,23 the brain-immune system cross-talk,24 the enteric nervous system of gastrointestinal tracts,25 related organs/tissues,26 airways/lungs,27,28 muscles including heart, skin,29 blood vessels/circulation,30 bioenergetics activity of mitochondria,31-34 and even the reproductive systems.35-39 Thereby, the effect of DMSO on AChE would be difficult to predict or control in different cell line-based experimental paradigms as a surrogate disease model, particularly for studying cholinergic signaling. Although the function of AChE in such cells/tissue/organs is not completely known, it is most likely related to autocrine and/or paracrine ACh signaling.25,28,40,41 Thus, the inclusion of DMSO in a cell culture experimental setup should be avoided by replacing it with methanol, or otherwise has to be carefully controlled, as it may alter the intrinsic cellular responses by inhibiting the activity of AChE. This issue is crucial in studies based on cellular models, which are known to be cholinergic 8

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and/or cholinoceptives, such as lymphocytes, astrocytes, and human embryonic stem cells.42,43 In our analysis, we found that DMSO could affect human AChE activity in a concentration-dependent manner with 16% DMSO showing ~ 98% inhibition of enzyme activity. Importantly, concentrations of 1 and 4 % DMSO; most commonly used during the majority of biochemical and cellular assays, showed ~ 37% and 79% inhibition, respectively (Table 1). Noteworthy, the same DMSO concentrations did not show any inhibitory effect on ChAT and BuChE enzymes, signifying high selectivity of DMSO for human AChE. Most importantly, based on our results, methanol may be the alternative choice for replacing DMSO as a solvent in cellular models with cholinergic and/or cholinoceptive phenotypes. Interestingly, a recent study published by Obregon et al. has also screened the effect of various solvents on rat AChE activity in different regions of the brain. In agreement with our findings on human enzyme, they showed that at 5-10% v/v concentrations, DMSO, acetone, and acetonitrile induced significant inhibition of rat AChE, but no inhibition by methanol.44 The mechanism of inhibition of AChE by DMSO and/or its binding sites on AChE is not known. Although our data suggest that DMSO behaves as a mixed-competitive ligand, meaning that, it binds to the active site of the enzyme. This is currently not possible to ascertain using for e.g. in silico analysis, since DMSO is a very small molecule. Nonetheless, to get further insights about DMSO mechanism of inhibition, we performed functional recovery assay to determine the dynamic of DMSO interaction with AChE (Figure 5). Intriguingly, this analysis indicated that DMSO behaves as an irreversible AChE inhibitor. This is because at 0.1-1% DMSO concentrations there was no recovery of the enzyme activity (Figure 5C). The analysis also suggested that the main effect of DMSO is unlikely to 9

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be resulting from unspecific conformational changes in the enzyme structure since at the highest DMSO concentrations (4 and 16%) there was actually 14-28% recovery of AChE activity (Figure 5C). In other words, we should expect DMSO-induced conformational changes in the enzyme structure at high concentrations but not at low DMSO concentrations. Thus, the most plausible conclusion is that DMSO behaves like a true irreversible inhibitor of AChE. However, the functional activity recovery assay provided some clues supporting the general view that DMSO can affect protein conformations at high concentrations by substituting the bonded water in the protein tertiary structure.9,45 The assay, as noted, showed that the enzyme recovered 14-28% of its activity at 4 and 16% DMSO concentrations. This is unlikely to occur if all the enzymes at these concentrations were irreversibly inhibited by DMSO, suggesting that some parts of the enzyme were in the inactivated state due to solventinduced changes in the enzyme structure, which upon removal of DMSO reverted back to its native conformation and regained its activity.

Conclusion Detailed enzyme kinetic analysis was performed to elucidate the effect of DMSO on the activity of various human cholinergic enzymes. The enzyme kinetic assessments showed that DMSO is a considerably potent and highly selective mixed-competitive inhibitor of human AChE as compared to all the other tested solvents and hence may bind to either enzyme or enzyme-substrate (ES) complex, thereby affecting both catalytic activity and efficiency of the enzyme. DMSO showed IC50 values in the lower millimolar range for human AChE. The result of the functional recovery assay however indicated that DMSO acts as an irreversible 10

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inhibitor of AChE. Therefore, careful controls and/or alternative solvents, such as methanol, have to be used when screening new hits as an inhibitor of AChE or when studying cellular models. The findings should also be useful for researchers working in the field of translational neuroscience and using cellular models with cholinergic and/or cholinoceptive phenotypes. The kinetic data provide crucial information in designing experiments with more stringent controls as well as enable more accurate analysis and interpretation of the findings.

