Materials and Reagents

Protein Purification

Poly-Prep chromatography column (Bio-Rad, catalog number: 7311550 )
Zeba^TM Spin Desalting Columns 40k MWCO 10 ml (Thermo Fisher Scientific, catalog number: 87772 )
BL21 (DE3) competent E. coli (NEB, catalog number: C2527I )
Rosetta^TM (DE3) competent E. coli (Millipore Sigma, catalog number: 70954 )
Isopropyl-β-D-thiogalactopyranoside (IPTG, Millipore Sigma, catalog number: I6758-10G )
Phosphate Buffered Saline (PBS, Lonza, catalog number: 17-516F )
Imidazole (Millipore Sigma, catalog number: I5513 )
Lysozyme from chicken egg white (Millipore Sigma, catalog number: 62971-10G-F )
Triton^TM X-100 (Millipore Sigma, catalog number: T8787-250ml )
Triton^TM X-114 (Millipore Sigma, catalog number: 93422-250ml )
Phenylmethylsulfonyl fluoride (PMSF, Millipore Sigma, catalog number: P7626-5G )
cOmplete^TM Mini, EDTA free (Roche, catalog number: 11836170001 )
Bovine Serum Albumin (BSA, Millipore Sigma, catalog number: A3608 )
HisPur^TM Cobalt Resin (Thermo Fisher Scientific, catalog number: 89964 )
1,4-Dithiothreitol (DTT, Millipore Sigma, catalog number: 10197777001 )
Sodium Phosphate Monobasic (Millipore Sigma, catalog number: S3139 )
Sodium Phosphate Dibasic (Millipore Sigma, catalog number: S3264 )
Sodium Chloride (Millipore Sigma, catalog number: S9888 )
HEPES (Millipore Sigma, catalog number: H3375 )
Glycerol (Millipore Sigma, catalog number: G5516 )
Protein Binding Buffer (see Recipes)
Protein Elution Buffer (see Recipes)
Protein Storage Buffer (see Recipes)

Cells and cell culture media

HEK293T and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC)
DC2.4 cells were obtained from Rock lab, University of Massachusetts Medical School, MA, USA
RAW-Blue ISG cells were obtained from Invivogen
Dulbecco’s modified Eagle’s medium (DMEM, Corning, catalog number: 10-041-CV )
Roswell Park Memorial Institute (RPMI) medium (Corning, catalog number: 10-013-CV )
0.25% Trypsin-EDTA (Gibco, catalog number: 25200-056 )
Fetal bovine serum (FBS, Gibco, catalog number: 10437-028 )
Penicillin-Streptomycin Solution, 100x (Corning, catalog number: 30-002-CI )

IFN-luciferase assay

96-well clear bottom white plate (Millipore Sigma, catalog number: CLS3610 )
pGL4.45[luc2p/ISRE/Hygro] Vector (Promega)
Firefly Luciferase Assay Kit (Biotium, catalog number: 30075-2 )
TransIT-X2^® Transfection Reagent (Mirus, catalog number: MIR 6004 )

RNA extraction, Reverse Transcription, and qPCR

LightCycler^TM 480 Multiwell Plate 96 clear with Sealing Foils (Roche, catalog number: 05102413001)
RNeasy^TM micro kit (Qiagen, catalog number: 74004 )
Beta-Mercaptoethanol (βME, Millipore Sigma, catalog number: M6250-10ML )
Reverse transcription kit (Thermo Fisher Scientific, catalog number: 4374966 )
SYBR^TM Green PCR Master Mix (Thermo Fisher Scientific, catalog number: 4309155 )
qPCR primers used for detection: mIFN-β-F: 5’-GCCTTTGCCATCCAAGAGATGC-3’, mIFN-β-R: 5’-ACACTGTCTGCTGGTGGAGTTC-3’, mActin-F: 5’-CATTGCTGACAGGATGCAGAAGG-3’, and mActin-R: 5’-TGCTGGAAGGTGGACAGTGAGG-3’ (ordered from IDT as custom oligo DNA)

mCXCL10 ELISA

Greiner Bio-One MICROLON^TM 600 High Binding 96-Well ELISA Assay Microplates (Fisher Scientific, catalog number: 07-000-627 )
Mouse CXCL10 ELISA kit (R&D, catalog number: DY466 )
TMB Substrate Set (BioLegend, catalog number: 421101 )
TWEEN 20 (Millipore Sigma, catalog number: P9416-100ml )

