Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography

Materials and Reagents

1. Material for buffer exchange (see Notes)

   1. Locking clips, 45 mm (Carl Roth, catalog number: H277.1 )

   2. Dialysis membrane Spectra/Por 7 MWCO 10,000, 45 mm (Carl Roth, catalog number: E872.1 )

   3. Glass pipettes: 5, 10, 20 and 25 ml (Labdirect, catalog numbers: 021.01.005 , 355.050.110 , 355.050.120 , 355.050.125 )

   4. Pipette tips (Sarstedt, catalog numbers: 70.760.012, 70.762, 70.3020 and Biozym, catalog number: VZ0001X )

   5. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001 )

   6. Double-distilled water (ddH₂O) (Kerndl, catalog number: 22501 )

   7. Magnesium chloride hexahydrate (MgCl₂) (Carl Roth, catalog number: 2189.1 )

   8. Manganese (II) chloride tetrahydrate (MnCl₂) (Carl Roth, catalog number: T881.1 )

   9. Sodium chloride (NaCl) (VWR, catalog number: 27810.295DB )

   10. Tris-base (Carl Roth, catalog number: 4855.2 )

   11. Glycerol (Carl Roth, catalog number: 3783.2 )

   12. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )

       
       

2. Material for protein concentration determination (see Notes)

   1. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001 )

   2. Spacer plates and short plates (Bio-Rad, catalog numbers: 1653310 and 1653308 )

   3. Pipette tips (Sarstedt, catalog numbers: 70.760.012, 70.762, 70.3020 and Biozym, catalog number: VZ0001X )

   4. Tris-base (Carl Roth, catalog number: 4855.2 )

   5. Glycine (Carl Roth, catalog number: 00 79.4 )

   6. Colored Prestained Marker (NEB, catalog number: P7719S )

   7. LMW marker (GE Healthcare, catalog number: 17-0446-01 )

   8. Double-distilled water (ddH₂O) (Kerndl, catalog number: 22501 )

   9. Rotiphorese (Carl Roth, catalog number: 3029.1 )

   10. Ammonium peroxydisulphate (Carl Roth, catalog number: 9178.1 )

   11. TEMED (Carl Roth, catalog number: 2367.3 )

   12. Glycerol (Carl Roth, catalog number: 3783.2 )

   13. Sodium lauryl sulphate (Carl Roth, catalog number: 4360.2 )

   14. Bromophenol blue (Carl Roth, catalog number: A512.1 )

   15. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )

   16. 2-propanol (Carl Roth, catalog number: 6752.5 )

   17. Acetic acid (Carl Roth, catalog number: 6755.1 )

   18. Coomassie blue R 250 (Carl Roth, catalog number: 3862.2 )

       
       

3. Thin-layer chromatography (TLC) of enzymatic assays with radiolabeled nucleotides

   1. Pipette tips (Sarstedt, catalog number: 70.760.012 and Biozym, catalog number: VZ0001X )

   2. Pipette filter tips (Sarstedt, catalog number: 70.1116.210 )

   3. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001 )

   4. Thin-layer plate (Macherey-Nagel, POLYGRAM CEL 300 PEI, catalog number: 801053 )

   5. Phosphor imaging plate (Fujifilm, BAS-IP SR 2025 )

   6. Plastic wrap (any type obtained from a grocery store)

   7. Exposure cassette (GE Healthcare, catalog number: 63-0035-44 )

   8. Purified DisA from S. venezuelae or B. subtilis (Witte et al., 2008; Latoscha et al., 2020)

   9. Purified inactive DisA variant (DisA_D86A) from S. venezuelae (Latoscha et al., 2020)

   10. Purified AtaC from S. venezuelae (Latoscha et al., 2020)

   11. 9.25 MBq [α-³²P]-ATP (Hartmann Analytik, catalog number: FP-207 )

   12. 9.25 MBq [³²P]-c-di-AMP (Hartmann Analytik, catalog number: FP-C517 ; a purified c-di-AMP-producing enzyme, e.g., Bacillus subtilis DisA, must be provided to the company for [³²P]-c-di-AMP synthesis upon request)

   13. For phosphodiesterase competition assays:

       1. c-di-AMP (BioLog, catalog number: C 088 )

