The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src‐family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR‐mediated ASM contraction and associated signalling events.20) and myosin phosphatase targeting subunit‐1 (MYPT‐1) was evaluated in cultured human ASM cells (hASMC). [Ca2+]i was evaluated in Fura‐2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined.Contraction was recorded in intact or α‐toxin permeabilized rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light‐chain‐20 (MLC20 and auto‐phosphorylation of SrcFK and FAK. MYPT‐1 phosphorylation was sensitive to inhibition of Rho‐kinase and SrcFK, but not FAK. Contraction induced by SR Ca2+ depletion and equivalent [Ca2+]i responses in hASMC were sensitive to inhibition of both SrcFK and FAK, while depolarization‐induced contraction was sensitive to FAK inhibition only. SrcFK auto‐phosphorylation was partially FAK‐dependent, while FAK auto‐phosphorylation was SrcFK‐independent.Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho‐kinase, while after α‐toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho‐kinase, but not FAK. CCh and BK increased phosphorylation of MYPT‐1 and MLC2+‐sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction and may be a viable target for modulating ASM tone in respiratory disease.SrcFK mediates Ca ASMairway smooth musclehASMCcultured human airway smooth muscle cellsKPSS+ for K+PSS with 80 mM equimolar substitution of Na20MLCmyosin light‐chain 20 KDa subunitMLCPmyosin light‐chain phosphataseMYPT‐1myosin phosphatase targeting subunit‐1ROCE2+ entryreceptor‐operated CaSOCE2+ entrystore‐operated CaVOCE2+ entryvoltage‐operated Caet al.,et al.,Airway smooth muscle (ASM) tone is subject to regulation by cholinergic, catecholamine and NANC neurotransmitters as well as local inflammatory mediators. In healthy airways, muscle tone is normally low, providing a low resistance path for airflow. However, contraction may be enhanced in response to chemical irritants or allergens, particularly in lower respiratory tract bronchioles  phosphorylation, which is in turn determined by the balance between Ca2+‐dependent activation of myosin light‐chain kinase (MLCK) and Ca2+‐independent inhibition of myosin light‐chain phosphatase (MLCP), as well as the formation and recruitment of myofilaments  and a combination of Ca2+ entry through receptor‐operated, store‐operated and voltage‐operated Ca2+ channels . Inhibition of MLCP occurs via phosphorylation of myosin phosphatase targeting subunit‐1 (MYPT‐1), primarily by Rho‐kinase  and focal adhesion kinase (FAK) are widely expressed non‐receptor tyrosine kinases (TKs) important in many aspects of cellular function, being activated in response to various stimuli including growth factors, GPCRs, reactive oxygen species and adhesion. SrcFK and FAK are often described as being mutually dependent or reciprocally activated, especially when associated with integrin engagement and/or growth factor receptor activation . We found that SrcFK, most likely c‐Src itself, modulate Rho‐kinase dependent Ca2+‐sensitization, but FAK does not, and that the two tyrosine kinases differentially regulate SOCE/ROCE and VOCE. We also suggest the existence of two subpopulations of GPCR‐activated SrcFK, one being FAK‐dependent and the other FAK‐independent.In this study, we hypothesized that SrcFK and FAK mediate GPCR‐induced ASM contraction via multiple signalling pathways and examined their influence on Rho‐kinase‐dependent MLCP inhibition/Caet al.,3; 1 MgSO4; 4 KCl; 5.56 glucose; 0.435 NaH2PO4; 1.8 CaCl2, pH 7.4), gassed with 95% air, 5% CO2 at 37°C. Bronchioles were incrementally stretched and alternately exposed to PSS containing 80 mM [K+]  until the point on the length tension curve at which muscle length was optimum for active tension development was achieved, as described previously  Act 1986 Amendment Regulations (SI 2012/3039) and were deemed to be as humane as possible. All results involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (McGrath 2+‐sensitization component of contraction was achieved by permeabilizing myograph‐mounted bronchioles with α‐haemolysin (α‐toxin). PSS was first exchanged for relaxing solution 2; 1000 KMS; 100 K2EGTA; 5 Na2ATP; 10 Na2creatine phosphate, pH 7.1), gassed with air at 26°C. α‐toxin (60 µg ml‐1) was then applied in relaxing solution with pCa raised to 6.7 for 30 min, permeabilization being confirmed by the development of active tension. pCa was adjusted via proportionate substitution of K2EGTA for CaEGTA, with 100 CaEGTA, 0 K2EGTA being equivalent to pCa4.5. Contractile responses to bronchoconstrictors were conducted at pCa 6.5 (~300 nM [Ca2+]), which induced a contraction equivalent to 10‐20% of that achieved by pCa 4.5. GTP 1 μM and 10 μM cyclopiazonic acid (CPA) were included to support G‐protein signalling and to prevent the influence of SR Ca2+ release on contraction respectively.Specific examination of the Rho‐kinase dependent Can = 11; 7 women, 4 males; age range 22–53 years; life‐long absence of respiratory symptoms; lung functions within normal limits) by deep endobronchial biopsy. ASM bundles were bathed in DMEM containing 10% FBS, l‐glutamine (2 mM), sodium pyruvate (1 mM), non‐essential amino acids and amphotericin B (2 µg ml‐1), and subjected to enzymatic digestion in nominally Ca2+‐free HEPES buffer containing: 5.56 mM glucose, 2 mg ml‐1 collagenase Type XI, 1 mg ml‐1 papaine, 1 mg ml‐1 trypsin inhibitor and 1 mM DTT, for 30 min at 37°C. Cells were then dispersed into culture flasks containing DMEM (plus supplements) and incubated at 37°C, pH 7.4. Smooth muscle phenotype was confirmed by positive staining with anti‐smooth muscle α‐actin, anti‐desmin and anti‐calponin, with Alexa Fluor®488 labelled secondary antibody (Lifetechnologies) and with TRITC‐labelled phalloidin to confirm the presence of stress fibres in resting cells  were designed as described previously  containing 1% phosphatase inhibitor cocktails 2 and 3 and 1% protease inhibitor cocktail . Cells were