SEAP-IFN assay

QUANTI-Blue^TM Solution (Invivogen, catalog code: rep-qbs )

Equipment

Misonix sonicator 3000
Real-time PCR system (Roche, model: LightCycler 480 )
Nanodrop spectrophotometer (Thermo Fisher, model: ND-1000 )
Thermal cycler (Bio-Rad, model: T100 )
Plate reader (Tecan, model: Infinite M200 )

Software

GraphPad Prism

Procedure

STINGΔTM protein purification
The DNA sequences (sources: Tmem173 NM_028261 Mouse Tagged ORF Clone, Origene Catalog number: MR227544, STING TMEM173 NM_198282 Human Tagged ORF Clone, Origene Catalog number: RC208418) of STINGΔTM protein (138 to 378 amino acids for mouse STING, 139 to 379 amino acids for human STING) were synthesized as gBlock DNA fragments (Integrated DNA Technologies) and cloned into the pSH200 expression vector (linearized via Nco I and Not I restriction enzymes) with a hexa-histidine-tag at the N-terminus (Figure 1). Plasmids for STINGΔTM mutants such as S365A were then generated via site-specific mutagenesis. DE3 Escherichia coli (E. coli) was used to express the proteins (mouse STINGΔTM in BL21 DE3, human STINGΔTM in Rosetta DE3). Lysogeny Broth (LB) containing the antibiotic ampicillin (100 mg/L) was used for bacteria culture, shaker conditions were 37 °C 220 rpm for growth and 18 °C 220 rpm for induction.

Figure 1. Plasmid maps of (A) pSH200_His6_TEV_human STING (139-379aa) and (B) pSH200_His6_TEV_mouse STING (138-378aa)

