/* * Improved Mathematical Model for the Primary Pacemaker Cell * * Model Status * * This model was created from Version 01 by Penny Noble of Oxford * University. Version 02 is known to run in COR and PCEnv. A PCEnv * session is also associated with this model. * * Model Structure * * The pacemaker activity of the sinoatrial (SA) node initiates * the spontaneous beating of the heart. Over the past decade a * large body of data on the ionic currents underlying this pacemaker * activity has been elucidated in patch-clamp experiments. On * the basis of this data, several mathematical models describing * the pace maker activity of a single rabbit SA node cell have * been developed. These include: Demir et al. Sinoatrial Node * Model, 1994; Modelling the Ion Currents Underlying Sinoatrial * Node Pacemaker Activity, Dokos et al., 1996; A Model of Sinoatrial * Node Vagal Control, Dokos et al., 1996; Demir et al. Sinoatrial * Node Model, 1999; Zhang et al. Sinoatrial Node Model, 2000; * and Boyett et al. Sinoatrial Node Model, 2001. * * With the elucidation of more experimental data, these models * have become increasingly complex, with equations that describe * calcium buffering, intracellular compartmentation, and additional * ionic currents through channels, pumps and exchangers. In the * study by Kurata et al. described here, the aim is to develop * an improved mathematical model of a single primary pacemaker * cell of the rabbit SA node, in order to investigate the mechanisms * underlying pacemaker generation. On the basis of the most recent * experimental data, Kurata et al. updated the previously published * models (see list above) in several ways: * * A sustained inward current has been added (Ist). * * The voltage- and Ca2+-dependent activation kinetics of the L-type * calcium channel current (ICa,L) have been reformulated. * * Expressions for the activation kinetics of the rapidly activating * delayed rectifier potassium current (IKr) have been updated. * * Revised kinetic equations for two 4-AP sensitive currents (Ito * and Isus) have been incorporated. * * Voltage- and concentration-dependent kinetics of the Na+-K+ * pump current (INaK) have been reformulated. * * The subsarcolemmal space (see below) as a diffusion barrier * for intracellular Ca2+ has been added. * * The model was validated by comparing simulation results with * experimental data, and also by comparing them with those of * previous mathematical models. The authors concluded that their * model represented a significant improvement over previous models * because it can: Simulate whole cell voltage-clamp data for ICa,L, * IKr, and Ist; Reproduce the wave shapes of spontaneous action * potentials and ionic currents during action potential clamp * recordings; and Mimic the effects of channel blockers or Ca * 2+ buffers on pacemaker activity more accurately than the previous * models. * * The model has been described here in CellML (the raw CellML * description of the Kurata et al. 2002 model can be downloaded * in various formats as described in ). * * The complete original paper reference is cited below: * * Dynamical description of