scraped into a tube and agitated before being placed on ice. Rat trachealis muscle was dissected free of adjoining cartilage, and epithelium was removed by scraping. Acute treatments were conducted in PSS/5% COSamples were boiled in NuPAGE LDS Sample Buffer (Invitrogen) at 95°C for 5 min before being loaded onto 4–12% NuPAGE Bis‐Tris gels (Invitrogen) for SDS‐PAGE. Sample protein content was determined using the bicinchoninic acid assay, calibrated against BSA protein standards, to enable loading of ~20 µg of protein per lane. Gels were run at 180 V for 1 h using an Xcell SureLock Mini‐Cell (Invitrogen) and MOPS running buffer (Invitrogen). Protein was transferred to a nitrocellulose membrane (Amersham) in 25 mM Tris, 192 mM glycine and 20% methanol, at 35 V for 1 h.Membranes were blocked with 5% skimmed milk in Tris buffered saline (TBS) for 1 h at room temperature, followed by incubation with specific anti‐phospho‐protein primary antibody  in TBS with 1% skimmed milk and 0.1% Tween‐20 (TBS‐T), overnight at 4°C. Following washes in TBS‐T, HRP‐conjugated secondary antibody  was applied for 1 h at room temperature, followed by a final wash in TBS‐T. ‘Phospho’ proteins were visualized with Super‐Signal West Femto chemi‐luminescent Substrate (Thermo scientific). Membranes were then stripped in Restore western blot stripping buffer (Thermo Scientific), re‐blocked and re‐incubated with corresponding ‘total’ antibody and appropriate secondary antibodies, as above. ‘Total’ proteins were visualized with either ECL plus or ECL prime . Images were captured and quantified using the ChemiDoc XRS+ gel‐imaging system (Biorad). An estimate of the proportion of target protein that was phosphorylated was calculated as a ratio of ‘phospho’ over ‘total’ signal for each protein band from each gel, and the effects of acute treatments on these ratios was expressed as a percentage of control (untreated samples run on the same gel).4, 4 KCl, 0.44 KH2PO4, 4.2 NaHCO3, 120 NaCl, 0.34 Na2HPO4, 20 HEPES and 2 CaCl) at room temperature for 40 min. Coverslips were mounted on an upright microscope and cells perfused with HBSS, containing test reagents as required. Changes in [Ca2+]i were measured as a ratio of 340 nm over 380 nm emission intensities with a ×20 oil immersion UV objective and a microspectrofluorimeter . For each coverslip, ratios obtained in zero [Ca2+]o and the absence of drug were taken as background fluorescence  and subtracted from all subsequent measurements.Cultured hASMCs were grown on glass cover‐slips until 70% confluent, followed by 7 days of serum starvation. Cells were loaded with 1 μM Fura PE‐3/AM in HBSS  cyclohexane carboxamide (Y27632); 6‐[4‐(3‐methanesulfonyl‐benzylamino)‐5‐trifluoromethyl‐pyrimidin‐2‐ylamino]‐3,4‐dihydro‐1H‐quinolin‐2‐one (PF‐573228); N‐[2‐[[[2‐[amino]‐5‐(trifluoromethyl)‐4‐pyrimidinyl]amino]methyl]phenyl]‐N‐methyl‐methanesulfonamide hydrate (PF‐431396) or Calbiochem: (4‐amino‐5‐(4‐chlorophenyl)‐7‐(dimethylethyl) pyrazolopyrimidine (PP2); 4‐amino‐7‐phenylpyrazolpyrimidine (PP3). Cell culture and western blot materials were obtained from Cell Signalling, Invitrogen, GE Healthcare or Thermo Scientific. Nifedipine, YM58483, and cyclopiazonic acid and α‐haemolysin were from Sigma.Antibodies were obtained from cell signalling (anti‐phospho‐Src (tyr416); anti‐Src; anti‐phospho‐FAK (Y397); anti‐phospho‐FAK (Y576/577); anti‐FAK; anti‐phospho‐MLC (S19); anti‐MYPT1; anti‐MLC), Millipore (anti‐phospho‐MYPT1 (T696)), Sigma . Kinase inhibitors were obtained from Sigma ‐4((R)‐1‐aminoethyl)‐2 (‐LogM EC50) and maximum response (Max). Bradykinin concentration responses were biphasic, and were best fitted using a two‐site saturation model, for the characterization of a high affinity component (PD2‐1 and Max‐1) and a low affinity component (PD2‐2 and Max‐2). Statistical analysis of data was by Student's paired or un‐paired t‐test , one‐way ANOVA  or two‐way ANOVA , with Holm–Sidak post tests where appropriate, and as indicated in figure or table legends, using SigmaPlot 10. Differences were considered significant if P < 0.05.All values are expressed as mean ± SEM. Non‐linear regression curve fitting was performed with SigmaPlot 10. Carbachol (CCh) concentration‐response curves were fitted using the Hill equation for the calculation of PDP < 0.01, paired t‐test, n = 8), Y27632  and PF‐573228 , and the PD2 was significantly increased by PP2 , Y27632  and PF‐573228   or maximum contraction . Conversely, PF‐431396 had similar effects as those of PF‐573228, causing a similar increase in PD2  and a similar reduction in maximum contraction  , but there was no significant change in PD2 .We examined the contractile responses to CCh and bradykinin (BK) in rat bronchioles, whereby the bronchoconstrictors were applied cumulatively at 5 min intervals. Two concentration‐response curves were performed in each bronchiole 0.01–100μM), the first acting as a control and the second after pre‐incubation with either the SrcFK inhibitor PP2 (30 μM), the Rho‐kinase inhibitor Y27632 (10 μM), the FAK inhibitor PF‐573228 (10 μM) or no inhibitor (control). In addition, to account for possible off‐target effects of PP2 and PF‐573228, key contractile responses were also repeated with PP3 (30 μM), the negative control for PP2 and a dual FAK/PYK2 inhibitor, PF‐431396 (10 μM). CCh caused a sustained contraction at each dose  . In timeP < 0.05, n = 11), while PD2‐1 and PD2‐2 were both unchanged  .Bradykinin caused a prominent transient contraction and a smaller sustained component at each dose Figure E. The co2+‐sensitization pathway, CCh or BK concentration‐response curves were repeated in α‐toxin‐permeabilized rat bronchioles, with [Ca2+]i fixed at pCa 6.5, in the absence or presence of PP2, Y27632 or PF‐573228. After α‐toxin‐permeabilization, bronchoconstictor concentration‐response curves were not repeatable (not shown), so controls and effects of antagonists were compared in separate bronchioles. CCh‐induced contraction was almost absent in the presence of Y27632 and