Day 1: Bacteria culture
1. Pick DE3 E. coli from glycerol stock and culture in 50 ml of LB with ampicillin at 37 °C overnight.
Day 2: Induction
1. Transfer 50 ml of overnight culture into a flask containing 1 L LB with ampicillin and culture for approximately 2 h at 37 °C until the culture’s OD₆₀₀ reaches 0.4. This OD value is critical for expressing human STINGΔTM in Rosetta DE3, as we have found that higher concentration can compromise protein purity; for the expression of mouse STINGΔTM in BL21 DE3, the OD600 can be in the range of 0.4-0.8 without compromising yield and purity.
2. Cool the 1 L culture on ice and add 0.5 ml 1 M IPTG, then shake at 18 °C for approximately 20 h.
Day 3: Protein purification
1. After induction, centrifuge bacteria cultures at 6,000 × g for 20 min.
2. Collect pellets and wash once with 30 ml PBS, then lyse in 20 ml protein binding buffer (Recipe 1) with 20 mg lysozyme, 200 μl Triton X-100, 1 mM PMSF (replenished every 30 min until Cobalt binding) and one tablet of cOmplete protease inhibitor cocktail tablets at room temperature for 20 min with gentle rotation at 20 rpm. After cells are lysed, the proteins should be kept ice-cold throughout the purification to minimize degradation (using cold protein binding buffer, protein elution buffer, and protein storage buffer).
3. Cool cell lysate on ice water and sonicate with Misonix sonicator 3000 at 18 W (with 3-s on and 5-s off intervals to prevent heat-deactivation of the protein) for a total of 5 min.
4. After sonication, centrifuge cell lysate at 14,000 × g at 4 °C for 30 min.
5. Wash 0.5 ml HisPur Cobalt Resin with 5 ml protein binding buffer, then add to the bacteria supernatant separated from the centrifugation, along with 20 μl Triton X-114 (to eliminate endotoxin) for Cobalt-HisTag binding (4 °C for 1 h with gentle rotation at 20 rpm).
6. After binding, carefully aspirate the supernatant and wash the Cobalt resin twice (4 °C for 30 min each time with gentle rotation at 20 rpm) with 5 ml protein binding buffer containing 5 μl Triton X-114.
7. Transfer washed Cobalt resin into a Poly-Prep chromatography column with 5 ml of protein binding buffer. After all the protein binding buffer has drained out under gravity, add 1.5 ml protein elution buffer (Recipe 2) and collect the elution.
8. Desalt the protein elution with Zeba^TM Spin Desalting Columns (40k MWCO 10 ml). Add 10 ml of protein storage buffer (Recipe 3) into the column and allow it to completely drain under gravity (until no liquid remains above the resin surface). Add the 1.5 ml protein elution dropwise onto the middle of the resin and allow it to drain completely. Finally, collect five 1 ml fractions with protein storage buffer (add 1 ml of storage buffer then collect the flow-through as “Fraction 1”, then add another 1 ml of storage buffer and collect “Fraction 2” … all the way till “Fraction 5”). Combine Fractions 3 and 4 (which contains the majority of the desalted protein) for SDS-PAGE characterization and BCA quantification of protein concentration. Fractions 1, 2 may contain minimal amounts of protein, fraction 5 may contain protein with imidazole. The concentrations of each fraction may also be quantified through the BCA assay.
9. For storage, 1 mM DTT was added and protein solution was aliquoted then stored in -80 °C. Protein function can be maintained for years under this storage condition, but can be compromised by multiple freeze-thaw cycles.
Cell lines used for in vitro STING activation
Human embryonic kidney 293T (HEK293T) cells are deficient in cGAMP synthase (cGAS) and STING proteins, but express other essential proteins in downstream STING signaling, including TANK-binding kinase 1 (TBK1) and Interferon regulatory factor 3 (IRF3). Therefore, it provides a good model for studying the function of cGAMP-STINGΔTM without interactions with endogenous cGAMP or STING proteins (Figure 2). Additionally, it can be transfected to overexpress full-length mutant STING proteins (for example HAQ STING). To detect STING activation, we generated an interferon (IFN) reporter cell line by integrating an IFN-stimulated response element (ISRE) that drives luciferase expression through the stable transfection of pGL4.45[luc2p/ISRE/Hygro] plasmid selected in hygromycin (200 μg/ml). HEK293T cells are not capable of uptaking cGAMP-STINGΔTM complex directly, so commercial transfection reagents (we primarily used TransIT-X2, though others like Lipofectamine also work) are required in order to deliver cGAMP-STINGΔTM complex into the cells. HEK293T cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
Mouse macrophage RAW264.7 and dendritic cell DC2.4 both express cGAS and STING, as well as TBK1 and IRF3 (Figure 2). They are also capable of internalizing cGAMP-STINGΔTM complex without the help of a transfection reagent. To detect STING activation, we can measure the cell secreted CXCL10 concentration in the medium with enzyme-linked immunosorbent assay (ELISA) or quantify the interferon-β mRNA level with qPCR. The aforementioned media contains no cells – medium that contains cells is denoted simply as ‘cells’. In addition, a reporter cell line derived from RAW264.7: RAW-Blue^TM ISG cells can be used to study the kinetics of STING activation in vitro, and since the Secreted embryonic alkaline phosphatase (SEAP) – IFN assay only requires 20 μl of media, it can be performed at multiple time points post treatment. RAW264.7 cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For SEAP-IFN assay, RAW-Blue^TM ISG cells are cultured in DMEM with 10% heat-inactivated (56 °C, 30 min) FBS and 1% penicillin/streptomycin. DC2.4 cells are cultured in Roswell Park Memorial Institute (RPMI) medium with 10% FBS and 1% penicillin/streptomycin.
All cells are cultured in a 37 °C, 5% CO₂ incubator, used at low passage number and tested negative for Mycoplasma contamination.

