Allosteric modulation of the substrate specificity of acyl-CoA wax alcohol acyltransferase 2
2017; Elsevier BV; Volume: 58; Issue: 4 Linguagem: Inglês
10.1194/jlr.m073692
ISSN1539-7262
AutoresJ.M. Arne, Made Airanthi K. Widjaja‐Adhi, Taylor Hughes, Kevin W. Huynh, Josie A. Silvaroli, Sylwia Chełstowska, Vera Y. Moiseenkova‐Bell, Marcin Golczak,
Tópico(s)Retinal Development and Disorders
ResumoThe esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-cis-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11-cis-retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11-cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis-retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols and thus enables preferential synthesis of 11-cis-retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity. The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-cis-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11-cis-retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11-cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis-retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols and thus enables preferential synthesis of 11-cis-retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity. In vertebrates, vision depends on photosensitive pigments composed of 11-cis-retinal bound to protein scaffolds (opsins), which are localized in the outer segments of specialized photoreceptor cells, rods and cones (1Wald G. The molecular basis of visual excitation.Nature. 1968; 219: 800-807Crossref PubMed Scopus (579) Google Scholar). Light captured by these photoreceptors photo-isomerizes 11-cis-retinylidene to its all-trans configuration, induces a conformational change in the protein scaffold, and enables G-coupled protein-mediated signal transduction events that lead to the perception of light (2Hubbard R. Wald G. Cis-trans isomers of vitamin A and retinene in the rhodopsin system.J. Gen. Physiol. 1952; 36: 269-315Crossref PubMed Scopus (235) Google Scholar, 3Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. 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Transcriptome analysis reveals rod/cone photoreceptor specific signatures across mammalian retinas. Hum. Mol. Genet. Epub ahead of print. August 9, 2016; doi:10.1093/hmg/ddw268.Google Scholar). This storage form of 11-cis retinoids can be readily converted into 11-cis-retinal to support cone function. Although most of the enzymes involved in the cone visual cycle remain to be uncovered, two enzymes, including dihydroceramide desaturase 1 (DES1) (23Kaylor J.J. Yuan Q. Cook J. Sarfare S. Makshanoff J. Miu A. Kim A. Kim P. Habib S. Roybal C.N. Identification of DES1 as a vitamin A isomerase in Muller glial cells of the retina.Nat. Chem. Biol. 2013; 9: 30-36Crossref PubMed Scopus (84) Google Scholar) and acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), (24Kaylor J.J. Cook J.D. Makshanoff J. Bischoff N. Yong J. Travis G.H. Identification of the 11-cis-specific retinyl-ester synthase in retinal Muller cells as multifunctional O-acyltransferase (MFAT).Proc. Natl. Acad. Sci. USA. 2014; 111: 7302-7307Crossref PubMed Scopus (38) Google Scholar) were recently proposed to be involved in this metabolic pathway. In the current model, DES1 catalyzes equilibrium isomerization of retinol yielding a mixture containing 9-cis, 11-cis, 9,13-di cis, and 13-cis-retinol, and all-trans-retinol in a ratio that closely resembles a thermodynamic equilibrium (23Kaylor J.J. Yuan Q. Cook J. Sarfare S. Makshanoff J. Miu A. Kim A. Kim P. Habib S. Roybal C.N. Identification of DES1 as a vitamin A isomerase in Muller glial cells of the retina.Nat. Chem. Biol. 2013; 9: 30-36Crossref PubMed Scopus (84) Google Scholar). Interestingly, 11-cis-retinol accounts for less than 1% of total retinol isomers in this mixture, yet only 11-cis-retinyl esters are accumulated in the ocular tissue. The enzyme that preferentially esterifies 11-cis-retinol has been proposed to be AWAT2 (24Kaylor J.J. Cook J.D. Makshanoff J. Bischoff N. Yong J. Travis G.H. Identification of the 11-cis-specific retinyl-ester synthase in retinal Muller cells as multifunctional O-acyltransferase (MFAT).Proc. Natl. Acad. Sci. USA. 2014; 111: 7302-7307Crossref PubMed Scopus (38) Google Scholar). Surprisingly, this integral membrane enzyme exhibits broad substrate specificity. In addition to vitamin A and its isomers, AWAT2 esterifies aliphatic fatty alcohols of various chain lengths (25Yen C.L. Brown C.H.t. Monetti M. Farese Jr, R.V. A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters.J. Lipid Res. 2005; 46: 2388-2397Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 26Turkish A.R. Henneberry A.L. Cromley D. Padamsee M. Oelkers P. Bazzi H. Christiano A.M. Billheimer J.T. Sturley S.L. Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily.J. Biol. Chem. 2005; 280: 14755-14764Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 27Miklaszewska M. Kawinski A. Banas A. Detailed characterization of the substrate specificity of mouse wax synthase.Acta Biochim. Pol. 2013; 60: 209-215Crossref PubMed Scopus (12) Google Scholar). Based on the published kinetic parameters of human AWAT2 and the cis-retinol composition resulting from DES1 activity, one would expect significant accumulation of retinyl ester isomers different than 11-cis, which is not observed in vivo. To scrutinize the function of AWAT2 in isomer-specific retinol esterification, we evaluated the enzymatic activity of mouse AWAT2 toward retinol isomers and examined the factors that affect substrate specificity of the enzyme. We show that AWAT2 enzymatic activity is allosterically modulated by 11-cis retinoids. This regulatory mechanism leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols. Furthermore, by using isotope-labeled substrates, we demonstrate that esterification of 11-cis-retinol does not depend on cellular retinaldehyde-binding protein (CRALBP). These data provide a mechanism by which AWAT2 gains substrate specificity as needed for the preferential accumulation of 11-cis-retinyl esters in the eye. The all-trans-retinal, deuterated all-trans-retinal (5D-all-trans-retinal), all-trans-retinol, 9-cis-retinal, 11-cis-retinal, and 13-cis-retinal, as well as 9-cis-retinyl palmitate and palmityl myristate, were purchased from Toronto Research Company. Alternatively, cis retinoids and their esters were synthetized and purified as described below. All-trans-retinyl palmitate, lauryl chloride, palmitoyl chloride, dodecanol, and tetradecanol were obtained from Sigma-Aldrich, whereas acyl-CoAs were purchased from Avanti Polar Lipids. HPLC grade solvents were purchased from Fisher Scientific. To obtain geometric isomers of retinal or deuterated retinal, a quartz cuvette containing 0.5 ml of 40 mM all-trans-retinal in acetonitrile was placed on ice and exposed to light emitted by a 250 watt halogen bulb equipped with UV light filter. The lamp was positioned 25 cm above the cuvette. After 15 min, the retinoids were extracted with 5 ml of hexane. Collected hexane fraction was washed twice with a saturated solution of NaCl and dried under a stream of nitrogen. The residual mixture of retinal isomers was resolubilized in 0.5 ml of hexane. Separation of the geometric isomers was achieved by HPLC using an Agilent 1100 series system (Agilent Technologies) equipped with a diode-array detector. Retinals were eluted from a Luna silica column (250 × 21.2 mm, 10 μm) (Phenomenex) with an isocratic flow of 10% ethyl acetate in hexane (v/v) at the flow rate of 5 ml/min. The elution profile of retinoids was monitored at 360 nm. Chromatographic peaks that corresponded to 9-cis, 11-cis, 13-cis, and all-trans isomers were collected into separate tubes and the organic solvents were evaporated in a SpeedVac (Eppendorf). Purified retinals were redissolved in 0.5 ml of ethanol and stored at −80°C. Concentrations of the ethanolic stock solutions of retinals were determined spectrophotometrically using the following molar extinction coefficients: 36,100, 24,935, 35,500, and 42,880 M−1cm−1 for 9-cis, 11-cis, 13-cis, and all-trans isomers, respectively (28Hubbard R. Brown P.K. Bownds D. Methodology of vitamin A and visual pigments.Methods Enzymol. 