was significantly smaller in the presence of PP2 , but in the presence of PF‐573228 was not different from controls , but was no different in PF‐573228. The underlying pCa 6.5 contraction was unaffected by either PP2 or PF‐573228, but was partially inhibited by Y27632 . BK also produced a modest (relative to CCh) concentration‐dependent contraction in permeabilized bronchioles  or CCh (100 μM), and whether this activation relates to subsequent activation of MLCK, we measured phosphorylation of MYPT‐1 at T696, a known phosphorylation target of Rho‐kinase  and MYPT‐1 (Y397), . We then examined the effects of PP2 and PF‐573228 on SOCE‐mediated contraction. SR Ca2+ was first depleted with cyclopiazonic acid  in the absence of extracellular Ca2+ and presence of 200 μM EGTA, then 2 mM Ca2+ was re‐applied. CPA was used here instead of a GPCR agonist because it would have been difficult to separate effects of the agonist on Ca2+ entry from those on Rho‐kinase‐mediated Ca2+ sensitization. In control experiments, re‐application of 2 mM Ca2+ induced a biphasic contraction, which peaked at ~2 min and slowly decayed to ~20% of 80 mM KPSS after 30 min. In the presence of either PP2 or PF‐573228, the sustained component was significantly smaller, decaying to <10% of 80 mM KPSS after 30 min  on the SOCE‐mediated contraction was also determined. Apart from a small residual transient contraction, the response was abolished by YM58483, while nifedipine was without effect, apart from a small reduction in the peak response  or PF‐573228 (10 μM) was applied between the first and second exposures, the contractile response was modestly but significantly reduced by PF‐573228, but not by PP2  caused a near instantaneous increase in [Ca2+]i. This decayed back to the baseline within ~2 min; a response typical of GPCR‐induced SR Ca2+‐release. 2 mM Ca2+ was then re‐applied for 20 min, with simultaneous washout of BK. This induced a biphasic rise in [Ca2+]i with a similar time course to the SOCE contractile responses. In the presence of either PP2 or PF‐573228, the sustained component was reduced by ~50%, while the initial transient component and the initial BK‐induced SR release were both unaffected  auto‐phosphorylation by BK was inhibited by PF‐573228 by ~50% , MYPT‐1 (T696), SrcFK (Y416) and FAK (Y576/577) phosphorylation in hASMC after transfection with c‐Src siRNA or scrambled siRNA. Specificity of c‐Src siRNA was verified by a ~70% reduction in c‐Src protein expression, while expressions of MLC20, MYPT‐1 and FAK were all unaffected , SrcFK (Y416) and FAK (Y576/577) to BK were all inhibited by ~60%, compared with matched scrambled siRNA transfected cells . RhoGEFs are known to be activated or modulated by various non‐receptor TKs , IP3‐dependent depletion of SR Ca2+ and subsequent STIM1/Orai1/TRP‐dependent influx (SOCE), and subsequent depolarization‐induced opening of l‐type Ca2+ channels (VOCE) , anti‐desmin (panel B) and anti‐calponin (panel C), visualised with Alexa Fluor®488 labelled secondary antibody (Lifetechnologies) and fluorescent microscopy. Cells were also stained with TRITC‐labelled phalloidin to confirm the presence of stress fibres in resting cells (Panel D). In Panel D, nuclei are visualised by staining with Hoechst. Scale bar =20μm.Figure S2 Effect of PP3 on contractile responses in rat bronchioles.Figure S3 Effect of PF‐431396 on contractile responses in rat bronchioles.Figure S4 Effects of SrcFK, Rho‐kinase and FAK inhibition on bradykinin‐induced contractile responses in rat bronchioles.Supporting info itemClick here for additional data file.


Background. In Mexico and other developing countries, few reports of the survival of children with acute leukaemia exist. Objective. We aimed at comparing the disease-free survival of children with acute myeloid leukaemia who, in addition to being treated with the Latin American protocol of chemotherapy and an autologous transplant, either underwent early intensified chemotherapy or did not undergo such treatment. Procedure. This was a cohort study with a historical control group, forty patients, less than 16 years old. Group A (20 patients), diagnosed in the period 2005–2007, was treated with the Latin American protocol of chemotherapy with an autologous transplant plus early intensified chemotherapy: high doses of cytarabine and mitoxantrone. Group B (20 patients), diagnosed in the period 1999–2004, was treated as Group A, but without the early intensified chemotherapy. Results. Relapse-free survival for Group A was 90% whereas that for Group B it was 60% (P = 0.041). Overall survival for Group A  was higher than that for Group B (60%). Complete remission continued for two years of follow-up. Conclusions. Relapse-free survival for paediatric patients treated with the Latin American protocol of chemotherapy with an autologous transplant plus early intensified chemotherapy was higher than that for those who did not receive early intensified chemotherapy. The importance of acute myeloid leukaemia (AML) is evident because, although comprising only 15%–20% of childhood leukaemias, the mortality rates of AML account for up to 30% of leukaemia-related deaths .When administered alone, chemotherapy in paediatric AML has improved the probability of event-free survival (EFS); however, even in the best series published in the last decade, EFS reached only 31%–54% with the probability of overall survival (OS) between 36% and 66% . When chAlthough the results with autologous transplants are similar to those with chemotherapy alone and their use is controversial, some groups that have obtained results either superior to those of chemotherapy alone or similar to those of allogeneic transplant retain their interest in the use of autologous transplants –30, beca ex vivo or purge in vivo have been developed  and that for Group A on AML-BFM 93. The treatment schedule was described with detail and the way as autologous transplant was done. In the table I the harvest of autologous haematopoietic stem cells is mentioned.