Figure 2. Immunoblotting of endogenous expression of cGAS, STING, TBK1, and IRF3 proteins from HEK293T, RAW264.7, and DC2.4 cells
IFN-luciferase assay with HEK293T-luc2p/ISRE/Hygro
Day 1: Preparation of Cells
1. Seed HEK293T-luc2p/ISRE/Hygro cells into 96-well plates (clear bottom flat white plates for luciferase assay) at a density of 3 × 10⁵ cells/ml in 100 μl media per well.
Day 2: Cell treatment
1. For each well, mix 1 μg of STINGΔTM protein with 0.025 μg cGAMP, then add 1 μl of TransIT-X2 in OptiMEM media to a total volume of 20 μl and incubate at room temperature for 15 min.
2. Add the mixture to the cell medium without pipetting up and down. At least 3 replicates should be performed for each treatment.
3. Incubate treated cells for 24 h.
Day 3: Luminescence measurement
1. Remove plates from the incubator, aspirate medium and add 25 μl lysis buffer (five-fold diluted with DI water from the provided 5× firefly luciferase lysis buffer) to each well, then incubate at room temperature for 15 min with orbital shaking.
2. Add 50 μl of luciferase assay buffer to each well (with D-luciferin freshly added at final concentration of 0.2 mg/ml), read plate for bioluminescence on a Tecan microplate reader.
mCXCL10 ELISA with RAW264.7 and DC2.4
Day 1: Preparation of Cells
1. Seed RAW264.7 or DC2.4 cells into 96-well plates at a density of 2 × 10⁵ cells/ml in 100 μl medium per well.
Day 2: Cell treatment
1. For each well, mix 1 μg of STINGΔTM protein with 0.025 μg cGAMP and 1 μl of TransIT-X2 in 20 μl OptiMEM medium, then incubate at room temperature for 15 min. Alternatively, for vehicle-free treatment, mix 5 μg of STINGΔTM protein with 0.125 μg cGAMP in 20 μl OptiMEM medium and incubate at room temperature for 15 min.
2. Add the mixture to the cell medium without pipetting up and down. At least 3 replicates should be performed for each treatment.
3. Incubate treated cells for 48 h.
Day 3: Preparation of ELISA plates
ELISA was performed according to the manufacturer’s protocol: Mouse CXCL10 ELISA kit (R&D, DY466 )
1. Dilute CXCL-10 capture antibody in PBS to 2 μg/ml.
2. Add 100 μl capture antibody solution to each well of the ELISA assay plates. Incubate at 4 °C overnight.
Day 4: ELISA
1. Aspirate each well of the ELISA assay plates, wash three times with Wash Buffer (PBS with 0.05% Tween 20).
2. Add 300 μl Reagent Diluent (PBS with 1% BSA) to each well to block plates. Incubate at room temperature for 1 h with orbital shaking at 220 rpm.
3. Aspirate plates and wash three times with Wash Buffer.
4. Add 100 μl media of RAW264.7 or DC2.4 cells 48 h post-treatment without dilution. Incubate at room temperature for 2 h with orbital shaking at 220 rpm.
5. Aspirate plates and wash three times with Wash Buffer.
6. Add 100 μl of the Detection Antibody at 100 ng/ml (diluted in Reagent Diluent) to each well. Incubate at room temperature for 2 h with orbital shaking at 220 rpm.
7. Aspirate plates and wash three times with Wash Buffer.
8. Add 100 μl of the provided Streptavidin-HRP stock solution 40-fold diluted in Reagent Diluent to each well. Incubate at room temperature for 20 min avoiding light with orbital shaking at 220 rpm.
9. Aspirate plates and wash three times with Wash Buffer.
10. Add 100 μl of TMB Substrate Solution, freshly prepared from mixing equal volumes of TMB Substrate A with TMB Substrate B. Incubate at room temperature for 20 min with orbital shaking at 220 rpm, avoiding light.
11. Add 50 μl of Stop Solution (2 N H₂SO₄) to each well. Gently tap the plate for mixing. Measure absorbance at 450 nm with a spectrophotometer/plate reader.
Quantification of mIFN-β expression by qPCR with RAW264.7 and DC2.4
Day 1: Preparation of Cells
1. Seed RAW264.7 or DC2.4 cells into 24-well plates at a density of 3 × 10⁵ cells/ml in 400 μl media per well.
Day 2: Cell treatment
1. For each well, mix 5 μg of STINGΔTM protein with 0.125 μg cGAMP and 5 μl of TransIT-X2 in 50 μl OptiMEM medium. Incubate at room temperature for 15 min. Alternatively, for vehicle-free treatment, mix 25 μg of STINGΔTM protein with 0.625 μg cGAMP in 50 μl OptiMEM medium and incubate at room temperature for 15 min.