sinoatrial node pacemaking: improved * mathematical model for primary pacemaker cell, Yasutaka Kurata, * Ichiro Hisatome, Sunao Imanishi, and Toshishige Shibamoto, 2002,American * Journal of Physiology, 283, H2074-H2101. PubMed ID: 12384487 * * reaction diagram * * [[Image file: kurata_2002.png]] * * Schematic diagram depicting the intracellular compartments for * Ca2+. */ import nsrunit; unit conversion on; unit per_millisecond=1E3 second^(-1); unit millimolar_per_millisecond=1E3 meter^(-3)*second^(-1)*mole^1; unit per_millimolar_millisecond=1E3 meter^3*second^(-1)*mole^(-1); // unit millisecond predefined // unit millivolt predefined unit per_millivolt=1E3 kilogram^(-1)*meter^(-2)*second^3*ampere^1; unit per_millivolt_millisecond=1E6 kilogram^(-1)*meter^(-2)*second^2*ampere^1; unit nanoS=1E-9 kilogram^(-1)*meter^(-2)*second^3*ampere^2; unit nanoS_per_picoF=1E3 second^(-1); unit picoF=1E-12 kilogram^(-1)*meter^(-2)*second^4*ampere^2; unit per_picoF=1E12 kilogram^1*meter^2*second^(-4)*ampere^(-2); unit picoA_per_millimolar_picoF=1 kilogram^1*meter^5*second^(-4)*ampere^(-1)*mole^(-1); unit picoA=1E-12 ampere^1; // unit millimolar predefined unit attojoule_per_millimole_kelvin=1E-15 kilogram^1*meter^2*second^(-2)*kelvin^(-1)*mole^(-1); unit femtocoulomb_per_millimole=1E-12 second^1*ampere^1*mole^(-1); math main { realDomain time millisecond; time.min=0; extern time.max; extern time.delta; real V(time) millivolt; when(time=time.min) V=-58.600291137693; real R attojoule_per_millimole_kelvin; R=8314400000000000; real T kelvin; T=310.15; real F femtocoulomb_per_millimole; F=96485000000000000; real Cm picoF; Cm=32; real i_CaT(time) picoA; real i_CaL(time) picoA; real i_Kr(time) picoA; real i_Ks(time) picoA; real i_to(time) picoA; real i_sus(time) picoA; real i_h(time) picoA; real i_st(time) picoA; real i_b_Na(time) picoA; real i_NaCa(time) picoA; real i_NaK(time) picoA; real i_K_ACh(time) picoA; real E_Na(time) millivolt; real E_K(time) millivolt; real Nai(time) millimolar; when(time=time.min) Nai=9.438646305915; real Nao millimolar; Nao=140; real Ki(time) millimolar; when(time=time.min) Ki=139.984146485614; real Ko millimolar; Ko=5.4; real g_CaL nanoS; g_CaL=0.58; real E_CaL millivolt; E_CaL=45; real Ca_sub(time) millimolar; when(time=time.min) Ca_sub=0.00019074741; real L_type_calcium_channel_current.d(time) dimensionless; when(time=time.min) L_type_calcium_channel_current.d=0.000602055134; real L_type_calcium_channel_current.f(time) dimensionless; when(time=time.min) L_type_calcium_channel_current.f=0.626999773853; real fCa(time) dimensionless; when(time=time.min) fCa=0.589580408056; real L_type_calcium_channel_current_d_gate.d_infinity(time) dimensionless; real L_type_calcium_channel_current_d_gate.tau_d(time) millisecond; real alpha_d(time) per_millisecond; real beta_d(time) per_millisecond; real L_type_calcium_channel_current_f_gate.f_infinity(time) dimensionless; real L_type_calcium_channel_current_f_gate.tau_f(time) millisecond; real alpha_fCa per_millisecond; real beta_fCa per_millimolar_millisecond; beta_fCa=60; real fCa_infinity(time) dimensionless; real tau_fCa(time) millisecond; real Km_fCa