       2. c-di-GMP (BioLog, catalog number: C 057 )

       3. cAMP (Sigma-Aldrich, catalog number: A6885 )

   14. Magnesium chloride hexahydrate (MgCl₂) (Carl Roth, catalog number: 2189.1 )

   15. Manganese (II) chloride tetrahydrate (MnCl₂) (Carl Roth, catalog number: T881.1 )

   16. Sodium chloride (NaCl) (VWR, catalog number: 27810.295DB )

   17. Tris-base (Carl Roth, catalog number: 4855.2 )

   18. Hydrochloric acid (Carl Roth, catalog number: 4625.2 )

   19. Glycerol (Carl Roth, catalog number: 3783.2 )

   20. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )

   21. Double-distilled water (ddH₂O) (Kerndl, catalog number: 22501 )

   22. 0.5 M EDTA, pH 8 (PanReac AppliChem, catalog number: A4892 )

   23. Ammonium sulfate ((NH₄)₂SO₄) (VWR, catalog number: 21333.296DB )

   24. Monopotassium phosphate (KH₂PO₄) (Carl Roth, catalog number: P018.2 )

   25. DisA cyclase buffer (see Recipes)

   26. PDE reaction buffer (see Recipes)

   27. TLC running buffer (see Recipes)

       
       

4. IEX activity assay

   1. Pipette tips (Sarstedt, catalog numbers: 70.1130.600 and 70.760.502 )

   2. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001 )

   3. Microcon-30kDa Centrifugal Filter Unit (Merck Millipore, catalog number: MRCF0R030 )

   4. Purified AtaC from S. venezuelae (Latoscha et al., 2020)

   5. Purified TmPDE from T. maritima (Drexler et al., 2017)

   6. c-di-AMP (BioLog, catalog number: C 088 )

   7. Manganese (II) chloride tetrahydrate (MnCl₂) (Carl Roth, catalog number: T881.3 )

   8. Sodium chloride (NaCl) (Merck Millipore, catalog number: 1.06404.5000 )

   9. Tris-base (Carl Roth, catalog number: 4855.2 )

   10. Hydrochloric acid, 25% (VWR, catalog number: 20257.296 )

   11. 10x AtaC reaction buffer (see Recipes)

   12. IEX Running buffer A (see Recipes)

   13. IEX Running buffer B (see Recipes)

Equipment

1. Equipment for buffer exchange (see Notes)

   1. Measuring beaker, 5,000 ml (Carl Roth, catalog number: 0 780.1 )

   2. Magnetic bar (Carl Roth, catalog number: C267.1 )

   3. Pipetting aid macro (Carl Roth, catalog number: X478.1 )

   4. Micropipettes (P20, P200, P1000)

   5. Magnetic stirrer (Heidolph, model: MR 2000 )

   6. Centrifuge (Eppendorf, model: 5427 R, catalog number: 5409000010 )

   7. 4 °C room

      
      

2. Equipment for protein concentration determination of your choice (see Notes)

   1. Mini-PROTEAN Tetra Cell (Bio-Rad, catalog number: 1658000EDU )

   2. Heat block (Labnet International, model: AccuBlock^TM Digital Dry Bath, catalog number: D1302-230V )

   3. Centrifuge (Eppendorf, model: 5427 R, catalog number: 5409000010 )

   4. Power supply (Bio-Rad, catalog number: 1645050 )

   5. Imager with CCD camera (GE Healthcare, model: ImageQuant LAS 4000 )

   6. Micropipette (P2, P10, P20)

      
      

3. TLC of enzymatic assays with radiolabeled nucleotides

   1. Micropipettes (P2, P10, P20)

   2. Heat block (Labnet International, model: AccuBlock^TM Digital Dry Bath, catalog number: D1302-230V )

   3. Centrifuge (Eppendorf, model: 5427 R, catalog number: 5409000010 )

   4. Contamination monitor (Berthold, model: LB 122 )

   5. Biomolecular imager (GE Healthcare, model: Typhoon FLA 7000 , catalog number: 28-9558-09)

   6. Image eraser (Molecular Dynamics, model: 410A )

   7. Acrylic Benchtop Beta Radiation Shield (ThermoFisher Scientific, catalog number: 6700-2418 )

   8. TLC chamber (Fisher Scientific, catalog number: 06-815-187 )

   9. Fume hood

      
      