2. Add the mixture to the cell medium without pipetting up and down.
3. Incubate treated cells for 24 h.
Day 3: RNA extraction, reverse transcription and qPCR
Perform RNA extraction following protocol provided by RNeasy Micro Kit:
1. Prepare fresh lysis buffer by adding 10 μl β-mercaptoethanol into 1 ml RLT buffer.
2. Aspirate medium, wash cells once with PBS, then add 350 μl lysis buffer per well. Incubate at room temperature for 5 min.
3. Transfer the cell lysate into Eppendorf tubes, vortex for 20 s, then add 350 μl 70% ethanol, pipette up and down, transfer to a spin column provided in the kit, and centrifuge for 15 s at 8,000 × g.
4. Discard flowthrough, add 350 μl RW1 buffer, and centrifuge for 15 s at 8,000 × g.
5. Prepare DNase buffer from DNase supplied in lyophilized form in glass vials. Use syringe to inject 500 μl water into the glass vial to dissolve the powder. Then open the vial and add 10 μl DNase solution + 70 μl RDD buffer to the column. Incubate at room temperature for 15 min. Then add 350 μl RW1 buffer to the spin column, centrifuge for 15 s at 8,000 × g and discard the collection tube.
6. Place the spin column in a new 2 ml collection tube as supplied. Add 500 μl RPE buffer, centrifuge for 15 s at 8,000 × g, and discard the flow through.
7. Add 500 μl 80% ethanol to the spin column, centrifuge for 2 min at 8,000 × g and discard the collection tube.
8. Place the spin column in another new 2 ml collection tube as supplied. Centrifuge at 8,000 × g for 5 min, discard the collection tube.
9. Place the spin tube in a new 1.5 ml collection tube as supplied, add 14 μl RNase-free water to the center of the spin column, and centrifuge for 1 min at 8,000 × g to elute the RNA.
10. Measure RNA concentration of each cell sample with Nanodrop spectrophotometer.
Reverse transcription
1. Dilute 1 μg extracted RNA with water to a total volume of 10 μl.
2. For each RNA sample, prepare below mixture in a PCR tube:
  Diluted RNA 10 μl
  10× RT buffer 2 μl
  dNTP 0.8 μl
  Random primer 2 μl
  RNase inhibitor 1 μl
  Enzyme 1 μl
  Water 3.2 μl
  Total volume 20 μl
3. Treat the sample with thermal cycler with the program below:
  25 °C 10 min
  37°C 2 h
  85 °C 5 min
  4 °C Infinite hold
qPCR
1. Dilute qPCR primers to 10 μM working concentration.
2. Add 80 μl water to 20 μl reverse transcription product from the previous step (this 5-fold dilution is denoted as ‘template’ in the following step)
3. Add the following to the qPCR 96-well plate for each reaction (add primer first, then add master mix):
  Template    1 μl
  2x SYBR mixture 10 μl
  Forward Primer    0.5 μl
  Reverse Primer 0.5 μl
  Water 8 μl
  Total volume   20 μl
IFN-SEAP assay with RAW-Blue ISG cells
Day 1: Preparation of Cells
1. Seed RAW264.7 or DC2.4 cells into 96-well plates at a density of 2 × 10⁵ cells/ml in 100 μl media per well.
Day 2: Cell treatment
1. 2 h prior to treatment, remove the medium and replenish it with 100 μl DMEM with 10% heat-inactivated FBS and 1% penicillin/streptomycin pre-warmed to 37 °C in order to reduce the noise level in subsequent QUANTI-Blue assay.
2. For each well, mix 1 μg of STINGΔTM protein with 0.025 μg cGAMP then 1 μl of TransIT-X2 in 20 μl OptiMEM medium, incubated at room temperature for 15 min. Alternatively, for vehicle-free treatment, mix 5 μg of STINGΔTM protein with 0.125 μg cGAMP in 20 μl OptiMEM medium and incubate at room temperature for 15 min.
3. Add the mixture to the cell medium without pipetting up and down. At least 3 replicates should be performed for each treatment.
4. Incubate treated cells in 37 °C incubator.
Days 3-4: IFN-SEAP assay
1. Take 20 μl of medium from treated cell wells and mix with 180 μl of QUANTI-Blue assay buffer in a separate 96-well plate. (Multiple time points between 12 to 48 h can be taken to study the kinetics.)
2. Incubate the plate at 37 °C for 6 to 10 h until a visible color difference is observed (Figure 3).
3. Determine the IFN-SEAP activity by measuring the absorbance at 635 nm with a spectrophotometer/plate reader.
  