Methods In vitro AChE and BuChE activity inhibition assay A modified version of Ellman’s colorimetric assay 16,17 was adapted to a high-throughput assay for the enzymatic activity of BuChE and AChE. The reagents, butyrylthiocholine iodide (BTC), acetylthiocholine iodide (ATC), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The modified assay details are as previously described. 46,47 The method was further optimized as a high-throughput assay for use in 384-well plates. Briefly, 25µL/well of a 1:400 diluted solution of a lysed pooled human RBC and 1:768 diluted (3.5 ng/ml final concentration) purified recombinant human AChE protein (Sigma, Cat no. C1682) was used for the measurement of AChE activity. The lysed RBC solution as the source of RBC AChE was prepared by lysing frozen RBC (separated from plasma by centrifugation) in an equal volume of phosphate buffer. This was then further diluted by an equal volume of glycerol, giving a four times diluted RBC stock solution, and stored at -20 oC as small volume aliquots. For BuChE, 25µL/well of a 1:450 diluted solution of a pooled human plasma was used for activity measurement. In the initial in vitro screening step, the wells were pre-incubated with 25µL/wells of DMSO for 10-30 minutes at room temperature. The final concentration of DMSO was 3.3% in each well. Finally, 25µL of a cocktail mix prepared in Na/K phosphate buffer, containing DTNB (final 11

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concentration 0.4 mM) and BTC, (final concentration 1 mM) or ATC (final concentration 0.5 mM), was added to each well and the changes in absorbance were monitored at 412 nm wavelength for 15-20 minutes with one-minute interval, using a microplate spectrophotometer reader (Infinite M1000, Tecan). The rate of the enzyme activity was determined from the linear part of the kinetic reaction curves as ∆OD/time.

In vitro ChAT activity inhibition fluorometric assay ChAT activity was measured using our newly developed fluorometric assay, using human recombinant ChAT (rChAT) protein. The reagents, choline chloride, acetyl coenzyme-A (ACoA, A2181) and 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ChAT assay can be run in either 96-well or 384-well plates. For 96-well plates, 50µL/well of 0.212 µg/ml (final concentration) of the recombinant ChAT was incubated with 3.3% DMSO (50µL/well) for 10-30 minutes at room temperature in dilution buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.0 mM EDTA, 0.05 % (v/v) Triton X-100). Then, 50 µL of a cocktail-A [dilution buffer containing choline chloride (final concentration 150 µM), ACoA (final concentration 13.3 µM) and CPM (final concentration 15 µM)] was added to each well. Immediately after adding cocktail-A, the changes in fluorescence were monitored kinetically at 479 nm after exciting at 390 nm at 1-2 minute intervals for 15-20 minutes using a microplate spectrophotometer reader (Infinite M1000, Tecan). On the 96-wells plate, several enzyme wells without inhibitor were also included during measurements as control and for estimating the inhibition level. Negative controls were wells without enzyme. The percentage inhibition was calculated based on the enzyme control value as a reference (100 % activity). Kinetic studies or in vitro estimation of Ki, IC50, and mode of action of DMSO 12

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For kinetic studies, a similar protocol as inhibition assay was followed; a dilution series of five different concentrations ranging from 10 -6 to 10 -3 M was prepared for DMSO. ATC was used as the substrate in the concentrations ranging from 0.03 to 1 mM. Absorbance measurements at each DMSO concentration were collected in duplicate. The rate of enzyme activity (as ∆OD/hr kinetic data) was calculated and processed using the GraphPad Prism 7 analysis software as previously described.15 The inhibitory constant (Ki) values were determined from the dose-response curve and the half-maximal inhibitory concentration (IC50) values were calculated by plotting the percentage enzyme activity vs. the log of the DMSO concentrations and fitting the data using the nonlinear regression Enzyme KineticsInhibition function. The Michaelis-Menten constant (Km), turnover number (Kcat), and maximal velocity (Vmax) values were calculated from substrate-velocity curves after fitting the data with nonlinear regression Michaelis-Menten kinetic function. The Km and Vmax values were further used to plot the Lineweaver-Burk plots; the plots were fitted using linear regression function using the GraphPad Prism 7 analysis software.15

