CXC chemokine ligand 12α-mediated increase in insulin secretion and survival of mouse pancreatic islets in response to oxidative stress through modulation of calcium uptake

Melita Vidakovic, Ernesto Caballero Garrido, Mirjana Mihailovic, Jelena Arambasic Jovanovic, Marija Sinadinovic, Jovana Rajic, Aleksandra Uskokovic, Svetlana Dinic, Nevena Grdovic, Milos Djordjevic, Anja Tolic, Goran Poznanovic


We examined whether CXCL12α improves insulin secretion by influencing the Ca2+ oscillation pattern and Ca2+ influx ([Ca2+]i), thereby enhancing the viability of pancreatic islet cells in oxidative stress. The islets of Langerhans were isolated from male OF1 mice and pretreated with 40 ng/mL of CXCL12α prior to exposure to 7.5 µM hydrogen peroxide, which served to induce oxidative stress. Incubation of islets with CXCL12α induced pancreatic β-cell proliferation and improved the ability of β-cells to withstand oxidative stress. Consecutive treatments of isolated islets with hydrogen peroxide caused a decline in β-cell functioning over time, while the CXCL12α pretreatment of islets exhibited a physiological response to high glucose that was comparable to control islets. The attenuated response of islets to a high D-glucose challenge was observed as a partial to complete abolishment of [Ca2+]i. Treatments with increasing concentrations of CXCL12α decreased the number of Ca2+ oscillations that lasted longer, thus pointing to an overall increase in [Ca2+]i, which was followed by increased insulin secretion. In addition, treatment of islets with CXCL12α enhanced the transcription rate for insulin and the CXCR4 gene, pointing to the importance of CXCL12/CXCR4 signaling in the regulation of Ca2+ intake and insulin secretion in pancreatic islet cells. We propose that a potential treatment with CXCL12α could help to remove preexisting glucotoxicity and associated temporary β-cell stunning that might be present at the time of diabetes diagnosis in vivo.

Received: July 11, 2017; Revised: October 23, 2017; Accepted: October 24, 2017; Published online: October 30, 2017

How to cite this article: Vidaković M, Garrido EC, Mihailović M, Arambašić-Jovanović J, Sinadinović M, Rajić J, Uskoković A, Dinić S, Grdović N, Đorđević M, Tolić A, Poznanović G. CXC chemokine ligand 12α-mediated increase in insulin secretion and survival of mouse pancreatic islets in response to oxidative stress through modulation of calcium uptake. Arch Biol Sci. 2018;70(1):191-204.


diabetes; calcium; CXC chemokine ligand 12α; insulin; pancreatic islet cells; voltage-gated calcium channels

Full Text:



Muoio DM, Newgard CB. Mechanisms of disease: Molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes. Nat Rev Mol Cell Biol. 2008;9(3):193-205.

Braun M, Ramracheya R, Bengtsson M, Zhang Q, Karanauskaite J, Partridge C, Johnson PR, Rorsman P. Voltage-gated ion channels in human pancreatic beta-cells: electrophysiological characterization and role in insulin secretion. Diabetes. 2008;57(6):1618-28.

Fridlyand LE, Jacobson DA, Philipson LH. Ion channels and regulation of insulin secretion in human beta-cells: a computational systems analysis. Islets. 2013;5(1):1-15.

Fujimoto W, Miki T, Ogura T, Zhang M, Seino Y, Satin LS, Nakaya H, Seino S. Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice. Diabetologia. 2009;52(5):863-72.

Proks P, Ashcroft FM. Modeling K(ATP) channel gating and its regulation. Prog Biophys Mol Biol. 2009;99(1):7-19.

Rorsman P, Eliasson L, Kanno T, Zhang Q, Gopel S. Electrophysiology of pancreatic beta-cells in intact mouse islets of Langerhans. Prog Biophys Mol Biol. 2011;107(2):224-35.

Henquin JC, Nenquin M, Ravier MA, Szollosi A. Shortcomings of current models of glucose-induced insulin secretion. Diabetes Obes Metab. 2009;11(Suppl.4):168-79.

Fridlyand LE, Jacobson DA, Kuznetsov A, Philipson LH. A model of action potentials and fast Ca2+ dynamics in pancreatic beta-cells. Biophys J. 2009;96(8):3126-39.

Nagasawa T, Kikutani H, Kishimoto T. Molecular cloning and structure of a pre-B-cell growth-stimulating factor. Proc Natl Acad Sci U S A. 1994;91(6):2305-9.