Under certain conditions, during binary mixture adsorption in nanoporous hosts, the concentration of one component may temporarily exceed its equilibrium value. This implies that, in contrast to Fick's Law, molecules must diffuse in the direction of increasing rather than decreasing concentration. Although this phenomenon of ‘overshooting' has been observed previously, it is only recently, using microimaging techniques, that diffusive fluxes in the interior of nanoporous materials have become accessible to direct observation. Here we report the application of interference microscopy to monitor ‘uphill' fluxes, covering the entire period of overshooting from initiation until final equilibration. It is shown that the evolution of the profiles can be adequately predicted from the single-component diffusivities together with the binary adsorption equilibrium data. The guest molecules studied  and the host material (ZSM-58 or DDR) are of practical interest in relation to the development of kinetically selective adsorption separation processes. During the transient adsorption of a binary mixture in a nanoporous host, the concentration of one component may temporarily exceed its equilibrium value, with molecules diffusing in the direction of increasing concentration. Here, the authors use microimaging to examine this process in a real system. Atoms and molecules are, as a consequence of their thermal energy, subject to permanent random movement (Brownian motion). This process, referred to as diffusion, is a universal phenomenon that occurs, on different timescales, in all states of matter13456During the initial stage of the uptake, the more mobile species diffuses rapidly into the interior of the nanoporous crystal, approaching a concentration dictated by the single-component equilibrium isotherm. With continuing molecular uptake, the slower component will eventually diffuse throughout the crystal and the concentrations of both components will approach uniform equilibrium levels determined by the binary isotherm. However, during this equilibration process, the slower diffusing molecules must compete with the already accommodated faster molecules, leading to a decrease in the local concentration of the latter. Consequently, during uptake, the concentration of the faster component ‘overshoots', exceeding its final equilibrium value. Hitherto experimental studies of this phenomenon were necessarily based only on measurements of the time-dependent average concentrations of both components in the adsorbed phase9102–C2H6  is high enough so that, on the timescale of our measurements, the local CO2 concentration attains its equilibrium value  essentially instantaneously. With C2H6–C3H6 mixtures, vice versa, the diffusivity of the slower component (C3H6) is small enough so that C2H6 (now the faster component!) equilibrates, with the C3H6 profiles remaining essentially unchanged. Finally, it is shown that the evolution of the concentration profiles, including both ‘overshooting' and final equilibration, can be predicted from the binary adsorption equilibrium isotherm and the single-component diffusivities.It was only with the introduction of infrared (IR) imaging12131412141617182H6 refs , 21, 22 3H6 refs , 24, 25 3H6 refs 27 turn o2 within the plane of observation. However, in most situations IFM can only provide information about overall loadings rather than the concentrations of individual components. In the present study, we succeeded in identifying and exploiting two special cases in which these limitations can be circumvented, making it possible to take advantage of the superior spatial resolution of IFM to study in detail the transient behaviour of a two-component adsorption system.By using characteristic frequencies of absorption as a ‘finger print' for a given molecular species, microimaging by IR microscopy is able to distinguish between different molecular species and adsorption sites282971431323312The application of IFM for microimaging is based on the fact that changes in guest concentration give rise to changes in the refractive index and therefore to changes in the phase shift between a light beam passing the crystal and a reference beam passing through the surroundings. To a first-order approximation, changes in the phase shift are related linearly to the concentration changes12ci denoting the change in the concentration of component i. ki stands for the proportionality factor between changes in the concentration of component i and phase changes and may be determined by comparison with the results of conventional adsorption measurements. It is evident that, even for a two-component system with known parameters ki , experimental measurements of changes Δ(Δϕ) in the phase shift (the primary parameters of IFM) do not provide sufficient information to determine the changes Δc1 and with Δ0 s ref. ).2–ethane by zeolite ZSM-58, the diffusion rate of one component (CO2) is fast enough to ensure essentially instantaneous equilibration of this component over the sample, with the local concentration therefore determined entirely by the partial pressure of this component in the surrounding atmosphere and the local concentration of the second component . Under such conditions, In one limiting case, realized in the present study for the uptake of CO2) and slow (ethane) components, respectively. Since, by assuming local equilibration of the fast component, the concentration c1 and, hence, its variation Δc1 are known functions of p1 and c2, Δc2 becomes the only unknown in where the indices 1 and 2 refer to the fast  is the other limiting case in which the uptake of one component is slow enough to be followed by IFM but still sufficiently fast that the concentration profiles of the other component may be assumed to remain essentially invariant during uptake of the fast component. For this type of experiment we have, once again, used ethane, now as the fast component, in a mixture with propene as the slow component  correlating the flux of species i with the concentration gradient of species j. Uphill diffusion, that is, diffusive fluxes (in this case of ethane) in the direction of increasing concentration, may thus be easily explained by the presence of a second component (propene) with a concentration profile that decreases sufficiently rapidly in the direction of the ethane flux.with the generalized diffusivities . Within this timescale, the concentration of ethane molecules would assume, essentially instantaneously, their equilibrium values determined by the local propene concentrations and the ethane pressure in the surrounding atmosphere. Overshooting terminates with the formation of a homogeneous propene distribution at which point the ethane molecules are also distributed homogeneously throughout the crystal, with both concentrations determined by their partial pressures in the surrounding