In vitro estimation of Ki for methanol, acetone, and acetonitrile For Ki estimation; a dilution series of five different concentrations ranging from 0 to 16% was prepared for all the solvents. ATC was used as the substrate in the concentrations ranging from 0.03 to 1 mM. Absorbance measurements at each solvent concentration were collected in duplicate. The rate of enzyme activity (as ∆OD/hr kinetic data) was calculated and processed using the GraphPad Prism 7 analysis software as previously described.15 The inhibitory constant (Ki) values were determined from the dose-response curve using the nonlinear regression Enzyme Kinetics-Inhibition function of GraphPad Prism 7 software.

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AChE functional recovery assay NuncTm Maxisorp 96-well plate (ThermoFisher Scientific) was incubated with 1:500 diluted monoclonal anti-AChE antibody (MAB 337, Chemicon International) overnight at 4 o

C. On the next day, the antibody solution was discarded and the plate was washed with Na/K

phosphate buffer, pH 7.4. The washed plate was incubated with 1:600 diluted human rAChE (Sigma, Cat no. C1682) for 3-4 hours at room temperature under gentle orbital shaking. Thereafter, human rAChE solution was discarded, and the plate was washed once with Na/K phosphate buffer, pH 7.4. Then 175 µL/well of different DMSO solutions (ranging from 0-16 %) was added to the assigned wells. In control wells, 175µL/well of Na/K phosphate buffer was used. Finally, 25µL of a cocktail mix was added to all wells and the changes in absorbance were monitored at 412 nm wavelength for 60 minutes with a two-minute interval, using a microplate spectrophotometer reader (Infinite M1000, Tecan). The cocktail mix was prepared in Na/K phosphate buffer, pH 7.4, containing DTNB (final concentration 0.4 mM) and ATC (final concentration 0.5 mM). After this run, the plate was emptied and washed 3-4 times with Na/K phosphate buffer, pH 7.4, to remove DMSO from the wells. After washing, the second run was performed by adding 200 µL/well of a cocktail mix. This cocktail contained 0.4 mM DTNB and 0.5 mM ATC. The changes in absorbance were monitored as before at 412 nm wavelength for 60 minutes with a two-minute interval. The rate of enzyme activity was calculated (as ∆OD/hr kinetic data) and processed using the GraphPad Prism 7 analysis software. Negative controls were wells without enzyme. The percentage inhibition was calculated based on the enzyme control value as a reference (100 % activity). Author Contributions Statement

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AK and TDS designed the experiments. AK performed all the experiments. Both authors performed the data analysis and wrote the manuscript. Both authors read and approved the final draft. Acknowledgements This study was supported by grants from Loo & Hans Osterman Foundation; KI Geriatrics Foundation; Olle Engkvist Byggmästare Foundation; Åke Wibergs Foundation; Åhlén-Foundation; Gunvor and Josef Anérs Foundation; Magnus Bergvalls Foundation; The Lars Hierta Memorial Foundation; Demens Foundation (Demensfonden); Gun and Bertil Stohnes Foundation; Foundation for Sigurd & Elsa Goljes Memory; Tore Nilsson Foundation; the Foundation for Old Servants; the Swedish Brain Foundation; Alzheimer Association, USA (2016-NIRG-391599), and the Swedish Research Council (project no 2016-01806). We are thankful to Dr. Rajnish Kumar and Dr. Prashant Murumkar (The MS University of Baroda) for their helpful discussions and to Maria Klebanova for proofreading the manuscript. Additional Information Competing financial interests: The authors declare no competing financial interests.