Rollins BJ. Chemokines. Blood. 1997;90(3):909-28.

Day CE, Guillen C, Willars GB, Wardlaw AJ. Characterization of the migration of lung and blood T cells in response CXCL12 in a three-dimensional matrix. Immunology. 2010;130(4):564-71.

Rabbany SY, Pastore J, Yamamoto M, Miller T, Rafii S, Aras R, Penn M. Continuous delivery of stromal cell-derived factor-1 from alginate scaffolds accelerates wound healing. Cell Transplant. 2010;19(4):399-408.

Sharp CD, Huang M, Glawe J, Patrick DR, Pardue S, Barlow SC, Kevil CG. Stromal cell-derived factor-1/CXCL12 stimulates chemorepulsion of NOD/LtJ T-cell adhesion to islet microvascular endothelium. Diabetes. 2008;57(1):102-12.

Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, Moepps B, Arenzana-Seisdedos F, Thelen M, Bachelerie F. The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem. 2005;280(42):35760-6.

Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, Sodroski J, Springer TA. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 1996;382(6594):829-33.

Yano T, Liu Z, Donovan J, Thomas MK, Habener JF. Stromal cell derived factor-1 (SDF-1)/CXCL12 attenuates diabetes in mice and promotes pancreatic beta-cell survival by activation of the prosurvival kinase Akt. Diabetes. 2007;56(12):2946-57.

Grdovic N, Dinic S, Mihailovic M, Uskokovic A, Jovanovic JA, Poznanovic G, Wagner L, Vidakovic M. CXC chemokine ligand 12 protects pancreatic beta-cells from necrosis through Akt kinase-mediated modulation of poly(ADP-ribose) polymerase-1 activity. PLoS One. 2014;9(7):e101172.

Liu Z, Stanojevic V, Avadhani S, Yano T, Habener JF. Stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival. Diabetologia. 2011;54(8):2067-76.

Nadal A, Rovira JM, Laribi O, Leon-quinto T, Andreu E, Ripoll C, Soria B. Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor. FASEB J. 1998;12(13):1341-8.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54.

Miller BA. Inhibition of TRPM2 function by PARP inhibitors protects cells from oxidative stress-induced death. Br J Pharmacol. 2004;143(5):515-6.

Cnop M, Welsh N, Jonas JC, Jorns A, Lenzen S, Eizirik DL. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes. 2005; 54(Suppl.2):S97-107.

Chang-Chen KJ, Mullur R, Bernal-Mizrachi E. Beta-cell failure as a complication of diabetes. Rev Endocr Metab Disord. 2008;9(4):329-43.

Robertson RP. Beta-cell deterioration during diabetes: what’s in the gun? Trends Endocrinol Metab. 2009;20(8):388-93.

Wajchenberg BL. beta-cell failure in diabetes and preservation by clinical treatment. Endocr Rev. 2007;28(2):187-218.

Ferrannini E. The stunned beta cell: a brief history. Cell Metab. 2010;11(5):349-52.

Kayali AG, Lopez AD, Hao E, Hinton A, Hayek A, King CC. The SDF-1alpha/CXCR4 axis is required for proliferation and maturation of human fetal pancreatic endocrine progenitor cells. PLoS One. 2012;7(6):e38721.

Kayali AG, Van Gunst K, Campbell IL, Stotland A, Kritzik M, Liu G, Flodstrom-Tullberg M, Zhang YQ, Sarvetnick N. The stromal cell-derived factor-1alpha/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas. J Cell Biol. 2003;163(4):859-69.

Sakai K, Matsumoto K, Nishikawa T, Suefuji M, Nakamaru K, Hirashima Y, Kawashima J, Shirotani T, Ichinose K, Brownlee M, Araki E. Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells. Biochem Biophys Res Commun. 2003;300(1):216-22.

Aley PK, Porter KE, Boyle JP, Kemp PJ, Peers C. Hypoxic modulation of Ca2+ signaling in human venous endothelial cells. Multiple roles for reactive oxygen species. J Biol Chem. 2005;280(14):13349-54.

Doan TN, Gentry DL, Taylor AA, Elliott SJ. Hydrogen peroxide activates agonist-sensitive Ca(2+)-flux pathways in canine venous endothelial cells. Biochem J. 1994;297(Pt 1):209-15.

Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer. 1996;32A(1):30-8.