atmosphere.2.With uptake and release times of several days  crystals to an atmosphere consisting of a mixture of these two species. CO2 is now the ‘fast' component  must have been able to diffuse ‘uphill' since otherwise intracrystalline concentrations exceeding those at the boundary would not be possible. Following the example given by 2. We note that CO2 diffuses so rapidly that the uphill diffusion process has already been accomplished before we were able to record the first profiles.Already in the very first CO2 flux ‘uphill' into the crystal interior. Already with the very first profile shown in 2 concentration indicates that, right from the beginning of our measurements, the CO2 fluxes are directed towards the crystal surface, that is, in the downhill direction for CO2. In our mixture uptake experiments with ethane and CO2, we are thus able to exactly follow this part of the history of overshooting that was inaccessible with mixtures of propene and ethane, that is, the approach to final equilibrium which, after overshooting, is controlled by the equilibration of the slower component.Considering ethane as the ‘slow' molecule, we are now able to follow the whole process of equilibration . The proOver the last few decades, benefitting from gains in the accuracy of experimental diffusion measurements, the prediction of diffusivities in zeolitic host–guest systems by molecular dynamics simulations444546474950The approach provided by transition state theory may be rationalized by exploiting the condition of dynamic equilibrium between the populations in the zeolite cavities and in the windows. Since the passage of a molecule through the window is considered as an ‘infrequent' event, any mutual interaction of molecules within the windows may be neglected. As a consequence, the mean transit time through a window is unaffected by the guest concentration and the relative window population increases in proportion to the pressure of the considered species in the surrounding atmosphere. The mean molecular lifetime within the individual cavities is thus immediately seen to be proportional to the ratio between the actual concentration and the partial pressure of the species under consideration. In this approach, a key quantity controlling the rate of molecular propagation is thus directly related to the equilibrium adsorption properties of the system. In the Methods section, we show that this correlation may be represented by the relationsandα) that is independent of the guest concentrations c1 and c2, and is proportional to the single-component diffusivity at zero loading.where the elements of the diffusion matrix are seen to be related to the partial derivatives of the partial pressures with respect to the local concentrations by a common factor  is the only free parameter in these calculations. For comparison, the corresponding experimental data are also indicated. The calculations using 522 and ethane, and ethane and propene, both in zeolite DDR), we succeeded in overcoming this limitation, thus making it possible to record the individual transient concentration profiles in a binary system with unprecedented spatial resolution. This new approach was applied to study two features of the ‘overshooting' phenomenon in which guest concentrations exceed their equilibrium values. First, it was shown that, in the presence of a non-uniform concentration of an essentially immobile component (propene) with its concentration decreasing towards the crystal interior, a second (faster) component (ethane) may diffuse ‘uphill' . To the best of our knowledge, this represents the first direct experimental demonstration of ‘uphill diffusion' (and the resultant ‘overshooting' behaviour) in a nanoporous adsorbent. A second set of experiments for the mixture ethane–CO2, in which ethane is now the slow component, allowed the attenuation and final disappearance of the overshoot to be studied in detail.The application of IFM, which allows the measurement of transient concentration profiles in nanoporous crystals with submicron spatial resolution, has hitherto been restricted to single-component systems since, in general, the profiles for the individual components of a mixed adsorbed phase cannot be resolved. However, by considering the adsorption of mixtures of two components with very different intracrystalline diffusivities  diffusivities at zero loading, without recourse to detailed molecular simulations.2 from natural gas and for separation of light hydrocarbons.In addition to the relevance of these results for fundamental research, the systems studied are also of particular technological interest due to the potential application of DDR as a kinetically selective adsorbent for removal of COϕ) between a light beam passing through the sample and a reference beam passing through the surrounding gas phase. By using an interferometer of Mach–Zehnder type with shearing mechanism and phase shifter, both light beams are superimposed and an interference pattern is generated. A change in the phase shift is caused by a change in the refractive index of the host material Δn when guest molecules are ad- or desorbed, leading to a change of this interference pattern. To a good approximation, the change in the phase shift Δ(Δϕ) is proportional to the change in the concentration of the guest molecules Δc:IFM makes it possible to record transient concentration profiles of guest molecules in nanoporous host systems with a high spatial and temporal resolution. It is based on the principle that the optical density of light passing through a transparent porous crystal depends on the nature and amount of guest molecules present in its pore system. The primary information recorded by IFM measurements is the change in phase shift Δ can be used to measure thickness, roughness, guest loading and other characteristics of films and membranes of nanoporous materials as demonstrated by Nair and Tsapatsiswith The measurements were carried out using an interference microscope of the type ‘Jenapol' (Carl Zeiss AG). Images were recorded by a CCD camera , which is, together with the phase shifter of the microscope, controlled by a personal computer. Monochromatic light at a wavelength of 589 nm is used in our microscope. Furthermore, the set-up includes a static vacuum system consisting of vacuum pump, reservoir tank for guest molecules, several pressure gauges and sensors for adjusting the pressure to the required level. A few dozen DDR crystallites under study are placed in an optical cell, which is connected to the vacuum system.Uptake or release experiments are initiated by step changes in the gas phase surrounding the crystals. IFM offers the potential to record maps of the intracrystalline distribution of guest molecules in large nanoporous crystals (10–500 μm) with a spatial resolution down to the pixel size of the CCD camera (0.45 μm × 