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References (1) WHO (2015) 10 facts on dementia, In WHO, World Health Organization. (2) Lu, P. H., Edland, S. D., Teng, E., Tingus, K., Petersen, R. C., Cummings, J. L., and Alzheimer's Disease Cooperative Study, G. (2009) Donepezil delays progression to AD in MCI subjects with depressive symptoms, Neurology 72, 2115-2121. (3) Colovic, M. B., Krstic, D. Z., Lazarevic-Pasti, T. D., Bondzic, A. M., and Vasic, V. M. (2013) Acetylcholinesterase inhibitors: pharmacology and toxicology, Curr Neuropharmacol 11, 315-335. (4) Janzen, W. P., and Bernasconi, P. (2009) High Throughput Screening, 2 ed., Humana Press. (5) Zheng, W., Thorne, N., and McKew, J. C. (2013) Phenotypic screens as a renewed approach for drug discovery, Drug Discov Today 18, 1067-1073. (6) Mozhaev, V. V., Khmelnitsky, Y. L., Sergeeva, M. V., Belova, A. B., Klyachko, N. L., Levashov, A. V., and Martinek, K. (1989) Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures. Alpha-chymotrypsin and laccase in mixed water/alcohol, water/glycol and water/formamide solvents, Eur J Biochem 184, 597-602. (7) Khmelnitsky, Y. L., Mozhaev, V. V., Belova, A. B., Sergeeva, M. V., and Martinek, K. (1991) Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis, Eur J Biochem 198, 31-41. (8) Gupta, M. N. (1992) Enzyme function in organic solvents, Eur J Biochem 203, 25-32. (9) Rammler, D. H., and Zaffaroni, A. (1967) Biological implications of DMSO based on a review of its chemical properties, Ann N Y Acad Sci 141, 13-23. (10) Di, L., and Kerns, E. H. (2006) Biological assay challenges from compound solubility: strategies for bioassay optimization, Drug Discov Today 11, 446-451. 16

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(11) Ben Trivedi, A., Kitabatake, N., and Doi, E. (1990) Toxicity of dimethyl sulfoxide as a solvent in bioassay system with HeLa cells evaluated colorimetrically with 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Agric Biol Chem 54, 29612966. (12) Chauret, N., Gauthier, A., and Nicoll-Griffith, D. A. (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes, Drug Metab Dispos 26, 1-4. (13) Wiggers, H. J., Cheleski, J., Zottis, A., Oliva, G., Andricopulo, A. D., and Montanari, C. A. (2007) Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays, Anal Biochem 370, 107-114. (14) Plummer, J. M., Greenberg, M. J., Lehman, H. K., and Watts, J. A. (1983) Competitive inhibition by dimethylsulfoxide of molluscan and vertebrate acetylcholinesterase, Biochem Pharmacol 32, 151-158. (15) Shlafer, M., Matheny, J. L., and Karow, A. M., Jr. (1976) Cardiac chronotropic mechanisms of dimethyl sulphoxide: inhibition of acetylcholinesterase and antagonism of negative chronotropy by atropine, Arch Int Pharmacodyn Ther 221, 21-31. (16) Moscone, D., Arduini, F., and Amine, A. (2011) A rapid enzymatic method for aflatoxin B detection, Methods Mol Biol 739, 217-235. (17) Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Feather-Stone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem Pharmacol 7, 88-95. (18) Prism version 7.02 for Windows, G. S., La Jolla California USA, www.graphpad.com”. Prism version 7.02 for Windows, GraphPad Software, La Jolla California USA.

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(19) Chen, Y., Sun, J., Fang, L., Liu, M., Peng, S., Liao, H., Lehmann, J., and Zhang, Y. (2012) Tacrine-ferulic acid-nitric oxide (NO) donor trihybrids as potent, multifunctional acetyl- and butyrylcholinesterase inhibitors, J Med Chem 55, 4309-4321. (20) Berg JM, T. J., Stryer L. (2002) Appendix: Vmax and KM Can Be Determined by Double-Reciprocal Plots., Biochemistry. 5th edition. New York: W H Freeman; 2002. (21) Strelow, J., Dewe, W., Iversen, P. W., Brooks, H. B., Radding, J. A., McGee, J., and Weidner, J. (2004) Mechanism of Action Assays for Enzymes, In Assay Guidance Manual (Sittampalam, G. S., Coussens, N. P., Brimacombe, K., Grossman, A., Arkin, M., Auld, D., Austin, C., Baell, J., Bejcek, B., Chung, T. D. Y., Dahlin, J. L., Devanaryan, V., Foley, T. L., Glicksman, M., Hall, M. D., Hass, J. V., Inglese, J., Iversen, P. W., Kahl, S. D., Kales, S. C., Lal-Nag, M., Li, Z., McGee, J., McManus, O., Riss, T., Trask, O. J., Jr., Weidner, J. R., Xia, M., and Xu, X., Eds.), Bethesda (MD). (22) Cornish-Bowden, A. (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors, Biochem J 137, 143144. (23) Fujii, T., and Kawashima, K. (2001) An independent non-neuronal cholinergic system in lymphocytes, Jpn J Pharmacol 85, 11-15. (24) Tracey, K. J., Czura, C. J., and Ivanova, S. (2001) Mind over immunity, Faseb J 15, 1575-1576. (25) Cheng, K., Samimi, R., Xie, G., Shant, J., Drachenberg, C., Wade, M., Davis, R. J., Nomikos, G., and Raufman, J. P. (2008) Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation, Am J Physiol Gastrointest Liver Physiol. 295, G591-597. (26) Thuong Nguyen, V., Hall, L. L., Gallacher, G., Ndoye, A., Jolkovsky, D. L., Webber, R. J., Buchli, R., and Grando, S. A. (2000) Choline Acetyltransferase, 18