4. IEX assay

   1. Micropipettes (P2, P20, P200, P1000)

   2. NanoDrop spectrophotometer

   3. Thermomixer (Eppendorf, catalog number: 5382000015 )

   4. Centrifuge (Eppendorf, catalog number: 5401000010 )

   5. Äkta (Cytiva former GE Healthcare)

   6. Resource-Q or Mono-Q (Cytiva, catalog number: 17117701 or 17516601 )

Software

1. ImageQuantTL (optional) (GE Healthcarre)

2. Typhoon FLA 7000 control software (GE Healthcare)

3. PhotoShop CS6 (Adobe)

4. Unicorn (Cytiva former GE Healthcare)

Procedure

1. Diadenylate cyclase assay with radiolabeled ATP coupled with TLC

   1. Until Step A6, work on ice.

   2. Exchange the buffer of purified proteins to DisA cyclase buffer (e.g., via dialysis, see Notes).

   3. Determine the molar concentration of proteins (see Notes).

   4. Setup of the reaction:

      1. Calculate the volume of DisA required to achieve a concentration of 5 µM in 20 µl.

      2. Pipette the required volume of DisA into a 1.5 ml reaction tube (i.e., if the DisA stock has a concentration of 50 µM, you will need to take 2 µl).

      3. Fill up to a total volume of 19.5 µl with DisA cyclase buffer.

   5. From now on, work behind a radiation shield and use filter tips (see Notes).

   6. Add 0.5 µl [³²P]-ATP to the reaction (final concentration: ~83 nM in 20 µl).

   7. Use 19.5 µl DisA cyclase buffer with 0.5 µl [³²P]-ATP as a control for ATP migration.

   8. Incubate reactions for 60 min at 30 °C in the heat block.

   9. Pipette 5 µl 0.5 M EDTA, pH 8 into 1.5 ml reaction tubes (one tube per reaction).

   10. Add 5 µl of each reaction to a tube containing EDTA to stop diadenylate cyclase reaction.

   11. Spot the mixture from Step A10 on the cellulose side of a thin-layer (TL) plate (each spot should be ~1.5 cm from the bottom and lateral edges of the TL plate as well as other spots; also see Notes).

   12. Let the spots dry completely at room temperature (RT).

   13. Fill TLC chamber with TLC running buffer (buffer should be ~1 cm deep).

   14. Place the TL plate in the TLC chamber with spotted samples close to the buffer without touching it.

   15. Incubate TLC until the liquid front is ~1 cm from the upper edge of the TL plate (this can take up to ~2.5 h).

   16. Remove TL plate from the chamber and let dry at RT (overnight or at least 45 min in fume hood).

   17. Place an erased imaging plate (IP) into an exposure cassette.

   18. Wrap TL plate completely into plastic wrap (see Notes) and place with the cellulose side down on top of the IP for exposure.

   19. The exposure time may vary depending on the optimal signal-to-noise ratio, but ~30 min exposure will suffice in most cases.

   20. Remove the TL plate from the cassette and check cassette and IP for radioactive contamination using the contamination monitor (see Notes).

   21. If no contamination is present, scan the IP in the Typhoon FLA 7000 using the laser at 650 nm and IP filter.

       
       

2. Phosphodiesterase assay with radiolabeled c-di-AMP coupled with TLC

   1. Until Step B7, work on ice.

   2. Exchange the buffer of purified AtaC to PDE reaction buffer (e.g., via dialysis, see Notes).

   3. Determine the molar concentration of AtaC (see Notes).

   4. If you want to perform competition with unlabeled nucleotides, proceed with Step B5, without competition proceed with Step B6.

   5. Setup of the reaction with competition:

      1. Calculate the volume of AtaC required to achieve a concentration of 100 nM in 20 µl.

      2. Pipette the required volume of AtaC into a 1.5 ml reaction tube (i.e., if the AtaC stock has a concentration of 1 µM, you will need to take 2 µl).