1971; 18C: 615-653Crossref Scopus (191) Google Scholar, 29Robeson C.D. Blum W.P. Dieterle J.M. Cawley J.D. Baxter J.G. Chemistry of vitamin A. XXV. Geometrical isomers of vitamin A aldehyde and an isomer of its α-ionone analog.J. Am. Chem. Soc. 1955; 77: 4120-4125Crossref Scopus (39) Google Scholar). This procedure yielded 9-cis, 11-cis, 13-cis, and all-trans isomers of retinal in 10.9/26.1/17.5/45.5 ratio. Retinals were reduced to the corresponding retinols by reacting them with ∼4 molar excess of sodium borohydride in 2 ml of ice-cold ethanol. Progress of these reactions was monitored by recording blue shift of the maximum absorbance of the reaction mixture from ∼360 to ∼325 nm, depending on the isomer. Upon completion, 2 ml of water were added to the reactions and retinoids were extracted with 4 ml of hexane. The organic phase was washed with 4 ml of saturated NaCl solution and dried in a SpeedVac. To verify purity of the final products, small aliquots were injected onto a Luna PREP silica column (250 × 4.6 mm, 10 μm) (Phenomenex) and eluted with 10% ethyl acetate in hexane (v/v) at the flow rate of 2 ml/min. Purified retinols were redissolved in 0.5 ml of ethanol and stored at −80°C. Retinyl esters were prepared by the reaction between retinols and 2 molar excess of either acetic anhydride, palmitoyl chloride, or lauryl chloride in anhydrous dichloromethane in the presence of N,N-dimethylaminopyridine at 4°C for 4 h. The reaction was terminated by the addition of water and the retinoids were extracted with hexane. Completeness of the reaction and purity of retinyl esters were checked chromatographically using a Luna PREP silica column (250 × 4.6 mm, 10 μm). Retinyl esters and their geometric isomers were separated in 1% ethyl acetate in hexane (v/v), whereas retinols were eluted by increasing ethyl acetate concentration to 10% (v/v). In both cases, the flow rate was 2 ml/min. The isomers were identified based on their characteristic shape and maxima of the absorbance spectra (4Kiser P.D. Golczak M. Palczewski K. Chemistry of the retinoid (visual) cycle.Chem. Rev. 2014; 114: 194-232Crossref PubMed Scopus (231) Google Scholar). cDNA of mouse AWAT2 (NCBI accession number NM_177746) was purchased from OriGene Technologies. To prepare an expression vector, the cDNA was amplified by PCR using the following primer pair: forward, GCAGATACTAGTGTTTAATTATCAAACAATATCAATAATGTTCTGGCCCACCAAGAAGGACC and reverse, CGTCTAGACGCGTTCAAACTATCACCAGCTCCTGGGTCTCTGAGATGCC. The PCR product was digested with SpeI and MluI restriction enzymes and sub-cloned into the YepM vector (30Figler R.A. Omote H. Nakamoto R.K. Al-Shawi M.K. Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein.Arch. Biochem. Biophys. 2000; 376: 34-46Crossref PubMed Scopus (87) Google Scholar). Saccharomyces cerevisiae strain BJ5457 (ATCC) was transfected by alkali-cation yeast transformation kit (MP Biomedicals) and the cells were plated on a leucine-deficient selection medium (−Leu) (MP Biomedicals). Colonies of yeast were picked up from the plate and inoculated into 25 ml of −Leu medium that contained 10% glycerol (v/v). The culture was incubated at 30°C for 16 h prior to transfer into 2 l of fresh −Leu/glycerol medium. Yeasts were grown until OD600 reached 1.2–1.4. The cells were harvested by centrifugation (6,000 g, 15 min), resuspended in 40 mM Tris/HCl (pH 8.0) with 250 mM sucrose, and disrupted by microfluidization at 100 psi (five cycles). Cell homogenate was spun to remove large cellular debris at 12,000 g for 20 min. The resulting supernatant was then centrifuged at 120,000 g for 1 h to collect the microsomal fraction, which was subsequently resuspended in 50 mM Tris/HCl (pH 8.0) and 250 mM sucrose, aliquoted, and stored at −80°C. Expression of AWAT2 in transfected yeasts was confirmed by Western blot analysis with anti-AWAT2 polyclonal antibody (NBP1-91574; Novus Biologicals). To generate glutathione S-transferase (GST)-AWAT2 construct, cDNA of mouse AWAT2 was first subcloned in pGex-2T vector (GE Healthcare) using the following primers: forward, GCAGATGGATCCTTCTGGCCCACCAAGAAGGACC; reverse, CGTCTAGAATTCTCAAACTATCACCAGCTCCTGGGTCTCTGAGATGC; and EcoRI/BamHI restriction sites. Then, cDNA encoding GST-AWAT2 fusion protein was amplified by PCR with the following primers: forward, GCAGATACTAGTGTTTAATTATCAAACAATATCAATAATGTTCTGGCCCACCAAGAAGGACC and reverse, CGTCTAGACGCGTTCAAACTATCACCAGCTCCTGGGTCTCTGAGATGCC. After digestion with SpeI and MluI restriction enzymes, the PCR product was ligated into YepM vector. Expression of the fusion protein and purification of the yeast microsomal fraction was conducted as described above. To purify