millimolar; Km_fCa=0.00035; real g_CaT nanoS; g_CaT=0.458; real E_CaT millivolt; E_CaT=45; real T_type_calcium_channel_current.d(time) dimensionless; when(time=time.min) T_type_calcium_channel_current.d=0.004571884917; real T_type_calcium_channel_current.f(time) dimensionless; when(time=time.min) T_type_calcium_channel_current.f=0.249637570396; real T_type_calcium_channel_current_d_gate.d_infinity(time) dimensionless; real T_type_calcium_channel_current_d_gate.tau_d(time) millisecond; real T_type_calcium_channel_current_f_gate.f_infinity(time) dimensionless; real T_type_calcium_channel_current_f_gate.tau_f(time) millisecond; real g_Kr nanoS; real paS(time) dimensionless; when(time=time.min) paS=0.629323128348; real paF(time) dimensionless; when(time=time.min) paF=0.3493633709533; real piy(time) dimensionless; when(time=time.min) piy=0.852396631172; real pa_infinity(time) dimensionless; real tau_paS(time) millisecond; real tau_paF(time) millisecond; real pi_infinity(time) dimensionless; real tau_pi(time) millisecond; real g_Ks nanoS; g_Ks=0.0259; real E_Ks(time) millivolt; real n(time) dimensionless; when(time=time.min) n=0.054409723782; real n_infinity(time) dimensionless; real tau_n(time) millisecond; real alpha_n(time) per_millisecond; real beta_n(time) per_millisecond; real g_to nanoS; g_to=0.18; real g_sus nanoS; g_sus=0.02; real q(time) dimensionless; when(time=time.min) q=0.531446952485; real r(time) dimensionless; when(time=time.min) r=0.005550489445; real q_infinity(time) dimensionless; real tau_q(time) millisecond; real r_infinity(time) dimensionless; real tau_r(time) millisecond; real i_h_Na(time) picoA; real i_h_K(time) picoA; real g_h_Na nanoS; g_h_Na=0.1437375; real g_h_K nanoS; g_h_K=0.2312625; real y(time) dimensionless; when(time=time.min) y=0.067156687129; real y_infinity(time) dimensionless; real tau_y(time) millisecond; real g_st nanoS; g_st=0.015; real E_st millivolt; E_st=37.4; real qa(time) dimensionless; when(time=time.min) qa=0.426018100136; real qi(time) dimensionless; when(time=time.min) qi=0.333330378068; real qa_infinity(time) dimensionless; real tau_qa(time) millisecond; real alpha_qa(time) per_millisecond; real beta_qa(time) per_millisecond; real qi_infinity(time) dimensionless; real tau_qi(time) millisecond; real alpha_qi(time) per_millisecond; real beta_qi(time) per_millisecond; real g_b_Na nanoS; g_b_Na=0.0054; real g_K_ACh picoA; real Km_Kp millimolar; Km_Kp=1.4; real Km_Nap millimolar; Km_Nap=14; real i_NaK_max picoA; i_NaK_max=3.6; real kNaCa picoA; kNaCa=125; real x1(time) dimensionless; real x2(time) dimensionless; real x3(time) dimensionless; real x4(time) dimensionless; real k41(time) dimensionless; real k34 dimensionless; real k23(time) dimensionless; real k21(time) dimensionless; real k32(time) dimensionless; real k43(time) dimensionless; real k12(time) dimensionless; real k14(time) dimensionless; real Qci dimensionless; Qci=0.1369; real Qn dimensionless; Qn=0.4315; real Qco dimensionless; Qco=0; real K3ni millimolar; K3ni=26.44; real Kci millimolar; Kci=0.0207; real K1ni millimolar; K1ni=395.3; real K2ni millimolar; K2ni=2.289; real Kcni millimolar; Kcni=26.44; real K3no