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Acetylcholinesterase, and Nicotinic Acetylcholine Receptors of Human Gingival and Esophageal Epithelia, J. Dent. Res. 79, 939-949. (27) Chernyavsky, A. I., Shchepotin, I. B., Galitovkiy, V., and Grando, S. A. (2015) Mechanisms of tumor-promoting activities of nicotine in lung cancer: synergistic effects of cell membrane and mitochondrial nicotinic acetylcholine receptors, BMC cancer 15, 152. (28) Brown, K. C., Perry, H. E., Lau, J. K., Jones, D. V., Pulliam, J. F., Thornhill, B. A., Crabtree, C. M., Luo, H., Chen, Y. C., and Dasgupta, P. (2013) Nicotine induces the upregulation of the alpha7-nicotinic receptor (alpha7-nAChR) in human squamous cell lung cancer cells via the Sp1/GATA protein pathway, J Biol Chem 288, 33049-33059. (29) Grando, S. A. (2006) Cholinergic control of epidermal cohesion, Exp Dermatol 15, 265282. (30) Grando, S. A., Kawashima, K., and Wessler, I. (2003) Introduction: the non-neuronal cholinergic system in humans, Life Sci 72, 2009-2012. (31) Gergalova, G., Lykhmus, O., Komisarenko, S., and Skok, M. (2014) alpha7 nicotinic acetylcholine receptors control cytochrome c release from isolated mitochondria through kinase-mediated pathways, Int. J. Biochem. Cell Biol 49, 26-31. (32) Lykhmus, O., Gergalova, G., Koval, L., Zhmak, M., Komisarenko, S., and Skok, M. (2014) Mitochondria express several nicotinic acetylcholine receptor subtypes to control various pathways of apoptosis induction, Int. J. Biochem. Cell Biol 53, 246-252. (33) Chernyavsky, A., Chen, Y., Wang, P. H., and Grando, S. A. (2015) Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis, Int Immunopharmacol. 29, 76-80.

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(34) Chernyavsky, A. I., Shchepotin, I. B., and Grando, S. A. (2015) Mechanisms of growthpromoting and tumor-protecting effects of epithelial nicotinic acetylcholine receptors, Int Immunopharmacol. 29, 36-44. (35) Sastry, B. V. (1997) Human placental cholinergic system, Biochem Pharmacol 53, 15771586. (36) Lips, K. S., Bruggmann, D., Pfeil, U., Vollerthun, R., Grando, S. A., and Kummer, W. (2005) Nicotinic acetylcholine receptors in rat and human placenta, Placenta 26, 735746. (37) Leventer, S. M., Rowell, P. P., and Clark, M. J. (1982) The effect of choline acetyltransferase inhibition on acetylcholine synthesis and release in term human placenta, J Pharmacol Exp Ther 222, 301-305. (38) Sastry, B. V., Janson, V. E., and Chaturvedi, A. K. (1981) Inhibition of human sperm motility by inhibitors of choline acetyltransferase, J Pharmacol Exp Ther 216, 378-384. (39) Bray, C., Son, J. H., and Meizel, S. (2005) Acetylcholine causes an increase of intracellular calcium in human sperm, Mol Hum Reprod 11, 881-889. (40) Xie, G., Cheng, K., Shant, J., and Raufman, J. P. (2009) Acetylcholine-induced activation of M3 muscarinic receptors stimulates robust matrix metalloproteinase gene expression in human colon cancer cells, Am J Physiol Gastrointest Liver Physiol. 296, G755-763. (41) Alessandrini, F., Cristofaro, I., Di Bari, M., Zasso, J., Conti, L., and Tata, A. M. (2015) The activation of M2 muscarinic receptor inhibits cell growth and survival in human glioblastoma cancer stem cells, Int Immunopharmacol. 29, 105-109. (42) Malmsten, L., Vijayaraghavan, S., Hovatta, O., Marutle, A., and Darreh-Shori, T. (2014) Fibrillar beta-amyloid 1-42 alters cytokine secretion, cholinergic signalling and neuronal differentiation, J Cell Mol Med 18, 1874-1888. 20

