Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans

Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans

Journal Pre-proof Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans Jasper Wattjes, Anna Niehues,...

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Journal Pre-proof Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans Jasper Wattjes, Anna Niehues, Bruno M. Moerschbacher

PII:

S0144-8617(19)31352-9

DOI:

https://doi.org/10.1016/j.carbpol.2019.115684

Reference:

CARP 115684

To appear in:

Carbohydrate Polymers

Received Date:

22 August 2019

Revised Date:

29 October 2019

Accepted Date:

26 November 2019

Please cite this article as: Wattjes J, Niehues A, Moerschbacher BM, Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115684

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans Jasper Wattjesa Anna Niehuesa1, and Bruno M. Moerschbachera* a

Institute for Biology and Biotechnology of Plants, University of Muenster, Schlossplatz 8, 48143 Münster, Germany Jasper Wattjes, [email protected] Anna Niehues, [email protected]

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Corresponding author: Prof. Bruno M. Moerschbacher, Institute for Biology and Biotechnology of Plants, University of Muenster, Schlossplatz 8, 48143 Münster, Germany. E-Mail: [email protected]; Tel: +49 251 8324794; Fax: +49 251 8328371 1

Present address: Center for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

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Graphical Abstract

Highlights -

Enzymatic-mass spectrometric fingerprinting analysis for chitosan FA determination Independent of pattern of acetylation and the intermolecular variation in FA Small sample amounts in the microgram scale are sufficient for analysis The method is as precise as other standard techniques like NMR and titration techniques

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Abstract We developed a rapid and precise method to determine the fraction of acetylation (FA) of unknown chitosan samples using a combination of enzymatic sample hydrolysis, isotopic labeling, and HILICESI-MS analysis. Chitosans are β-(1,4)-linked, partially N-acetylated and linear polyglucosamines representing an interesting group of functional biopolymers with a broad range of applications. For a better understanding of their structure-function relationships, it is key to have sensitive, accurate structural analysis tools available to determine parameters like the degree of polymerization (DP), fraction of acetylation (FA), or pattern of acetylation (PA). Here, we describe an improved enzymatic / mass spectrometric method for FA analysis of chitosan polymers. In contrast to the original chitinosanase-based mass spectrometric fingerprinting analysis of FA, the new method is independent of the PA and the intermolecular variation in FA (ĐFA) of the chitosan sample. This allows accurate analysis of heterogeneously de-N-acetylated samples representing the majority of commercially available chitosans1 2

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Abbreviations: DP, degree of polymerization; FA, fraction of acetylation; PA, pattern of acetylation; ĐFA , intermolecular variation in FA

Keywords

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Chitosan, FA determination, chitosan hydrolases, enzymatic fingerprinting, mass spectrometry Introduction

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Chitosans are among the most promising functional biopolymers, with superb material properties and versatile biological functionalities, making them ideal candidates for a plethora of applications in e.g. agriculture and biomedicine, food industry and material sciences (Younes & Rinaudo, 2015). Efficient exploitation of this potential requires understanding of structure-function relationships on the molecular or nanoscale level. Chitosans are linear co-polymers and oligomers consisting of β(1,4)-linked 2-acetamido-2-deoxy-D-glucopyranosyl (GlcNAc, A) and 2-amino-2-deoxy-Dglucopyranosyl (GlcN, D) units. The free amino groups of GlcN units convey a unique polycationic character to chitosans at slightly acidic pH values, while the methyl groups of the GlcNAc units convey hydrophobicity. This partly cationic - partly hydrophobic nature of chitosans leads to specific molecular interactions with partly anionic - partly hydrophobic biological macromolecules and structures, such as many proteins, nucleic acids, and membranes. As a consequence, the biological activities of chitosan oligomers and polymers are dependent on a number of structural parameters, most importantly the degree of polymerization (DP), the fraction of acetylation (FA), and the pattern of acetylation (PA). While the influence of DP and FA on bioactivities is fairly well understood (El Gueddari & Moerschbacher, 2004; Tokura, Ueno, Miyazaki, & Nishi, 1996; Younes, Sellimi, Rinaudo, Jellouli, & Nasri, 2014), that of PA is only recently being studied (Wattjes et al., 2019). In addition to biological functionalities, the physico-chemical characteristics of chitosans, such as viscosity, gelling behavior or nanoparticle formation are strongly influenced by DP, FA, and most likely PA (SantanderOrtega, Peula-García, Goycoolea, & Ortega-Vinuesa, 2011; Schatz, Viton, Delair, Pichot, & Domard, 2003; Sreekumar, Goycoolea, Moerschbacher, & Rivera-Rodriguez, 2018). Hence, reliable, accurate, and sensitive analytical methods are required for investigating molecular structure-function relationships of chitosans. In particular, FA has been shown to exert a strong influence on the physico-chemical properties (Schatz et al., 2003) and biological activities of chitosan polymers (El Gueddari & Moerschbacher, 2004). We recently described a method for enzymatic-mass spectrometric fingerprinting analysis for FA determination (Niehues, Wattjes, Bénéteau, RiveraRodriguez, & Moerschbacher, 2017). In contrast to NMR, the current gold standard for FA 2