millimolar; K3no=4.663; real K1no millimolar; K1no=1628; real K2no millimolar; K2no=561.4; real Kco millimolar; Kco=3.663; real do(time) dimensionless; real di(time) dimensionless; real Cao millimolar; Cao=2; real j_Ca_dif(time) millimolar_per_millisecond; real j_rel(time) millimolar_per_millisecond; real j_up(time) millimolar_per_millisecond; real j_tr(time) millimolar_per_millisecond; real tau_dif_Ca millisecond; tau_dif_Ca=0.04; real tau_tr millisecond; tau_tr=60; real K_rel millimolar; K_rel=0.0012; real P_up millimolar_per_millisecond; P_up=0.005; real P_rel per_millisecond; P_rel=0.5; real K_up millimolar; K_up=0.0006; real Ca_up(time) millimolar; when(time=time.min) Ca_up=1.462338380106; real Cai(time) millimolar; when(time=time.min) Cai=0.000312494921; real Ca_rel(time) millimolar; when(time=time.min) Ca_rel=0.296742023718; real Mgi millimolar; Mgi=2.5; real V_i litre; V_i=0.0000000000015835; real V_rel litre; V_rel=0.0000000000000042223; real V_up litre; V_up=0.000000000000040816; real V_sub litre; V_sub=0.000000000000035098; real TMC_tot dimensionless; TMC_tot=0.062; real CM_tot dimensionless; CM_tot=0.045; real TC_tot dimensionless; TC_tot=0.031; real CQ_tot dimensionless; CQ_tot=10; real delta_fTMC(time) millimolar_per_millisecond; real delta_fCMi(time) millimolar_per_millisecond; real delta_fCMs(time) millimolar_per_millisecond; real delta_fTC(time) millimolar_per_millisecond; real delta_fCQ(time) millimolar_per_millisecond; real delta_fTMM(time) millimolar_per_millisecond; real fTMM(time) millimolar; when(time=time.min) fTMM=0.350600895635; real fCMi(time) millimolar; when(time=time.min) fCMi=0.116947220413; real fCMs(time) millimolar; when(time=time.min) fCMs=0.074631965653; real fTC(time) millimolar; when(time=time.min) fTC=0.059206293446; real fTMC(time) millimolar; when(time=time.min) fTMC=0.602955114871; real fCQ(time) millimolar; when(time=time.min) fCQ=0.260317260703; real kf_TC per_millimolar_millisecond; kf_TC=88.8; real kf_TMM per_millimolar_millisecond; kf_TMM=2.277; real kf_TMC per_millimolar_millisecond; kf_TMC=227.7; real kf_CM per_millimolar_millisecond; kf_CM=227.7; real kf_CQ per_millimolar_millisecond; kf_CQ=0.534; real kb_TC per_millisecond; kb_TC=0.446; real kb_TMC per_millisecond; kb_TMC=0.00751; real kb_TMM per_millisecond; kb_TMM=0.751; real kb_CM per_millisecond; kb_CM=0.542; real kb_CQ per_millisecond; kb_CQ=0.445; // // V:time=((-1)*(i_CaL+i_CaT+i_Kr+i_Ks+i_to+i_sus+i_h+i_st+i_b_Na+i_K_ACh+i_NaK+i_NaCa)/(1 picoF)); // E_Na=(R*T/F*ln(Nao/Nai)); E_K=(R*T/F*ln(Ko/Ki)); // i_CaL=(g_CaL*(V-E_CaL)*L_type_calcium_channel_current.d*L_type_calcium_channel_current.f*fCa); // L_type_calcium_channel_current_d_gate.d_infinity=(1/(1+exp((-1)*(V+(14.1 millivolt))/(6 millivolt)))); L_type_calcium_channel_current_d_gate.tau_d=(1/(alpha_d+beta_d)); alpha_d=((-1)*(.02839 per_millivolt_millisecond)*(V+(35 millivolt))/(exp((-1)*(V+(35 millivolt))/(2.5 millivolt))-1)-(.0849 per_millivolt_millisecond)*V/(exp((-1)*V/(4.808 millivolt))-1)); beta_d=((.01143 per_millivolt_millisecond)*(V-(5 millivolt))/(exp((V-(5 millivolt))/(2.5 