GST-AWAT2, the yeast microsomes were incubated with 20 mM n-dodecyl-β-D-maltoside (DDM) in 5.4 mM sodium phosphate dibasic, 1.3 mM potassium phosphate monobasic, 137 mM NaCl, and 2.7 mM KCl solution (PBS) for 16 h at 4°C and spun at 140,000 g for 1 h at 4°C. The supernatant was diluted with PBS to lower the concentration of DDM to 2 mM and subsequently incubated with glutathione-Sepharose (GE Healthcare) for 3 h at 4°C. The resin was then placed in a chromatography column and washed with 10 column volumes of PBS. GST-AWAT2 was eluted with 10 mM reduced glutathione in PBS. Fractions containing the protein were pooled together, concentrated in a Centricon (Amicon) cutoff of 100 kDa, and loaded onto a Superdex 200 Increase 10/300 GL size exclusion column (GE Healthcare) equilibrated with 2 mM DDM in PBS. The protein was eluted with isocratic flow of the equilibration buffer (1 ml/min). A purified GST-AWAT2 sample at 0.2 mg/ml concentration was applied onto a glow-discharged carbon-coated copper Quantifoil R2/1 grid (Quantifoil Micro Tools; 400 mesh) and stained using 2% (w/v) uranyl acetate. Briefly, the grid was blotted with purified sample and washed twice with distilled water prior to uranyl acetate staining (1 min). Micrographs were collected on an FEI Tecnai F20 TWIN microscope with a Tietz TemCam-F416 complementary metal-oxide semiconductor (CMOS)-based camera (4k × 4k). The negative stained micrographs were processed with RELION 1.4 software (31Scheres S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination.J. Struct. Biol. 2012; 180: 519-530Crossref PubMed Scopus (3026) Google Scholar). Briefly, 1,603 particles were manually picked to generate reference-free 2D classes (32Scheres S.H. Semi-automated selection of cryo-EM particles in RELION-1.3.J. Struct. Biol. 2015; 189: 114-122Crossref PubMed Scopus (242) Google Scholar). After expanding the dataset to 4,509 particles, different trials of 3D classification were performed with multiple initial models of GST and GST-AWAT2 without imposing symmetry restraints. The 3D classes revealed the particle size distribution of the negative stained dataset. The 3D classification trial with reasonable 3D volume classes (using volume/mass conversion of 0.81 Da/Å) was used to reconstruct a 3D structure. All 3D maps were visualized in UCSF Chimera (33Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera–a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (28163) Google Scholar). A larger dataset of the same negative stain conditions (12,846 particles) was used to confirm the particle size distribution. A clone of human CRALBP (NCBI accession number P12271.2) incorporated into pET19b vector was obtained from Dr. J. W. Crabb (Cole Eye Institute) (34Crabb J.W. Chen Y. Goldflam S. West K. Kapron J. Methods for producing recombinant human cellular retinaldehyde-binding protein.Methods Mol. Biol. 1998; 89: 91-104PubMed Google Scholar). Bl21(DE3) Escherichia coli competent cells (Invitrogen) were transfected with this construct and a single colony was used to inoculate 25 ml of LB medium (Thermo Fisher) containing 50 μM ampicillin. Bacterial culture was grown overnight at 37°C and subsequently transferred to 2 l of fresh medium. The culture was shaken at 30°C for around 4 h until OD600 reached 0.4–0.6. Then, the temperature was lowered to 25°C and expression of CRALBP was induced by adding isopropyl-1-thio-D-galactopyranoside to a final concentration of 0.15 mM. At the same time, ampicillin concentration was increased to 100 μM. Four hours later, bacteria were harvested by centrifugation at 6,000 g for 15 min at 4°C. Bacteria pellet was resuspended in water and frozen at −80°C. The cells were lysed by osmotic shock (35Burger A. Berendes R. Voges D. Huber R. Demange P. A rapid and efficient purification method for recombinant annexin V for biophysical studies.FEBS Lett. 1993; 329: 25-28Crossref PubMed Scopus (67) Google Scholar) and sonicated (three times for 1 min). The resulting crude lysate was spun down at 36,000 g for 20 min at 4°C. The soluble fraction, after adjustment of its buffer composition to 50 mM Tris/HCl (pH 8.0), 250 mM NaCl, and 5 mM imidazole, was loaded onto a HisTrap HP, 5 ml column (GE Healthcare) equilibrated with loading buffer [50 mM Tris/HCl (pH 8.0) that