Material and methods Chitosan samples.

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determination, this technique requires only microgram amounts of sample and allows a mediumthroughput approach. The method is based on the absolute cleavage specificity of a novel chitosan hydrolase, chitinosanase (Kohlhoff et al., 2017), that leads to a dependency of the enzymatic fingerprints on the FA of the substrate. Using simulated enzymatic fingerprints of chitosans with random PA, it was possible to build a partial least squares regression (PLSR) model able to predict FA values of unknown chitosan samples prepared by partial chemical N-acetylation, and the results were validated using 1H-NMR measurements. As the method is based on the sequence specificity of chitinosanase, we expected it to be sensitive not only to FA but also to PA. Indeed, using chitinosanase-based fingerprinting analyses, we showed that partial enzymatic de-N-acetylation of highly acetylated random PA-chitosans can yield chitosans with low FA and different non-random PA, depending on the chitin deacetylase enzyme used (Wattjes et al., 2019). However, as a consequence of this PA dependency, chitinosanase-based FA determination as described previously can be expected to be limited to chitosans with random PA and might not be reliable for chitosans that have a non-random PA. The situation is further complicated by the fact that chitosan polymer samples are invariably mixtures of molecules with different DP and different FA. Hence, to fully describe a chitosan sample, even one with known random PA, it would be required to not only indicate its average DP and average FA, but also its dispersity in both DP (ĐDP) and FA (ĐFA) (Cord-Landwehr, Niehues, Wattjes, & Moerschbacher, 2019). Commercially available chitosans are mostly produced by a heterogeneous alkaline de-N-acetylation process, most likely resulting in products that indeed have a high dispersity ĐFA. Theoretical considerations suggest and in silico simulations confirm that chitinosanase-based fingerprinting is also influenced by ĐFA. We here present a new enzymatic-mass spectrometric fingerprinting method for the analysis of FA of an unknown chitosan sample that is independent of both PA and ĐFA. Based on previously published approaches (Nanjo, Katsumi, & Sakai, 1991) we chose two well-characterized chitosanolytic enzymes, namely chitinase ChiB from Serratia marcescens (Vaaje-Kolstad, Horn, Sørlie, & Eijsink, 2013) and chitosanase CSN-174 from Streptomyces sp. N174 (Fukamizo, Honda, Goto, Boucher, & Brzezinski, 1995). In contrast to chitinosanase with its unusually high sequence specificity (DA│XX) (Kohlhoff et al., 2017), these enzymes are characterized by rather low sequence specificities (ChiB XA│XX (Cord-Landwehr et al., 2017); CSN-174 DX│XX (Weikert, Niehues, Cord-Landwehr, Hellmann, & Moerschbacher, 2017), where X can be either A or D). The resulting paCOS were chemically N-acetylated using deuterated acetic anhydride and finally analyzed using HILIC-ESI-MS, as previously described (Cord-Landwehr et al., 2017).

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Heterogeneously de-N-acetylated chitosans were obtained from the three companies Primex ehf. (Siglufjörður, Iceland), Heppe Medical Chitosan GmbH (Halle, Germany), and Gillet Chitosan eurl (Plumaudan, France). Homogeneously N-acetylated chitosan samples of varying FA were produced from fully de-N-acetylated chitosan (FA = 0) provided by Mahtani Chitosan Pvt. Ltd. (Veraval, Gujarat, India). Chemical N-acetylation was carried out using acetic anhydride as previously described (Lamarque, Lucas, Viton, & Domard, 2005). 2.2

Recombinant enzymes.