Gen W, Tani M, Takeshita J, Ebihara Y, Tamaki K. Mechanisms of Ca2+ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes. Basic Res Cardiol. 2001;96(6):623-9.

Kumasaka S, Shoji H, Okabe E. Novel mechanisms involved in superoxide anion radical-triggered Ca2+ release from cardiac sarcoplasmic reticulum linked to cyclic ADP-ribose stimulation. Antioxid Redox Signal. 1999;1(1):55-69.

Suzuki YJ, Forman HJ, Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med. 1997;22(1-2):269-85.

Yan Y, Wei CL, Zhang WR, Cheng HP, Liu J. Cross-talk between calcium and reactive oxygen species signaling. Acta Pharmacol Sin. 2006;27(7):821-6.

Bielefeldt K, Whiteis CA, Sharma RV, Abboud FM, Conklin JL. Reactive oxygen species and calcium homeostasis in cultured human intestinal smooth muscle cells. Am J Physiol. 1997;272(6Pt1):G1439-50.

Kaneko M, Matsumoto Y, Hayashi H, Kobayashi A, Yamazaki N. Oxygen free radicals and calcium homeostasis in the heart. Mol Cell Biochem. 1994;139(1):91-100.

Klonowski-Stumpe H, Schreiber R, Grolik M, Schulz HU, Haussinger D, Niederau C. Effect of oxidative stress on cellular functions and cytosolic free calcium of rat pancreatic acinar cells. Am J Physiol. 1997;272(6Pt1):G1489-98.

Leist M, Nicotera P. Calcium and neuronal death. Rev Physiol Biochem Pharmacol. 1998;132:79-125.

Rojanasakul Y, Wang L, Hoffman AH, Shi X, Dalal NS, Banks DE, Ma JK. Mechanisms of hydroxyl free radical-induced cellular injury and calcium overloading in alveolar macrophages. Am J Respir Cell Mol Biol. 1993;8(4):377-83.

Clague JR, Langer GA. The pathogenesis of free radical-induced calcium leak in cultured rat cardiomyocytes. J Mol Cell Cardiol. 1994;26(1):11-21.

Nicotera P, Rossi AD. Nuclear Ca2+: physiological regulation and role in apoptosis. Mol Cell Biochem. 1994;135(1):89-98.

Herson PS, Lee K, Pinnock RD, Hughes J, Ashford ML. Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line. J Biol Chem. 1999;274(2):833-41.

Krippeit-Drews P, Britsch S, Lang F, Drews G. Effects of SH-group reagents on Ca2+ and K+ channel currents of pancreatic B-cells. Biochem Biophys Res Commun. 1994;200(2):860-6.

Baumgartner-Parzer SM, Wagner L, Pettermann M, Grillari J, Gessl A, Waldhausl W. High-glucose--triggered apoptosis in cultured endothelial cells. Diabetes. 1995;44(11):1323-7.

Du XL, Sui GZ, Stockklauser-Farber K, Weiss J, Zink S, Schwippert B, Wu QX, Tschope D, Rosen P. Induction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species. Diabetologia. 1998;41(3):249-56.

Tanaka Y, Gleason CE, Tran PO, Harmon JS, Robertson RP. Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants. Proc Natl Acad Sci U S A. 1999;96(19):10857-62.

Tanaka Y, Tran PO, Harmon J, Robertson RP. A role for glutathione peroxidase in protecting pancreatic beta cells against oxidative stress in a model of glucose toxicity. Proc Natl Acad Sci U S A. 2002;99(19):12363-8.

Ihara Y, Toyokuni S, Uchida K, Odaka H, Tanaka T, Ikeda H, Hiai H, Seino Y, Yamada Y. Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. Diabetes. 1999;48(4):927-32.

Talchai C, Xuan SH, Lin HV, Sussel L, Accili D. Pancreatic beta Cell Dedifferentiation as a Mechanism of Diabetic beta Cell Failure. Cell. 2012;150(6):1223-34.

Dean PM, Matthews EK. Electrical activity in pancreatic islet cells. Nature. 1968;219(5152):389-90.

Henquin JC. Triggering and amplifying pathways of regulation of insulin secretion by glucose. Diabetes. 2000;49(11):1751-60.

Misler S, Barnett DW, Gillis KD, Pressel DM. Electrophysiology of stimulus-secretion coupling in human beta-cells. Diabetes. 1992;41(10):1221-8.

Rorsman P, Braun M, Zhang Q. Regulation of calcium in pancreatic alpha- and beta-cells in health and disease. Cell Calcium. 2012;51(3-4):300-8.