millivolt))-1)); L_type_calcium_channel_current.d:time=((L_type_calcium_channel_current_d_gate.d_infinity-L_type_calcium_channel_current.d)/L_type_calcium_channel_current_d_gate.tau_d); // L_type_calcium_channel_current_f_gate.f_infinity=(1/(1+exp((V+(30 millivolt))/(5 millivolt)))); L_type_calcium_channel_current_f_gate.tau_f=((44.3 millisecond)+(257.1 millisecond)*exp((-1)*((V+(32.5 millivolt))/(13.9 millivolt))^2)); L_type_calcium_channel_current.f:time=((L_type_calcium_channel_current_f_gate.f_infinity-L_type_calcium_channel_current.f)/L_type_calcium_channel_current_f_gate.tau_f); // alpha_fCa=(Km_fCa*beta_fCa); fCa_infinity=(Km_fCa/(Km_fCa+Ca_sub)); tau_fCa=(fCa_infinity/alpha_fCa); fCa:time=((fCa_infinity-fCa)/tau_fCa); // i_CaT=(g_CaT*(V-E_CaT)*T_type_calcium_channel_current.d*T_type_calcium_channel_current.f); // T_type_calcium_channel_current_d_gate.d_infinity=(1/(1+exp((-1)*(V+(26.3 millivolt))/(6 millivolt)))); T_type_calcium_channel_current_d_gate.tau_d=((1 millisecond)/(1.068*exp((V+(26.3 millivolt))/(30 millivolt))+1.068*exp((-1)*(V+(26.3 millivolt))/(30 millivolt)))); T_type_calcium_channel_current.d:time=((T_type_calcium_channel_current_d_gate.d_infinity-T_type_calcium_channel_current.d)/T_type_calcium_channel_current_d_gate.tau_d); // T_type_calcium_channel_current_f_gate.f_infinity=(1/(1+exp((V+(61.7 millivolt))/(5.6 millivolt)))); T_type_calcium_channel_current_f_gate.tau_f=((1 millisecond)/(.0153*exp((-1)*(V+(61.7 millivolt))/(83.3 millivolt))+.015*exp((V+(61.7 millivolt))/(15.38 millivolt)))); T_type_calcium_channel_current.f:time=((T_type_calcium_channel_current_f_gate.f_infinity-T_type_calcium_channel_current.f)/T_type_calcium_channel_current_f_gate.tau_f); // g_Kr=((.025 nanoS)*(Ko/(1 millimolar))^.59); i_Kr=(g_Kr*(V-E_K)*(.6*paF+.4*paS)*piy); // pa_infinity=(1/(1+exp((-1)*(V+(23.2 millivolt))/(10.6 millivolt)))); tau_paS=((.84655354 millisecond)/(.0042*exp(V/(17 millivolt))+1.5E-4*exp((-1)*V/(21.6 millivolt)))); tau_paF=((.84655354 millisecond)/(.0372*exp(V/(15.9 millivolt))+9.6E-4*exp((-1)*V/(22.5 millivolt)))); paS:time=((pa_infinity-paS)/tau_paS); paF:time=((pa_infinity-paF)/tau_paF); // pi_infinity=(1/(1+exp((V+(28.6 millivolt))/(17.1 millivolt)))); tau_pi=((1 millisecond)/(.1*exp((-1)*V/(54.645 millivolt))+.656*exp(V/(106.157 millivolt)))); piy:time=((pi_infinity-piy)/tau_pi); // E_Ks=(R*T/F*ln((Ko+.12*Nao)/(Ki+.12*Nai))); i_Ks=(g_Ks*(V-E_Ks)*n^2); // n_infinity=(alpha_n/(alpha_n+beta_n)); tau_n=(1/(alpha_n+beta_n)); alpha_n=((.014 per_millisecond)/(1+exp((-1)*(V-(40 millivolt))/(9 millivolt)))); beta_n=((.001 per_millisecond)*exp((-1)*V/(45 millivolt))); n:time=((n_infinity-n)/tau_n); // i_to=(g_to*(V-E_K)*q*r); i_sus=(g_sus*(V-E_K)*r); // q_infinity=(1/(1+exp((V+(49 millivolt))/(13 millivolt)))); tau_q=(.6*(65.17/((.57 per_millisecond)*exp((-1)*(.08 per_millivolt)*(V+(44 millivolt)))+(.065 per_millisecond)*exp((.1 per_millivolt)*(V+(45.93 millivolt))))+(10.1 millisecond))); q:time=((q_infinity-q)/tau_q); // r_infinity=(1/(1+exp((-1)*(V-(19.3 millivolt))/(15 millivolt)))); tau_r=(.66*1.4*((15.59 millisecond)/(1.037*exp((.09 per_millivolt)*(V+(30.61 millivolt)))+.369*exp((-1)*(.12 per_millivolt)*(V+(23.84 millivolt))))+(2.98 