0.45 μm) and a time resolution of about 10 s. The crystal under study should be transparent and exhibit nearly parallel and plane surfaces at top and bottom. Further details of the IFM technique may be found in literature14For mixtures of guest molecules, the change in the recorded signal contains contributions from both components which cannot be measured separately. However, the concentration profiles of both components can be easily calculated if the mobility of both components differs substantially. This is in particular true if the pore size is in the range of the critical diameter of the guest molecules—a condition that is usually found in technical applications based on the molecular sieving effect. Then, small differences in the guest size lead to dramatic differences in the diffusivities of both components. This opens the field for detailed investigations of various guest mixtures in different host systems of practical relevance.The DDR zeolites of the batch under study were very uniform in size. The crystallites had a diameter of 34 μm, the height was estimated to be about 20 μm. For further details of the synthesis, we refer to literature22−1) for about 10 h under vacuum. Subsequently, the sample was cooled down to room temperature (23 °C) and the cell was fixed under the microscope. All measurements presented here were carried out at this temperature. In the case of the ethane/CO2 experiment .The crystals were activated at 200 °C . Then, the sample was immediately exposed to an atmosphere of 200 mbar ethane.ϕ) as the primary experimental data provided by IFM on recording the uptake of ethane–CO2 mixtures on a crystal of zeolite ZSM-58. k2)ethane isotherms with the relative isotherms from the IFM signals for the pure components. 25L of the crystals must be accounted for. With the crystals used in this study, however, this precaution turned out to be unnecessary as the crystal height was very uniform. The almost-perfect agreement between the shape of the macroscopically determined adsorption isotherms  and The factors ki in ci of either component to be calculated from the measured changes Δ(Δϕ) in the phase of the light beam passing through the crystal under study. The required second equation is provided by the assumption of local equilibrium for the fast diffusing component (CO2). At equilibrium, the guest concentration of each component is determined by the pressures pi of both components in the gas phase . Equivalently, the mutual dependence of the two concentrations and their gas pressures at equilibrium may be expressed in the form ci=ci where now the concentration of component j is understood as the second independent variable which, together with the pressure pi of the other component (which is assumed to be equilibrated over the sample), determines the concentration of component j. Our assumption of local equilibrium of CO2 throughout the crystal makes this choice of the notation more appropriate for our further discussion in which we consider the concentration of CO2 as a function of the local ethane concentration and the externally applied pressure of CO2:Knowledge of the factors 2 pressure known for a given sorption experiment, by 2 is seen to be a function of only the local ethane concentration. Hence, with the functional relationship between the local concentrations of ethane and carbon dioxide, shown to exist by virtue of With the value of the COcethane varying from 0 up to about 1 mol kg−1 . The relation between the two concentrations is thus seen to yield a simple linear dependencyIASTcethane provided by By inserting the respective expression for et al.kpropene=10.67 rad/(mol kg−1) (or kpropene=6.06 × 10−3 rad/(mol m−3), with a crystal density Conversion of the phase shifts into absolute single-component concentration values was based on the adsorption isotherms of Hedin Since, close to the boundary, the crystals are not transparent, the recorded profiles do not extend right to the crystal surface. The propene concentration directly at the crystal boundaries is assumed to correspond to the total propene loading  (or kethane=7.42 rad/(mol kg−1)).The maximum ethane concentration for this crystal has been estimated in the following way: in the centre part of the propene profile after presorption see , the propethane=200 mbar, an ethane concentration of c10. This concentration of ethane is maintained at the crystal border (at r=R) for all times t>0 , where we imply that there are no barriers. With these considerations, we arrive at the concentration profiles in absolute concentration values as presented in Furthermore, the ethane concentration at the crystal surface had to be estimated, since this parameter plays an important role as one of the boundary conditions for solving Fick's second law of diffusion. Applying IAST for a propene concentration of We have to solve Fick's second law for two-component diffusionin cylindrical symmetryc1, is formulated as follows:For a solution, the equation must be regularized and appropriate initial and boundary conditions need to be defined. For both experiments presented in the manuscript, the problem that needs to be solved for achieving the evolution of the ethane concentration with time, ɛ>0 be small and R<R0 close to R. The problem is to find c1 on  ×  ofLet withc1 according to c2 is independent of time, it can easily be replaced by a fit curve depending on only the radius r, as it would be the case for propene. Second, if c2 can directly be expressed by a function of c1. This is the case for CO2, which is assumed to be instantaneously equilibrated. Furthermore, eligible models for D11 and D12 are needed. These models will be provided by analysis of the data maintained by the approaches made for D11 and D12 =c10 is valid.For solving D11 and D12 from transition state theory. Given the existence of narrow windows between the individual cages, the diffusion of guest molecules in the ZSM-58 pore system may be described by a simple jump model40We exploit the special geometry of the pore structure of ZSM-58 see  to derivλ from, say, left to right is given by:The total number of molecules jumping, per unit time, between two adjacent cages of separation and, correspondingly, byfor molecules jumping from right to left.