Hoenig M, MacGregor LC, Matschinsky FM. In vitro exhaustion of pancreatic beta-cells. Am J Physiol. 1986;250(5Pt1):E502-11.

Martin F, Ribas J, Soria B. Cytosolic Ca2+ gradients in pancreatic islet-cells stimulated by glucose and carbachol. Biochem Biophys Res Commun. 1997;235(3):465-8.

Basu S, Broxmeyer HE. CCR5 ligands modulate CXCL12-induced chemotaxis, adhesion, and Akt phosphorylation of human cord blood CD34+ cells. J Immunol. 2009;183(11):7478-88.

Drury LJ, Ziarek JJ, Gravel S, Veldkamp CT, Takekoshi T, Hwang ST, Heveker N, Volkman BF, Dwinell MB. Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways. Proc Natl Acad Sci U S A. 2011;108(43):17655-60.

Kim CH, Hangoc G, Cooper S, Helgason CD, Yew S, Humphries RK, Krystal G, Broxmeyer HE. Altered responsiveness to chemokines due to targeted disruption of SHIP. J Clin Invest. 1999;104(12):1751-9.

Agle KA, Vongsa RA, Dwinell MB. Calcium mobilization triggered by the chemokine CXCL12 regulates migration in wounded intestinal epithelial monolayers. J Biol Chem. 2010;285(21):16066-75.

Saiman Y, Bansal M. Hepatic Stellate Cell-derived CXCL12 promotes T cell adhesion to Sinusoidal Endothelial Cells. Hepatology. 2012;56:258a-259a.

Schajnovitz A, Itkin T, D'Uva G, Kalinkovich A, Golan K, Ludin A, Cohen D, Shulman Z, Avigdor A, Nagler A, Kollet O, Seger R, Lapidot T. CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions. Nat Immunol. 2011;12(5):391-8.

Quesada I, Martin F, Soria B. Nutrient modulation of polarized and sustained submembrane Ca2+ microgradients in mouse pancreatic islet cells. J Physiol. 2000; 525(Pt1):159-67.

Santos RM, Rosario LM, Nadal A, Garcia-Sancho J, Soria B, Valdeolmillos M. Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets. Pflugers Arch. 1991;418(4):417-22.

Soria B, Tuduri E, Gonzalez A, Hmadcha A, Martin F, Nadal A, Quesada I. Pancreatic islet cells: a model for calcium-dependent peptide release. HFSP J. 2010;4(2):52-60.

Valdeolmillos M, Nadal A, Contreras D, Soria B. The relationship between glucose-induced K+ATP channel closure and the rise in [Ca2+]i in single mouse pancreatic beta-cells. J Physiol. 1992;455:173-86.

Martin F, Sanchez-Andres JV, Soria B. Slow [Ca2+]i oscillations induced by ketoisocaproate in single mouse pancreatic islets. Diabetes. 1995;44(3):300-5.

Ammala C, Ashcroft FM, Rorsman P. Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells. Nature. 1993;363(6427):356-8.

Arkhammar P, Juntti-Berggren L, Larsson O, Welsh M, Nanberg E, Sjoholm A, Kohler M, Berggren PO. Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage-activated Ca2+ channels. J Biol Chem. 1994;269(4):2743-9.

Huang LP, Bhattacharjee A, Taylor JT, Zhang M, Keyser BM, Marrero L, Li M. Ca2+](i) regulates trafficking of Ca(v)1.3 (alpha(1D) Ca2+ channel) in insulin-secreting cells. Am J Physiol-Cell Ph. 2004;286(2):C213-21.

Ji JZ, Yang SN, Huang XH, Li XD, Shen L, Diamant N, Berggren PO, Gaisano HY. Modulation of L-type Ca2+ channels by distinct domains within SNAP-25. Diabetes. 2002;51(5):1425-36.

Kang Y, Huang X, Pasyk EA, Ji J, Holz GG, Wheeler MB, Tsushima RG, Gaisano HY. Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines. Diabetologia. 2002;45(2):231-41.

Passafaro M, Codignola A, Rogers M, Cooke I, Sher E. Modulation of N-type calcium channels translocation in RINm5F insulinoma cells. Pharmacol Res. 2000;41(3):325-34.

Plant TD. Properties and calcium-dependent inactivation of calcium currents in cultured mouse pancreatic B-cells. J Physiol. 1988;404:731-47.