  Figure 3. Example IFN-SEAP assay of mixing medium of treated RAW-Blue ISG cells with QUANTI-Blue assay buffer showing color differences due to STING signaling

Data analysis

Data can be analyzed with GraphPad Prism and statistical analyses performed with one-way analysis of variance (ANOVA) followed by Student’s t-test for statistical significance.

Recipes

Protein Binding Buffer
50 mM sodium phosphate
0.5 M NaCl
10 mM imidazole
pH 7.4
Protein Elution Buffer
50 mM sodium phosphate
0.5 M NaCl
150 mM imidazole
pH 7.4
Protein Storage Buffer
20 mM HEPES
150 mM NaCl
10% glycerol
pH 7.4

Acknowledgments

This work was supported by the Department of Defense Congressionally Directed Medical Research Program’s (CDMRP) Ovarian Cancer Research Program, Cancer Center Support Grant (CCSG) Pilot Awards at the David H. Koch Institute for Integrative Cancer Research at MIT, the Institute for Soldier Nanotechnologies (ISN) at MIT, the Marble Center for Cancer Nanomedicine, Northeastern University Faculty start-up funding, and the Peer Reviewed Medical Research Program from the Department of Defense’s Congressionally Directed Medical Research Programs (W81XWH18PRMRPDA).

Competing interests

The authors declare that they have no competing interests.

References

Ahn, J., Konno, H. and Barber, G. N. (2015). Diverse roles of STING-dependent signaling on the development of cancer. Oncogene 34(41): 5302-5308.
Fu, J., Kanne, D. B., Leong, M., Glickman, L. H., McWhirter, S. M., Lemmens, E., Mechette, K., Leong, J. J., Lauer, P. and Liu, W. (2015). STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade. Sci Transl Med 7(283): 283ra252-283ra252.
He, Y., Hong, C., Yan, E. Z., Fletcher, S. J., Zhu, G., Yang, M., Li, Y., Sun, X., Irvine, D. J. and Li, J. (2020). Self-assembled cGAMP-STINGΔTM signaling complex as a bioinspired platform for cGAMP delivery. Sci Adv 6(24): eaba7589.
Jin, L., Xu, L., Yang, I. V., Davidson, E. J., Schwartz, D. A., Wurfel, M. M. and Cambier, J. C. (2011). Identification and characterization of a loss-of-function human MPYS variant. Genes Immun 12(4): 263-269.
Ohkuri, T., Kosaka, A., Ishibashi, K., Kumai, T., Hirata, Y., Ohara, K., Nagato, T., Oikawa, K., Aoki, N. and Harabuchi, Y. (2017). Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site. Cancer Immunol Immunother 66(6): 705-716.
Patel, S., Blaauboer, S. M., Tucker, H. R., Mansouri, S., Ruiz-Moreno, J. S., Hamann, L., Schumann, R. R., Opitz, B. and Jin, L. (2017). The common R71H-G230A-R293Q human TMEM173 is a null allele. J Immunol 198(2): 776-787.
Shae, D., Becker, K. W., Christov, P., Yun, D. S., Lytton-Jean, A. K., Sevimli, S., Ascano, M., Kelley, M., Johnson, D. B. and Balko, J. M. (2019). Endosomolytic polymersomes increase the activity of cyclic dinucleotide STING agonists to enhance cancer immunotherapy. Nat Nanotechnol 1.
Sivick, K. E., Surh, N. H., Desbien, A. L., Grewal, E. P., Katibah, G. E., McWhirter, S. M. and Dubensky, T. W. (2017). Comment on“The Common R71H-G230A-R293Q Human TMEM173 Is a Null Allele. J Immunol 198(11): 4183-4185.
Xia, T., Konno, H. and Barber, G. N. (2016). Recurrent loss of STING signaling in melanoma correlates with susceptibility to viral oncolysis. Cancer Res 76(22): 6747-6759.