Chitinase ChiB from Serratia marcescens and chitosanase CSN-174 from Streptomyces sp. N174 were produced as recombinant enzymes as previously described (Hamer, Moerschbacher, & Kolkenbrock, 2014; Nampally, Moerschbacher, & Kolkenbrock, 2012). 2.3 FA determination by hydrophilic interaction liquid chromatography – electrospray ionization – mass spectrometry (HILIC-ESI-MS). 3

𝑫𝑷

𝑫𝑷=𝟐 𝒊=𝟎

𝟓

𝑫𝑷

𝒊 𝑰 }⁄{ ∑ ∑ 𝑰𝒊 } 𝑫𝑷 𝒊

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𝐹𝐴 = { ∑ ∑

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For FA determination, we hydrolyzed 30 µg of chitosan (1 µg/µl) by incubation for 2 h with 1 µg each of ChiB and CSN-174. The reaction was carried out in 150 mM ammonium acetate buffer (pH 4.3) at 37°C. Samples were vacuum-dried. For FA determination, samples were chemically N-acetylated and isotopically labeled as previously described (Cord-Landwehr et al., 2017). For MS measurements, 1 µl of each sample (1 µg/µl) was injected into the system. Samples were measured on a Dionex Ultimate 3000RS UHPLC system (Thermo Scientific, Milford, USA) equipped with an Acquity UPLC BEH Amide column (1.7 µm, 2.1 mm x 150 mm; Waters Corporation, Milford, MA, USA) and a VanGuard precolumn (1.7 µm, 2.1 mm x 5 mm; Waters Corporation, Milford, MA, USA), and coupled to an amaZon speed ESI-MSn detector (Bruker Daltonik, Bremen, Germany). Oligomers were separated over a 20.5 min gradient elution profile: 0-3 min isocratic 100% A (80:20 ACN:H2O with 10 mM NH4HCO2 and 0.1% (v/v) HCOOH); 3-12.5 min linear from 0% to 17% B (20:80 ACN:H2O with 10 mM NH4HCO2 and 0.1% (v/v) HCOOH); 12.5-14.5 min linear from 17% B to 75% B; 14.5-15.5 min isocratic 75% B; column re-equilibration: 15.5-16.5 min linear from 75% B to 100% A; 16.5-20.5 min isocratic 100% A. Samples were measured in positive ion mode. MS data were evaluated as previously described (Cord-Landwehr et al., 2017). Finally, the ratio of relative intensities of acetylated (GlcNAc, A) and isotopically labeled N-acetylated units ([2H3] N-acetyl-D-glucosamine, R) were used for FA determination. To this end, the total intensity of each oligomer was multiplied by their corresponding FA values, all values were summed up and finally divided by the total intensity of all oligomers to yield final FA values. Therefore FA values can be calculated as follows

𝑫𝑷=𝟐 𝒊=𝟎

High-performance size-exclusion chromatography mass spectrometry (HP-SEC-MS)

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where 𝒊 represents the number of acetylated units and I the intensity measured by HILIC-ESI-MS for a given oligomer.

2.5

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Samples before and after enzymatic hydrolysis were analyzed using HP-SEC-MS. Separation was achieved based on a method previously described for the analysis of pectins (Voxeur et al., 2019). Therefore samples were diluted at 1 mg/ml in ammonium formate 50 mM, formic acid 0.1%. Chromatographic separation was performed on an ACQUITY UPLC Protein BEH SEC Column (125 Å, 1.7 μm, 4.6 mm X 300 mm, Waters Corporation, Milford, MA, USA) and elution was carried out using 50 mM ammonium formate, formic acid 0.1% at a flow rate of 400 µl/min and a column oven temperature of 40°C. The injection volume was set to 10 µl. Samples were measured on the same UPLC-system and MS data were evaluated as described in 2.3. H-NMR measurements.

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Proton NMR was used to investigate the FA of chitosan samples as described by Hirai et al. (Hirai, Odani, & Nakajima, 1991). Briefly, approximately 15 mg of chitosan were dissolved overnight in 1 ml acidic solution of D2O (1 ml 99.9% D2O and 2 µl DCl) and were subsequently lyophilized. Then, samples were redissolved in D2O and 1H-NMR spectra were recorded using an Agilent DD2 NMR Spectrometer (Agilent, Santa Clara, USA). Samples were measured at 500 MHz and 348 K. NMR measurements were carried out at the Institute for Organic Chemistry, University of Münster. 3.