      3. Fill up to a total volume of 17.5 µl with PDE reaction buffer.

      4. Set up 1 mM solutions of each unlabeled nucleotide (c-di-AMP, c-di-GMP, cAMP) in ddH₂O.

      5. Add 2 µl of each nucleotide per reaction in Step B5c.

      6. Set up a reaction with 2 µl ddH₂O as negative control for competition.

      7. Proceed with Step B7.

   6. Setup of the reaction without competition:

      1. Calculate the volume of AtaC required to achieve a concentration of 100 nM in 20 µl.

      2. Pipette the required volume of AtaC into a 1.5 ml reaction tube (i.e., if the AtaC stock has a concentration of 1 µM, you will need to take 2 µl).

      3. Fill up to a total volume of 19.5 µl with PDE reaction buffer.

      4. Proceed with Step B7.

   7. From now on, work behind a radiation shield and use filter tips (see Notes).

   8. Dilute [³²P]-c-di-AMP 1:20 in PDE reaction buffer and add 0.5 µl to the reaction (final concentration: ~2 nM in 20 µl).

   9. Use 19.5 µl PDE reaction buffer with 0.5 µl of 1:20 diluted [³²P]-c-di-AMP as a control for c-di-AMP migration.

   10. Incubate reactions for 60 min at 30 °C in the heat block.

   11. To stop enzymatic reaction, incubate at 95 °C for 5 min.

   12. Centrifuge reactions at maximal speed for 3 min.

   13. Spot 5 µl supernatant of each reaction on the cellulose side of a thin-layer (TL) plate (each spot should be ~1.5 cm from the bottom and lateral edges of the TL plate and other spots).

   14. Let the spots dry completely at room temperature (RT).

   15. Fill TLC chamber with TLC running buffer (buffer should be ~1 cm high).

   16. Place the TL plate in the TLC chamber with spotted samples close to the buffer without touching it.

   17. Incubate TLC until the liquid front is ~1 cm from the upper edge of the TL plate (this can take up to ~2.5 h).

   18. Remove TL plate from the chamber and let dry at RT (overnight or at least 45 min in fume hood).

   19. Place an erased imaging plate (IP) into an exposure cassette.

   20. Wrap TL plate completely into plastic wrap (see Notes) and place with the cellulose side down on top of the IP for exposure.

   21. The exposure time may vary depending on the optimal signal-to-noise ratio, but ~60 min exposure will suffice in most cases.

   22. Remove the TL plate from the cassette and check cassette and IP for radioactive contamination using the contamination monitor.

   1. If no contamination is present, scan the IP in the Typhoon FLA 7000 using the laser at 650 nm and IP filter.

      
      

3. IEX AtaC activity assay

   1. Set up 100 µl reactions containing 100 nM protein, 62.5 µM-2,000 µM substrate (e.g., c-di-AMP) and 1x reaction buffer. Control reactions containing protein or substrate only are necessary to exclude nonspecific results.

   2. Incubate the reaction at 37 °C for 1 h.

   3. Stop the reaction by transferring the complete reaction mix into a Microcon-30kDa Centrifugal Filter Unit and spinning for 10 min at 14,000 x g. This ultrafiltration step separates the protein from its reaction products, which are obtained in the filtrate.

   4. Repeat Step C3 if there is still liquid in the filter.

   5. Wash the filter by adding 100 µl running buffer A and repeating Step C3.

   6. Fill up the filtrate with running buffer A to 500 µl.

   7. Equilibrate an ion exchange column (1 ml Resource-Q or Mono-Q) in running buffer A.

   8. Inject the sample from step 6 and use a linear gradient from running buffer A to B (i.e., 0-40% B, 20 CV). Set the detection wavelength on 260 nm or 280 nm, if 260 nm is not possible.

Data analysis

1. TLC assay

   Images scanned in the Typhoon FLA 7000 sometimes require adjustment of contrast. If necessary, open the scanned file in PhotoShop and modify spot intensity for the whole picture via “Image” → “Adjustments” → “Levels” until an optimal contrast is reached. Exemplary DAC and PDE assays separated by TLC are shown in Figure 2.