τ(c), within the individual cavities is proportional to the ratio between the actual concentration and the partial pressure of the species. Thus, one may noteThe mean molecular lifetime, denoted as or, in more detail:andτ-terms' in Replacing the ‘andCombing the last two relations yieldswhere we have taken account of the proportionality between the net number of jumps and the flux into a given direction.Comparison with Fick's first law of diffusionresults, finally, in the expressionsandα denoting a factor of proportionality, which is seen to be equal to the single-component diffusivity at zero loading divided by the Henry constant.with Lij=0 for i≠j) correlating the fluxes with the gradients of their chemical potentials of different molecular species. Such cross-correlations may in fact be neglected, given the low occupation probability of ‘window' positions as assumed in our simplified model.One may immediately arrive at these relations by inserting the expression of the self-diffusivity as derived by transition state theory (Equation (4.42) in ref. p1 of the pressure of the relevant diffusant on the concentrations of both components under equilibrium. In co-adsorption with ethane, CO2 may be assumed to be instantaneously equilibrated throughout the crystal. The CO2 concentration at any position in the crystal is therefore determined by the local ethane concentration and the fixed value of p2=200 mbar for the CO2 pressure. Correspondingly, also the equilibrium ethane pressure p1 at any position corresponding to the local concentrations of the two components is fixed and determined by these concentrations. IAST was applied for either constant c2 and a fine-meshed list of p1 values, or for constant c1 and a fine-meshed list of p2 values. Subsequently, numerical differentiation of the respective lists was performed to achieve the list of values for c1 and c2. To determine the partial derivatives appearing in used in the numerical solution.as illustrated by t1…t2 are easily seen to be correlated with the evolution of the intracrystalline concentrations by the relationBy recording the transient concentration profiles during molecular uptake and release, microimaging also provides all information necessary for the measurement of the intracrystalline diffusive fluxes. Thus, for the system under study, molecular fluxes entering the individual crystal in a time interval 2, with the fluxes resulting via D11 and D12 as predicted with α=4.8 × 10−16. α=4 × 10−16 .  × 10−16 . This siHow to cite this article: Lauerer, A. et al. Uphill diffusion and overshooting in the adsorption of binary mixtures in nanoporous solids. Nat. Commun. 6:7697 doi: 10.1038/ncomms8697 (2015).Supplementary Figures 1-9, Supplementary Notes 1-2 and Supplementary References


Bcl-2) was cloned 30 years ago and associated with B-cell follicular lymphoma. A number of Bcl-2 homologs were identified later on. Importantly, the Bcl-2 family was found to control the mitochondrial outer membrane permeabilization: a key step of the mitochondrial pathway of apoptosis.1 Bcl-2 homologs are evolutionarily conserved throughout metazoans and considered as the hallmarks of multicellularity. Genetic manipulation in nematodes and mice demonstrated that the bcl-2 family has a pivotal role in tissue homeostasis by controlling cell death; however, an increased number of in vitro studies have identified additional non-apoptotic functions, suggesting that Bcl-2 proteins are in fact multitask factors , the Ca2+-ATPase (SERCA) pump, the ryanodine receptor, the Bax inhibitor-1 channel, as well as, the voltage-dependent anion channel (VDAC) at the mitochondria . It was reported that overexpression of Bcl-2 may lead to a decrease of the ER Ca2+ load,5 and the ability of Bcl-2 proteins to regulate intracellular Ca2+ homeostasis was linked to non-apoptotic functions . In fact, a number of them contribute to apoptosis regulation though the control of Caunctions .2+ fluxes regulation, we took advantage of the zebrafish. This model possesses all key components of the vertebrate cell death machinery and represents a suitable model for studying Ca2+ trafficking during embryogenesis.7 Different studies already highlighted the role of zebrafish Bcl-2 proteins in apoptosis. However, their roles on Ca2+ homeostasis and their precise functions during early development are still unknown. We paid attention to two Bcl-2 homologs: Nrz (Nr-13 ortholog in zebrafish) and Bcl-wav, a new evolutionary conserved Bcl-2 homolog in bony fishes and anurans. These genes exhibit opposite effects regarding apoptosis, but both orchestrate the actin cytoskeleton dynamics via the regulation of Ca2+ homeostasis . Epiboly is characterized by migration of the embryonic cells from the top of the yolk sac towards the vegetal pole of the embryo to envelop the entire yolk . It is ibclwav, a pro-apoptotic member, leads to marked developmental defects, including shortening and deviation of the antero–posterior axis.6 This phenotype is owing to the major alterations of convergence and extension (CE) movements during gastrulation, independently of cell death. Whereas during CE, embryonic stem cells migrate synchronously to establish the major embryonic axis  and giving insight into the role of mitochondrial Ca2+ oscillations in vertebrate development.6 Unexpectedly, mcu knockout in the mouse results in a rather mild phenotype with overall size reduction, decreased effort capacities and metabolic alterations, but no major developmental defects.9It is important to note that 2+ oscillations may have variable importance during embryogenesis, depending on the egg type and size. Indeed, during the development of teleosteans, including zebrafish, eggs have huge amounts of yolk stocks and undergo incomplete cleavage. The cells located at the animal pole of the embryo need to undergo a complex set of morphogenic movements including epiboly and CE , a phenotype also observed in mcu-KO mice. Another example is the apoptosis inhibitor mcl-1 whose invalidation leads to a lethal peri-implantation phenotype characterized by implantation defaults of the blastocyst . Interestingly, this process is mediated by trophoblast cells, which form cytoplasmic lamelipodia like projections that pass through the zona pellucida and interact with the external environment.12 Ca2+ signaling seems to be required for trophoblast adhesion as chelation of intracellular Ca2+ or inhibition of the Ca2+-dependent proteins significantly decreases trophoblast-binding activity. Conversely, blastocyst cavitation is accelerated by elevation of intracellular Ca2+ and occurred predominantly via IP3R.13 Moreover, isolated mcl-1-deficient blastocysts showed no increased apoptosis, suggesting that mcl-1 might have additional uncharacterized functions. Interestingly, Mcl-1 was described to regulate intracellular Ca2+ homeostasis by interacting with IP3R.14 Recently, it was shown that Mcl-1 may also interact with VDAC to increase mitochondrial Ca2+ level and promote cell migration.15 This raises the possibility that a physiological function of Mcl-1 may be linked to intracellular Ca2+ trafficking to permit blastocyst adhesion during mouse embryonic development.Knockouts (KO) in mice, confirmed that most of Bcl-2 proteins are primarily involved in cell death regulation. However some phenotypes cannot be explained by this sole effect. For instance bcl-2 KO mice phenotypes focusing on early developmental steps. Thus research performed on zebrafish confirms that Bcl-2 proteins are not only involved in cell death but may control cell migration in a Ca2+-dependent manner during embryonic development, contributing to extend our knowledge about the multiple roles of this fascinating family of proteins.In light of the multiple functions of these proteins beyond apoptosis and results obtained with the zebrafish model it may be worthwhile to revisit the