Results and Discussion

In principle, the dependency on PA and ĐFA could be resolved by simply hydrolyzing the chitosan polymer completely into monomers, then quantifying the molar ratio of GlcN and GlcNAc units. However, complete chemical hydrolysis is difficult to achieve without at least partial loss of acetyl 4

groups and moreover, GlcN monomers are not very stable under the hydrolysis conditions. Therefore, we opted for an enzymatic approach. We initially tried to achieve complete hydrolysis using different chitinases and chitosanases followed by glucosaminidases and Nacetylglucosaminidases based on previously published work (Nanjo et al., 1991). However, it turned out that the partially acetylated chitosan oligomers produced by the former enzymes were not quantitatively converted into monomers by the latter two enzymes. It was, therefore, necessary to determine the molar ratio of GlcN and GlcNAc units within the oligomers, using quantitative mass spectrometry as described previously.

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The newly developed FA determination method, thus, is based on enzymatic hydrolysis followed by isotopic labeling and HILIC-ESI-MS analysis. It allows FA determination of unknown chitosan samples at microgram scale (Figure 1). Chitosan polymers are hydrolyzed enzymatically to yield oligomeric products that in contrast to the parental, polymeric material can easily be analyzed using LC-MS approaches. For the enzymatic degradation, we used two well studied chitosanolytic enzymes, namely chitinase ChiB from Serratia marcescens and chitosanase CSN-174 from Streptomyces sp. N174. The combination of a chitinase and a chitosanase allows to cover a broad range of FA. In fact, the method is only limited by the solubility of the sample in aqueous solutions. In the case of chitosan polymers, this covers FA values ranging from 0 to approximately 0.6-0.7, where the upper limit is dependent on the DP of the polymers. To facilitate general sample handling, we tested both enzymes under different conditions such as salt concentration, pH, and temperature, aiming to identify conditions under which both enzymes work efficiently. Reasonable activity for both enzymes was found in 150 mM ammonium acetate buffer, pH 4.3 at 37°C. To ensure that no polymeric material remained in the sample after enzymatic treatment, we carried out HP-SEC-MS analyses (supplementary Figure S-3). After the treatment, the peak representing polymeric material completely disappeared indicating a complete hydrolysis of the starting material. After hydrolysis, GlcN units in the resulting oligomers were submitted to chemical N-acetylation and isotopic labeling using [2H6]-acetic anhydride to produce [2H]3- N-acetylglucosamine units (R), as previously described (Cord-Landwehr et al., 2017). Since fully acetylated COS with a DP larger than six become insoluble during this process, it was important to ensure that after hydrolysis, only products with a DP ≤ 5 remained to avoid loss of information. We validated the absence of such COS using HILIC-ESI-MS (supplementary Figure S-2). After chemical N-acetylation and labeling, we measured the samples again by HILIC-ESI-MS for the final FA determination.

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Figure 1. Overview FA determination. Chitinase ChiB from Serratia marcescens and chitosanase CSN-174 from Streptomyces sp. N174 were used to hydrolyze chitosan samples to oligomers of DP 2-5. Samples were N-acetylated and isotopically labeled using [2H6] acetic anhydride. HILIC-ESI-MS measurements were used to determine the ratio of N-acetylated and non-acetylated units that finally were used for FA calculation.

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The process of deuterated N-acetylation (converting D-units into R-units) equalizes retention and ionization behavior of the oligomers with identical DP, and at the same time maintains discriminability of acetylated and de-N-acetylated units. The step is necessary since it allows the calculation of the percentage of N-acetylated and de-N-acetylated units in the total amount of DP 2-5 oligomers. This ratio between A and R (former D) units can be used to calculate FA values based on the total ion intensities of all oligomers. The total composition of the N-acetylated and labeled oligomers is shown in Figure 2.

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Figure 2. Relative abundancies of DP 2-5 oligomers after chemical N-acetylation by [2H6]-acetic anhydride. Samples were measured in duplicates. The ratios of acetylated units (A) and N-acetylated units (R, former D) were used to calculate FA values.

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To validate the approach, we determined FA values of a broad range of chitosan samples with both the new method and 1H-NMR measurements (supplementary figure S-1). In addition, we also used the chitinosanase based FA determination method previously described by our group (Kohlhoff et al., 2017). Since PA and ĐFA of chitosan preparations are influenced by the production routes used, we included commercially available chitosans produced from chitin by heterogeneous de-N-acetylation, and lab-produced chitosans derived from polyglucosamine by homogeneous chemical N-acetylation. The results (Table 1) clearly show that both the current gold-standard for FA-determination, NMR, and the new fingerprinting method give very similar results. Commercial producers typically determine FA using titration as a method requiring less costly infrastructure. Though this method is more prone to yield erroneous results, the values provided by the manufactures are in good agreement with those obtained by the other two methods. On average, the deviation of our method from NMR values is < 0.02, and < 0.04 from titration values (supplementary table S-1), which is within the accuracy of NMR of ca. ± 0.05.