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(43) Vijayaraghavan, S., Karami, A., Aeinehband, S., Behbahani, H., Grandien, A., Nilsson, B., Ekdahl, K. N., Lindblom, R. P., Piehl, F., and Darreh-Shori, T. (2013) Regulated Extracellular Choline Acetyltransferase Activity- The Plausible Missing Link of the Distant Action of Acetylcholine in the Cholinergic Anti-Inflammatory Pathway, PloS one 8, e65936. (44) Obregon, A. D., Schetinger, M. R., Correa, M. M., Morsch, V. M., da Silva, J. E., Martins, M. A., Bonacorso, H. G., and Zanatta, N. (2005) Effects per se of organic solvents in the cerebral acetylcholinesterase of rats, Neurochem Res 30, 379-384. (45) Henderson, T. R., Henderson, R. F., and York, J. L. (1975) Effects of dimethyl sulfoxide on subunit proteins, Ann N Y Acad Sci 243, 38-53. (46) Darreh-Shori, T., Brimijoin, S., Kadir, A., Almkvist, O., and Nordberg, A. (2006) Differential CSF butyrylcholinesterase levels in Alzheimer's disease patients with the ApoE epsilon4 allele, in relation to cognitive function and cerebral glucose metabolism, Neurobiol Dis 24, 326-333. (47) Darreh-Shori, T., Kadir, A., Almkvist, O., Grut, M., Wall, A., Blomquist, G., Eriksson, B., Langstrom, B., and Nordberg, A. (2008) Inhibition of acetylcholinesterase in CSF versus brain assessed by 11C-PMP PET in AD patients treated with galantamine, Neurobiol Aging 29, 168-184.

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Figure Legends Figure 1. Concentration-dependent inhibition of human recombinant AChE by different solvents. The graphs represent the substrate-velocity inhibition curves of human recombinant AChE (rAChE) activity at different concentrations of the substrate acetylthiocholine and DMSO (A), methanol (B), acetonitrile (C), and acetone (D). Nonlinear regression analysis was used to determine the inhibition constant (Ki) of all the solvents for human AChE, as described in the Method section. These analyses indicate that DMSO is the most potent mixed-competitive inhibitor compared to the other three solvents. Methanol, on the other hand, shows the negligible effect on AChE activity. The symbols show percentages of different solvents. The values are shown as the mean ± SEM of two experiments performed in duplicate. Figure 2. The Lineweaver-Burk illustration of the mode of inhibition of AChE by DMSO. The plot was calculated from the kinetic rate of the human rAChE enzyme activity as described in the Method section at different substrate concentrations (ranging from 0.03 to 1 mM) with or without DMSO at specified concentrations. This plot illustrates that DMSO acts as a mixed-competitive inhibitor. The symbols show percentages of DMSO. The Lineweaver-Burk plots were fitted using the linear regression analysis function of GraphPad Prism 7 software. The values are shown as mean of two individual experiments performed in duplicate. Figure 3. Dose-response inhibition curves of DMSO against human rAChE. The inhibition curves of human rAChE activity were determined at specified substrate concentrations in the presence of different concentrations of DMSO. The symbols show the molar concentration of the substrate acetylthiocholine. The IC50 values were calculated after fitting the curves using nonlinear regression function of GraphPad Prism 7. The values are shown as mean of two individual experiments performed in duplicate. * IC50 value calculated at the substrate concentration of 0.5 mM. 22