contained 250 mM NaCl and 5 mM imidazole]. After loading, the column was washed with 10 column volumes of the loading buffer and CRALBP was eluted with gradient of imidazole in the loading buffer (5–300 mM, flow rate 1 ml/min for 30 min). Fractions containing protein were pooled together, concentrated on a Centricon (Amicon) cutoff 10 kDa to about 5 ml, diluted 10-fold with 10 mM Tris/HCl (pH 8.0), and loaded onto a UNO Q1 (BioRad) ion exchanger column. CRALBP was eluted with gradient of NaCl (0–0.5 M) over 30 min at a flow rate of 0.5 ml/min. Collected fractions were examined by SDS-PAGE, and those containing purified apo-CRALBP were pooled together, concentrated to 3 mg/ml, and 0.2 ml aliquots were stored at −80°C. The holo-CRALBP was prepared by incubation 2 mg of the apolipoprotein with 2 molar excess of 11-cis-retinol delivered in DMSO (2%, v/v) for 30 min on ice in 10 mM Tris/HCl (pH 8.0), 10% glycerol (v/v). To remove unbound retinoid, the protein solution was diluted 10-fold with 10 mM Tris/HCl (pH 8.0), centrifuged at 36,000 g for 15 min at 4°C, and holo-CRALBP was repurified on a UNO Q1 column, as described above. The effectiveness of holo-CRALBP formation was verified spectrophotometrically by recording UV/Vis absorbance spectrum of the purified protein sample. The complex of CRALBP with 11-cis-retinol revealed a maxima absorbance ratio A344/A278 of 0.9 that corresponded to molar ratio of retinoid to the protein of 0.87, as determined by HPLC-based quantification of 11-cis-retinol extracted from holo-CRALBP. To test substrate specificity of AWAT2 toward geometric isomers of retinol, 9-cis, 11-cis, 13-cis, or all-trans-retinol was added to a reaction mixture containing 20 mM Tris/HCl (pH 7.0), 0.4 mM acyl-CoA, and 2 μl of microsomes isolated from yeast expressing AWAT2 (8 μg of total protein). The retinoids were delivered in 2 μl of N,N-dimethylformamide to a final concentration of 100 μM. The total volume of the reaction mixture was 0.2 ml. Although C12:0-, C16:0-, and C18:1-CoA species were tested, myristoyl-CoA (C14:0) was chosen as a primary source of acyl moiety in most of the experiment because of the highest AWAT2 activity in the presence of this acyl-CoA as well as better HPLC separation of 9-cis-retinyl myristate from 11-cis-retinyl palmitate that was used in further experiments. The enzymatic reaction was carried out at 37°C for up to 150 min and quenched with 0.2 ml of ethanol. The retinoids were extracted with 0.3 ml of hexane. The retinoid composition was examined by normal phase HPLC using a Luna PREP silica column (250 × 4.6 mm, 10 μm) (Phenomenex) with a step gradient of 1% (v/v) ethyl acetate in hexane over 10 min followed by 20 min of 10% (v/v) ethyl acetate in hexane at a flow rate of 2 ml/min. Retinyl esters were detected at 325 nm and quantified by correlating peak areas with known quantities of synthetic standards. To determine the respective Km and Vmax values for each retinol isomer, the initial velocity of the enzymatic reaction was calculated in the presence of increasing substrate concentration. The substrate concentrations ranged between 1 and 140 μM. The reaction was carried out for 5 min for 11-cis-retinol and 10 min for each of the other retinol isomers. GraphPad software was used to calculate the kinetic parameters by fitting the experimental data to the Hill model (equation 1) via nonlinear regression. υ=Vmax×[Sh]/(Km)h+[S]h(Eq. 1) To evaluate the substrate preference of AWAT2 in the presence of a mixture of the different retinol isomers, equimolar concentrations of isomers (25 μM each) were added to the same reaction. The reaction was incubated for 30 min prior to the retinoid extraction. All other conditions and analytical procedures were as described above. The initial velocity of AWAT2-dependent esterification was measured in the presence of 0, 2, 4, 6, or 10 μM of 11-cis-retinyl palmitate and variable concentrations of 9-cis-retinol. The inhibition constant (Ki) values were estimated by calculating the x-intercept from the equation resulting from the linear regression of the Dixon (36Dixon M. The determination of enzyme inhibitor constants.Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3288) Google Scholar) and C
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