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Table 1. Comparison of the FA values determined for a range of chitosan samples using 1H-NMR, chitinosanase-based FA determination (“MSold”) as well as the new method described in this work (“MSnew”). MS measurements were carried out as four replicates from two batches of dissolved sample (rounded mean values are given; for original data, see supplementary tables S-1 and S-2). Moreover FA deviation values (Δ FA) of the new method from both 1H-NMR and titration are given as well as the average deviation. FA

FA

FA

FA

Δ FA1

Δ FA2

titration*

1H-NMR

MSnew

MSold

│NMR-MSnew│

│titr.-MSnew│

HMC 1

0.28*

0.30

0.26

0.39

0.04

0.02

HMC 2

0.28*

0.20

0.19

0.31

0.01

0.09

Gillet 1

0.20*

0.20

0.22

0.44

0.02

0.02

Gillet 2

0.10*

0.14

0.12

0.35

0.02

0.02

Sample

Primex 1

0.20*

0.20

0.20

0.42

0.00

0.00

Primex 2

0.12*

0.13

0.16

0.34

0.03

0.04

ChemAc 1

-

0.56

0.56

0.62

0.00

-

ChemAc 2

-

0.28

0.30

0.32

0.02

-

7

ChemAc 3

-

0.22

0.21

0.23

average

0.01

-

0.017

0.032

* titration, FA value given by manufacturer, determined using titration; MSnew, FA determined by new method; MSold, FA determined using chitinosanase-based fingerprinting, Δ FA1 │NMR-MSnew│, absolute deviation of the new method compared to 1H-NMR, Δ F 2 │titr.-MSnew│, absolute deviation of the new method compared to titration. A

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In contrast, the previously described fingerprinting method based on chitinosanase hydrolysis was only able to correctly determine the FA of homogeneously N-acetylated samples. Due to the sensitivity of this method to a non-random PA and a broad ĐFA, this fact may be taken as a strong indication that the commercial chitosans either have a non-random PA, or a broader ĐFA compared to the lab-produced chitosans generated by chemical N-acetylation, which have random PA. Given that a comparative analysis of a broad range of heterogeneously de-N-acetylated chitosan samples has shown that all of them appear to have more or less random PA (Weinhold, Sauvageau, Kumirska, & Thöming, 2009), our results support the idea that the commercial chitosan samples indeed possess a high ĐFA, i.e. a high intermolecular variation in FA that is influencing the chitinosanase-based method. This conclusion might also be supported by the observation (see supplementary tables S-1 and S-2) that two different batches taken from the same sample ‘Primex 2’ gave rather different FA values (while the duplicate determinations on both batches were highly reproducible). These differences in FA distribution have already been discussed and can be attributed to the heterogeneous production process of these samples (Chang, Tsai, Lee, & Fu, 1997). When working with heterogeneously de-Nacetylated samples, this fact should be taken into consideration since they still represent most of the commercially available chitosan samples today

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The fingerprinting method described here represents a rapid and precise alternative to currently existing methods for FA determination of unknown chitosan samples which is as precise as the current gold standard, NMR. It is highly sensitive, requiring only microgram amounts of sample, unlike NMR methods which require milligrams of chitosans. It is applicable to all samples that can be dissolved in aqueous solutions (FA 0 - ca. 0.6-0.7). In contrast to the previously described chitinosanase-based fingerprinting method, it is not dependent on the availability of a specific chitosan hydrolase but can be carried out using easily available recombinant chitinases and chitosanases. Moreover, the method is not affected by different PA or ĐFA of the chitosan polymers analyzed. Acknowledgements

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We would like to thank Dr. Dominique Gillet (Gillet Chitosan eurl, Mahtani Chitosan Pvt. Ltd.), Dr. Katja Richter (Heppe Medical Chitosan GmbH), and Dr. Hélène Liette Lauzon (Primex ehf.) for providing chitosan samples as well as Dr. Klaus Bergander (Institute for Organic Chemistry, University of Münster) for NMR measurements. Financial support from the German Bundesministerium für Ernährung und Landwirtschaft (BMEL) and its Fachagentur Nachwachsende Rohstoffe (FNR) under project number 22031315 is gratefully acknowledged. 6.

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