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Figure 4. Concentration-dependent inhibition of human red blood cell AChE activity by DMSO. (A) Shows the substrate-velocity inhibition curves of human red blood cell AChE (RBC AChE) activity at different concentrations of DMSO and the substrate acetylthiocholine. Nonlinear regression analysis was used to determine the inhibition constant (Ki) of DMSO for human RBC AChE, as detailed in the Method section. These analyses also indicated that DMSO behaves as a mixed-competitive inhibitor as was found for rAChE. The symbols show percentages of DMSO. The values are shown as the mean ± SEM of two experiments performed in duplicate. (B) The table shows the Km and Vmax values for DMSO at different concentrations obtained from non-linear regression analysis using GraphPad Prism 7. The values are shown as the mean ± SEM of two experiments. * Since 98% of the enzyme was inhibited by DMSO, no reliable Km could be calculated at 16.6% DMSO. Figure 5. DMSO behaves like a true irreversible AChE inhibitor in the functional recovery assay. The mode of AChE inhibition (reversible or irreversible) by DMSO was determined using functional recovery assay for human rAChE, as described in the Method section. (A) The immobilized human rAChE was incubated with different concentrations of DMSO in a 96-well plate and the changes in the activity of rAChE were monitored (Run 1). The graphs in (A) show the enzymatic rate of AChE at the depicted DMSO percentages. (B) The same plate was then washed 3-4 times to remove DMSO from the wells. The activity of the immobilized rAChE was reassessed as before by adding the Ellman’s reagent cocktail (Run 2). The graphs show the measured rate of the rAChE activity in the wells following removal of DMSO. (C) Shows the percentage activity of rAChE after Run 1 and 2 at indicated DMSO concentrations. The bar charts were calculated based on the enzyme control value (0% DMSO) as 100 % activity reference. The data shows that DMSO at 0.1-1% 23

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concentrations behaves like a pure irreversible inhibitor. However, at 4 and 16% DMSO concentrations 14-28 % of AChE activity was recovered, most likely indicating that DMSO at these high concentrations may also induce conformational changes in the enzyme structure, perhaps due to a solvent-induced effect. The substrate, acetylthiocholine (ATC) was used at 0.5 mM concentration. The values are shown as the mean ± SEM of ≥ 2 experiments.

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Table 1. In vitro inhibitory activity of different solvents on human rAChE. Solvent concentration

Enzyme Inhibition (%)

% (v/v)

DMSO

Methanol

Acetonitrile

Acetone

0

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

0.06

10.1 ± 0.26

0.0 ± 0.00

7.8 ± 0.00

0.0 ± 0.00

0.26

19.0 ± 1.94

0.0 ± 0.00

9.9 ± 0.63

0.0 ± 0.00

1

36.7 ± 3.28

2.0 ± 6.39

21.2 ± 1.03

12.5 ± 0.00

4.1

78.7 ± 0.69

6.2 ± 4.63

54.3 ± 2.94

54.4 ± 8.32

16.6

98.0 ± 0.00

24.7 ± 0.30

79.7 ± 1.46

76.2 ± 1.62

The level of Enzyme Inhibition (EI) is shown at various concentrations of different solvents for human rAChE. The substrate concentration was 0.5 mM. Percentage EI values are shown as the mean ± SEM of two individual experiments performed in duplicate. Results are expressed as percentage inhibition compared to control.

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Table 2. IC50 values of DMSO at different substrate concentrations. Substrate Conc. (mM)

IC50 (mM)

DMSOa (%)

0.03 133.4 0.94 0.06 124.4 0.88 0.12 152.9 1.08 0.25 170.6 1.20 0.50 258.4 1.83 1.00 366.6 2.60 The IC50 values are shown as molar concentration of the DMSO. a Indicates the concentration in terms of percentages of DMSO corresponding to the IC50 values. The substrate was acetylthiocholine. The values are shown as the mean of two experiments.

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Table 3. The Km, Kcat and Vmax values for DMSO at different concentrations obtained from non-linear regression analysis of the substrate-velocity curve of AChE activity. Vmax (mM/hr) 1.16 ± 0.075

Km (mM) 0.17 ± 0.03

Kcat 103 (s-1) 6.30 ± 0.40

R squared 0.98

0.06

1.03 ± 0.061

0.16 ± 0.03

5.63 ± 0.33

0.98

0.26

0.98 ± 0.043

0.18 ± 0.02

5.32 ± 0.23

0.99

1

0.94 ± 0.037

0.35 ± 0.03

5.12 ± 0.20

0.99

4.1

0.51 ± 0.019

0.84 ± 0.05

2.79 ± 0.10

0.99

16.6

*

*

*

-

DMSO Conc. %(v/v) 0

* Since 98% of the enzyme was inhibited by DMSO, no reliable Km and Kcat could be calculated. The values are shown as the mean ± SEM of two experiments. Analysis was done using GraphPad Prism 7.