Wiser O, Trus M, Hernandez A, Renstrom E, Barg S, Rorsman P, Atlas D. The voltage-sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. P Natl Acad Sci USA. 1999;96(1):248-53.

Yang SN, Larsson O, Branstrom R, Bertorello AM, Leibiger B, Leibiger IB, Moede T, Kohler M, Meister B, Berggren PO. Syntaxin 1 interacts with the L(D) subtype of voltage-gated Ca(2+) channels in pancreatic beta cells. Proc Natl Acad Sci U S A. 1999;96(18):10164-9.

Henquin JC, Dufrane D, Nenquin M. Nutrient control of insulin secretion in isolated normal human islets. Diabetes. 2006;55(12):3470-7.

Taylor JT, Huang L, Keyser BM, Zhuang H, Clarkson CW, Li M. Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release. Am J Physiol Endocrinol Metab. 2005;289(5):E900-8.

Dolphin AC. The G.L. Brown Prize Lecture. Voltage-dependent calcium channels and their modulation by neurotransmitters and G proteins. Exp Physiol. 1995;80(1):1-36.

Barg S, Ma X, Eliasson L, Galvanovskis J, Gopel SO, Obermuller S, Platzer J, Renstrom E, Trus M, Atlas D, Striessnig J, Rorsman P. Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells. Biophys J. 2001;81(6):3308-23.

Smith PA, Aschroft FM, Fewtrell CM. Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells. J Gen Physiol. 1993;101(5):767-97.

Scharenberg AM. TRPM2 and pancreatic beta-cell responses to oxidative stress. Islets. 2009;1(2):165-6.

Perraud AL, Fleig A, Dunn CA, Bagley LA, Launay P, Schmitz C, Stokes AJ, Zhu Q, Bessman MJ, Penner R, Kinet JP, Scharenberg AM. ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature. 2001;411(6837):595-9.

Hara Y, Wakamori M, Ishii M, Maeno E, Nishida M, Yoshida T, Yamada H, Shimizu S, Mori E, Kudoh J, Shimizu N, Kurose H, Okada Y, Imoto K, Mori Y. LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell. 2002;9(1):163-73.

Perraud AL, Takanishi CL, Shen B, Kang S, Smith MK, Schmitz C, Knowles HM, Ferraris D, Li W, Zhang J, Stoddard BL, Scharenberg AM. Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels. J Biol Chem. 2005;280(7):6138-48.

Chen J, Gusdon AM, Thayer TC, Mathews CE. Role of increased ROS dissipation in prevention of T1D. Ann N Y Acad Sci. 2008;1150:157-66.

Lenzen S. Oxidative stress: the vulnerable beta-cell. Biochem Soc Trans. 2008; 36 (Pt 3): 343-7.

Duncanson S, Sambanis A. Dual factor delivery of CXCL12 and Exendin-4 for improved survival and function of encapsulated beta cells under hypoxic conditions. Biotechnol Bioeng. 2013;110(8): 2292-300.

Habener JF, Stanojevic V. alpha-cell role in beta-cell generation and regeneration. Islets. 2012;4(3):188-98.

Yang SN, Berggren PO. Beta-cell CaV channel regulation in physiology and pathophysiology. Am J Physiol Endocrinol Metab. 2005;288(1):E16-28.

Princen K, Hatse S, Vermeire K, De Clercq E, Schols D. Evaluation of SDF-1/CXCR4-induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry. Cytometry A. 2003;51(1):35-45.

Rubin JB. Chemokine signaling in cancer: one hump or two? Semin Cancer Biol. 2009;19(2):116-22.

Teicher BA, Fricker SP. Cxcl12 (Sdf-1)/Cxcr4 Pathway in Cancer. Clin Cancer Res. 2010;16(11):2927-31.

Hsu WH, Xiang HD, Rajan AS, Kunze DL, Boyd AE, 3rd. Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell. J Biol Chem. 1991;266(2):837-43.

Renstrom E, Ding WG, Bokvist K, Rorsman P. Neurotransmitter-induced inhibition of exocytosis in insulin-secreting beta cells by activation of calcineurin. Neuron. 1996;17(3):513-22.

Roe MW, Worley JF, Tokuyama Y, Philipson LH, Sturis J, Tang JP, Dukes ID, Bell GI, Polonsky KS. NIDDM is associated with loss of pancreatic beta-cell L-type Ca2+ channel activity. Am J Physiol-Endoc M. 1996;270(1):E133-40.


  • There are currently no refbacks.


Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.