   
   

   
   Figure 2. Thin-layer chromatography (TLC) of a diadenylate cyclase (A) and a c-di-AMP phosphodiesterase assay (B). A. Diadenylate cyclase (DAC) assay with 5 µM of purified S. venezuelae wild-type DisA (DisA_Sven, lanes 2 and 3) and inactive DisA_Sven mutant variant (DisA_D86A, lanes 4 and 5) incubated with 83 nM [³²P]-ATP at 30 °C. After 30 and 60 min samples were taken and inactivated by mixing with equal volume of 0.5 M EDTA, pH 8. Inactivated reactions were spotted on a thin-layer (TL) plate and separated via TLC. After drying, the TL plate was exposed for 30 min to an imaging plate which was subsequently scanned using Typhoon FLA 7000 . B. subtilis DisA (DisA_Bsu, lane 6) was used as a positive control for DAC reaction. Lane 1 shows migration of [³²P]-ATP in absence of enzymes. All samples were separated on the same TL plate and the picture was cropped to the relevant lanes. In contrast to the inactive DisA_D86A, DisA_Sven synthesizes [³²P]-c-di-AMP out of [³²P]-ATP as indicated by formation of a product that has the similar size like [³²P]-c-di-AMP produced by DisA_Bsu, which is a characterized DAC. B. Phosphodiesterase assay of S. venezuelae AtaC with competition with unlabeled nucleotides (lanes 3 to 5). 100 nM of purified protein was supplemented with 100 µM unlabeled c-di-AMP, c-di-GMP and cAMP (or ddH₂O as control, lane 2) before incubation with 2 nM [³²P]-c-di-AMP at 30 °C. After 60 min samples were taken and heat-inactivated. Precipitated protein was removed by centrifugation and supernatants of the inactivated reactions were spotted on a TL plate and separated via TLC. After drying, the TL plate was exposed for 60 min to an imaging plate which was subsequently scanned using Typhoon FLA 7000 . Lane 1 shows migration of [³²P]-c-di-AMP in absence of enzyme. AtaC cleaves [³²P]-c-di-AMP to [³²P]-AMP using [³²P]-pApA as an intermediate cleavage product (lane 2). This reaction is specific since unlabeled c-di-AMP significantly inhibits, and thus outcompetes [³²P]-c-di-AMP turnover (lane 3) whereas c-di-GMP and cAMP have no effect (lanes 4 and 5).

   
   

2. IEX assay

   The IEX assay can also be used for quantification of the hydrolysis turnover for c-di-AMP. In this case, a peak integration of the respective nucleotide peaks has to be performed using the Äkta software Unicorn (Cytiva former GE Healthcare) or any other mathematical analysis software. From the ratio of the peak areas of substrate and product, a percentage of hydrolyzed substrate or formed product can be calculated. Figure 3 shows exemplary IEX separation of c-di-AMP, 5′-pApA and AMP standards. Figure 4 demonstrates the interfering peaks of c-di-AMP and 5′-pApA after IEX separation of a c-di-AMP PDE assay with AtaC and the removal of 5′-pApA from the reaction by TmPDE.

   
   

   
   Figure 3. Ion-Exchange Chromatography (IEX) standards for 100 µl solutions containing 200 µM c-di-AMP, 5′-pApA or AMP

   
   

   
   Figure 4. Ion-Exchange Chromatography (IEX) of a c-di-AMP phosphodiesterase assay. A. A 100 µl reaction containing 1 µM S. venezuelae AtaC + 400 µM c-di-AMP incubated for 1 h at 37 °C was stopped by ultrafiltration and separated via IEX. B. A similar reaction in a coupled assay with TmPDE to separate the peaks of c-di-AMP and 5′-pApA. A 100 µL reaction containing 1 µM S. venezuelae AtaC + 400 µM c-di-AMP incubated for 1 h at 37 °C was stopped by ultrafiltration, followed by the addition of 100 nM TmPDE which was stopped by ultrafiltration after incubation for 1 h at 37 °C and separated via IEX.

Notes

1. General comments on TLC assays

   1. Buffer exchange is needed to replace the elution buffer in protein eluates to the proper reaction buffers (DisA cyclase buffer or PDE reaction buffer) and can be performed with many methods: e.g., size exclusion chromatography, ultrafiltration or dialysis. In our lab, we use dialysis, which however has no advantages or disadvantages compared to other methods for buffer exchange. You can choose a method that suits you best.