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Figure 1. Concentration-dependent inhibition of human recombinant AChE by different solvents. The graphs represent the substrate-velocity inhibition curves of human recombinant AChE (rAChE) activity at different concentrations of the substrate acetylthiocholine and DMSO (A), methanol (B), acetonitrile (C), and acetone (D). Nonlinear regression analysis was used to determine the inhibition constant (Ki) of all the solvents for human AChE, as described in the Method section. These analyses indicate that DMSO is the most potent mixed-competitive inhibitor compared to the other three solvents. Methanol, on the other hand, shows the negligible effect on AChE activity. The symbols show percentages of different solvents. The values are shown as the mean ± SEM of two experiments performed in duplicate. 258x208mm (300 x 300 DPI)

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Figure 2. The Lineweaver-Burk illustration of the mode of inhibition of AChE by DMSO. The plot was calculated from the kinetic rate of the human rAChE enzyme activity as described in the Method section at different substrate concentrations (ranging from 0.03 to 1 mM) with or without DMSO at specified concentrations. This plot illustrates that DMSO acts as a mixed-competitive inhibitor. The symbols show percentages of DMSO. The Lineweaver-Burk plots were fitted using the linear regression analysis function of GraphPad Prism 7 software. The values are shown as mean of two individual experiments performed in duplicate. 150x98mm (300 x 300 DPI)

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Figure 3. Dose-response inhibition curves of DMSO against human rAChE. The inhibition curves of human rAChE activity were determined at specified substrate concentrations in the presence of different concentrations of DMSO. The symbols show the molar concentration of the substrate acetylthiocholine. The IC50 values were calculated after fitting the curves using nonlinear regression function of GraphPad Prism 7. The values are shown as mean of two individual experiments performed in duplicate. * IC50 value calculated at the substrate concentration of 0.5 mM. 91x77mm (300 x 300 DPI)

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Figure 4. Concentration-dependent inhibition of human red blood cell AChE activity by DMSO. (A) Shows the substrate-velocity inhibition curves of human red blood cell AChE (RBC AChE) activity at different concentrations of DMSO and the substrate acetylthiocholine. Nonlinear regression analysis was used to determine the inhibition constant (Ki) of DMSO for human RBC AChE, as detailed in the Method section. These analyses also indicated that DMSO behaves as a mixed-competitive inhibitor as was found for rAChE. The symbols show percentages of DMSO. The values are shown as the mean ± SEM of two experiments performed in duplicate. (B) The table shows the Km and Vmax values for DMSO at different concentrations obtained from non-linear regression analysis using GraphPad Prism 7. The values are shown as the mean ± SEM of two experiments. * Since 98% of the enzyme was inhibited by DMSO, no reliable Km could be calculated at 16.6% DMSO.

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Figure 5. DMSO behaves like a true irreversible AChE inhibitor in the functional recovery assay. The mode of AChE inhibition (reversible or irreversible) by DMSO was determined using functional recovery assay for human rAChE, as described in the Method section. (A) The immobilized human rAChE was incubated with different concentrations of DMSO in a 96-well plate and the changes in the activity of rAChE were monitored (Run 1). The graphs in (A) show the enzymatic rate of AChE at the depicted DMSO percentages. (B) The same plate was then washed 3-4 times to remove DMSO from the wells. The activity of the immobilized rAChE was reassessed as before by adding the Ellman’s reagent cocktail (Run 2). The graphs show the measured rate of the rAChE activity in the wells following removal of DMSO. (C) Shows the percentage activity of rAChE after Run 1 and 2 at indicated DMSO concentrations. The bar charts were calculated based on the enzyme control value (0% DMSO) as 100 % activity reference. The data shows that DMSO at 0.1-1% concentrations behaves like a pure irreversible inhibitor. However, at 4 and 16% DMSO concentrations 1428 % of AChE activity was recovered, most likely indicating that DMSO at these high concentrations may also induce conformational changes in the enzyme structure, perhaps due to a solvent-induced effect. The substrate, acetylthiocholine (ATC) was used at 0.5 mM concentration. The values are shown as the mean ± SEM of ≥ 2 experiments. 414x125mm (300 x 300 DPI)

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