millisecond))); r:time=((r_infinity-r)/tau_r); // i_h_Na=(g_h_Na*(V-E_Na)*y^2); i_h_K=(g_h_K*(V-E_K)*y^2); i_h=(i_h_Na+i_h_K); // y_infinity=(1/(1+exp((V+(64 millivolt))/(13.5 millivolt)))); tau_y=((.7166529 millisecond)/(exp((-1)*(V+(386.9 millivolt))/(45.302 millivolt))+exp((V-(73.08 millivolt))/(19.231 millivolt)))); y:time=((y_infinity-y)/tau_y); // i_st=(g_st*(V-E_st)*qa*qi); // qa_infinity=(1/(1+exp((-1)*(V+(57 millivolt))/(5 millivolt)))); tau_qa=(1/(alpha_qa+beta_qa)); alpha_qa=(1/((.15 millisecond)*exp((-1)*V/(11 millivolt))+(.2 millisecond)*exp((-1)*V/(700 millivolt)))); beta_qa=(1/((16 millisecond)*exp(V/(8 millivolt))+(15 millisecond)*exp(V/(50 millivolt)))); qa:time=((qa_infinity-qa)/tau_qa); // qi_infinity=(alpha_qi/(alpha_qi+beta_qi)); tau_qi=(6.65/(alpha_qi+beta_qi)); alpha_qi=(1/((3100 millisecond)*exp(V/(13 millivolt))+(700 millisecond)*exp(V/(70 millivolt)))); beta_qi=(1/((95 millisecond)*exp((-1)*V/(10 millivolt))+(50 millisecond)*exp((-1)*V/(700 millivolt)))+(2.29E-4 per_millisecond)/(1+exp((-1)*V/(5 millivolt)))); qi:time=((qi_infinity-qi)/tau_qi); // i_b_Na=(g_b_Na*(V-E_Na)); // g_K_ACh=((.0011 picoA)*(Ko/(1 millimolar))^.41); i_K_ACh=(g_K_ACh*(Ki-Ko*exp((-1)*V*F/(R*T)))/(1 millimolar)); // i_NaK=(i_NaK_max*(1+(Km_Kp/Ko)^1.2)^((-1)*1)*(1+(Km_Nap/Nai)^1.3)^((-1)*1)*(1+exp((-1)*(V-E_Na+(120 millivolt))/(30 millivolt)))^((-1)*1)); // i_NaCa=(kNaCa*(x2*k21-x1*k12)/(x1+x2+x3+x4)); x1=(k41*k34*(k23+k21)+k21*k32*(k43+k41)); x2=(k32*k43*(k14+k12)+k41*k12*(k34+k32)); x3=(k14*k43*(k23+k21)+k12*k23*(k43+k41)); x4=(k23*k34*(k14+k12)+k14*k21*(k34+k32)); k43=(Nai/(K3ni+Nai)); k12=(Ca_sub/Kci*exp((-1)*Qci*V*F/(R*T))/di); k14=(Nai/K1ni*Nai/K2ni*(1+Nai/K3ni)*exp(Qn*V*F/(2*R*T))/di); k41=exp((-1)*Qn*V*F/(2*R*T)); di=(1+Ca_sub/Kci*(1+exp((-1)*Qci*V*F/(R*T))+Nai/Kcni)+Nai/K1ni*(1+Nai/K2ni*(1+Nai/K3ni))); k34=(Nao/(K3no+Nao)); k21=(Cao/Kco*exp(Qco*V*F/(R*T))/do); k23=(Nao/K1no*Nao/K2no*(1+Nao/K3no)*exp((-1)*Qn*V*F/(2*R*T))/do); k32=exp(Qn*V*F/(2*R*T)); do=(1+Cao/Kco*(1+exp(Qco*V*F/(R*T)))+Nao/K1no*(1+Nao/K2no)*(1+Nao/K3no)); // j_Ca_dif=((Ca_sub-Cai)/tau_dif_Ca); j_rel=(P_rel*(Ca_rel-Ca_sub)/(1+(K_rel/Ca_sub)^2)); j_up=(P_up/(1+K_up/Cai)); j_tr=((Ca_up-Ca_rel)/tau_tr); // Nai:time=((-1)*(i_h_Na+i_st+i_b_Na+3*i_NaK+3*i_NaCa)*Cm/((1 picoF)*F*(V_i+V_sub))); Ki:time=((-1)*(i_Kr+i_Ks+i_to+i_sus+i_h_K+i_K_ACh+(-1)*2*i_NaK)*Cm/((1 picoF)*F*(V_i+V_sub))); Cai:time=((j_Ca_dif*V_sub-j_up*V_up)/V_i-(CM_tot*delta_fCMi+TC_tot*delta_fTC+TMC_tot*delta_fTMC)); Ca_sub:time=(((-1)*(i_CaL+i_CaT-2*i_NaCa)*Cm/((1 picoF)*2*F)+j_rel*V_rel)/V_sub-(j_Ca_dif+CM_tot*delta_fCMs)); Ca_up:time=(j_up-j_tr*V_rel/V_up); Ca_rel:time=(j_tr-(j_rel+CQ_tot*delta_fCQ)); // fTC:time=delta_fTC; delta_fTC=(kf_TC*Cai*((1 millimolar)-fTC)-kb_TC*fTC); fTMC:time=delta_fTMC; delta_fTMC=(kf_TMC*Cai*((1 millimolar)-(fTMC+fTMM))-kb_TMC*fTMC); fTMM:time=delta_fTMM; delta_fTMM=(kf_TMM*Mgi*((1 millimolar)-(fTMC+fTMM))-kb_TMM*fTMM); fCMi:time=delta_fCMi; delta_fCMi=(kf_CM*Cai*((1 millimolar)-fCMi)-kb_CM*fCMi); fCMs:time=delta_fCMs; delta_fCMs=(kf_CM*Ca_sub*((1 millimolar)-fCMs)-kb_CM*fCMs); fCQ:time=delta_fCQ; delta_fCQ=(kf_CQ*Ca_rel*((1 millimolar)-fCQ)-kb_CQ*fCQ); }