   2. The exact method to determine the protein concentration is not crucial to perform the TLC assays and can be achieved by many methods: e.g., photometric measurement at 280 nm, Bradford assay or 1D SDS gel densitometry. In our lab, we routinely use the latter, which however has no advantages or disadvantages compared to other methods. Feel free to use any method that is convenient for you.

   3. Enzymatic assays with radiolabeled nucleotides should be performed only by trained individuals with sufficient protection equipment.

   4. Usage of filter tips is strongly recommended to avoid contamination of micropipettes with radioactive material.

   5. The given enzyme concentrations were optimized for purified DisA (S. venezuelae and B. subtilis) and purified AtaC (S. venezuelae). Enzymes from other bacteria may require different concentrations. If you work with a new protein, first test enzymatic activity with 1 to 5 µM purified protein and subsequently determine the optimal protein concentrations by titration.

   6. Procedure section, Step A11: spotting the whole 10 µl mixture at once may result in blending of the single spots before drying. To avoid that, you can spot 3 µl + 3 µl + 4 µl with drying of spots between each volume spotted.

   7. The TL plate can be stored at RT for at least 3 days without significant loss of signal quality and can be used multiple times for exposure to erased imaging plates. If you are working with substrates with lower radioactive activity than indicated in the materials section, it might be necessary to prolong the exposure times of the imaging plate.

   8. The use of plastic wrap for the TL plate reduces the risk of radioactive contamination of the imaging plate and cassette. You may place an additional layer of plastic wrap on the imaging plate to further reduce the risk.

   9. Imaging plates can be erased by exposure to a light source (e.g., for 10 min using the image eraser, Molecular Dynamics, model: 410A ) and reused. Consequently, after removing the TL plate from the cassette, avoid long exposure of the imaging plate to ambient light prior to scanning.

      
      

2. General comments on IEX assay

   1. c-di-AMP forms long non-symmetrical Peaks on IEX columns and can therefore overlay with other Peaks (see also comment 3). Thus, if a RP-HPLC system is available, it is the preferred method to use instead.

   2. Basic knowledge of Äkta systems is required.

   3. If the c-di-AMP conversion has to be quantified by peak integration, the overlay of the peaks from 5′-pApA and c-di-AMP has to be considered. In this case, a second enzyme, which can only degrade 5′-pApA but not c-di-AMP (e.g., TmPDE [Drexler et al., 2017]), has to be added after ultrafiltration. Add TmPDE to a final concentration of 100 nM and incubate at 37 °C for 1 h. Repeat the procedure section paragraph C starting from step 3.

   4. The precise elution point of the nucleotides on an IEX column is very sensitive to salt, pH, temperature and also the Äkta system. For optimal comparison, assays should be performed with the same buffer and on the same Äkta system.

Recipes

1. DisA cyclase reaction buffer, modified from Christen et al. (2005)

   25 mM Tris-HCl pH = 8

   250 mM NaCl

   10 mM MgCl₂

   5 mM β-mercaptoethanol

   10% glycerol

2. PDE reaction buffer, modified from Huynh et al. (2015)

   20 mM Tris-HCl pH = 7.5

   50 mM NaCl

   1 mM MnCl₂

3. TLC running buffer, modified from Christen et al. (2005)

   Saturated (NH₄)₂SO₄ and 1.5 M KH₂PO₄ pH = 3.6 (mixed in a ratio of 1:1.5 v/v)

4. 10x AtaC reaction buffer

   500 mM Tris pH = 7.5

   1,000 mM NaCl

   1 mM MnCl₂

5. IEX running buffer A

   50 mM Tris pH = 9

6. IEX running buffer B

   50 mM Tris pH = 9

   1,000 mM NaCl

Acknowledgments

Research in the Witte lab is funded by DFG Grant GRK1721 and the DFG Priority Program SPP 1879 (Grant WI 3717/3-1). Research in the Tschowri lab is funded by the DFG Emmy Noether Program (Grant TS 325/1-1) and the DFG Priority Program SPP 1879 (Grants TS 325/2-1 and TS 325/2-2).

Competing interests

The authors declare no competing interests.

References

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