Increases were fatty acid specific, dose-dependent and different for each cell type. The arachidonic acid effects on [Ca2+]i were higher in spermatids than in spermatocytes and persisted when residual extracellular Ca2+ was chelated by EGTA, indicating that the increase in [Ca2+]i originated from release of intracellular calcium stores. At the concentrations required for these increases, unsaturated fatty acids produced no significant changes in the plasma membrane potential of or non-specific permeability in spermatogenic cells. For the case of arachidonic acid, the [Ca2+]i increases were not caused by its metabolic conversion to eicosanoids or anandamide; thus we attribute this effect to the fatty acid itself. As estimated with fluorescent probes, unsaturated fatty acids did not affect the intracellular pH but were able to induce a progressive decrease in the mitochondrial membrane potential. The association of this decrease with reduced reactive oxygen species (ROS) production strongly suggests that unsaturated fatty acids induced mitochondrial uncoupling. This effect was stronger in spermatids than in spermatocytes. As a late event, arachidonic acid induced caspase 3 activation in a dose-dependent manner both in the absence and presence of external Ca2+. The concurrent but differential effects of unsaturated fatty acids on [Ca2+]i and mitochondrial functions are additional manifestations of the metabolic changes that germ cells undergo during their differentiation.The aim of this work was to explore the ability of free arachidonic acid, palmitic acid and the unsaturated fatty acids oleic acid and docosahexaenoic acid to modify calcium homeostasis and mitochondrial function in rat pachytene spermatocytes and round spermatids. In contrast to palmitic acid, unsaturated fatty acids produced significant increases in intracellular calcium concentrations ([Ca We chose two polyunsaturated FA, AA and docosahexaenoic acids (DHA) as well as oleic acid (OA) and palmitic acid (PA) because they are abundant in membrane glycerophospholipids of all cells in the seminiferous epithelium i determinations were carried out using a ratiometric method as described i vs. time data between 30 and 200 s after FA addition. Statistical differences between treatment and control rates of increase in [Ca2+]i were analysed by applying a t-test with Wellch’s correction or an ANOVA followed by a post-test when different experimental conditions were compared. When the data was expressed as a percentage increase in [Ca2+]i by each FA or when the F-test indicated significant differences in group variances, the comparison between treatment and control was performed using non-parametrical Mann-Whitney analysis [The initial rate of FA-induced Caanalysis . In all 2+]i after addition of arachidonic (AA), docosahexaenoic (DHA), oleic (OA) and palmitic acid (PA) in round spermatids. [Ca2+]i changes induced by FA in KH-lactate-EGTA media were dose-dependent. At 8 μM, OA showed a biphasic curve that was not studied further and could be interpreted either as Ca2+ reuptake into intracellular calcium stores (ICaS) or as Ca2+ exit from the cells after 700 s of OA addition. The rates of increase in [Ca2+]i induced by different concentrations of AA, DHA, OA and PA in pachytene spermatocytes and round spermatids are compared in 2+]i in pachytene spermatocytes . The glucose-dependent effect on initial rate of AA-induced [Ca2+]i rise observed in round spermatids was not observed in pachytene spermatocytes, which is in agreement with the known metabolic properties of these two cell types and their differences in [Ca2+]i homeostasis i when added after AA in round spermatids and pachytene spermatocytes under all the conditions tested . These results demonstrate that the rates of [Ca2+]i changes induced by UFAs were not limited by complete depletion of ICaS in the conditions tested in this work.As a positive control, cyclopiazonic acid (CPA), which can deplete the ICaS by inhibiting the sarcoplasm-endoplasmic reticulum Ca2+]i could be mediated by cyclooxygenase (COX) or lipoxygenase (LOX) metabolites of AA, inhibitors of COX or LOX were tested . The presence of the COX inhibitor indomethacin (10 μM), but not the LOX inhibitors NDGA (5 μM) or 2-TEDC (5 μM), induced a significant and partial reduction in the kinetics of the changes induced by 4 μM AA  into round spermatids was used to determine whether the FAs in this study affected germ cell integrity . AA was mes Figs –4 were n2+]i in round spermatids or pachytene spermatocytes (data not shown).Given that unsaturated FAs at > 8 μM concentrations were able to induce entry of PI into spermatogenic cells, and because PI can permeate connexin hemichannels and pannexin channels that play a role in ATP secretion see ,44), we , we 44])2+]i induced by FA in spermatogenic cells were not associated with entry of Ca2+, the existence of a described voltage-dependence of some GPCRs i rise was not mediated by a loss of membrane integrity, by changes in plasma membrane potential, or by activation of ATP release from spermatogenic cells.The regulatory mechanisms that allow SC to control mammalian spermatogenesis are still poorly understood. It is known that SC release a number of soluble factors that could affect proliferation and differentiation of spermatogenic cells ,57,58. H2+]i were apparently specific to UFAs, as shown by the lack of effect of PA and by the different responses of spermatids to AA and DHA. The effect of OA was intermediate between PA and polyunsaturated FA, as higher concentrations of OA were required to increase [Ca2+]i to levels similar to those induced by AA or DHA. The effects may be ascribed directly to the fatty acids rather than to some of their bioactive metabolites, as shown in the case of AA by the negligible effect of adding anandamide (an AA-derived endo-cannabinoid metabolite) in similar concentrations as AA to the cells, or by pharmacologically blocking the conversion of AA via cyclooxygenase or lipoxygenase inhibitors.Increases in [Ca2+]i they were ascribed to the ATP-dependent maintenance of Ca2+ homeostasis. This agrees with the results of the present study, in which UFA, in spermatids more so than in spermatocytes, induces a relatively monotonic increase in the rate of Ca2+ release from ICaS and a concomitant decrease in mitochondrial membrane potential as concentrations of UFAs increase.Rhodamine 123 in cells permeabilized with digitonin allowed us to determine the effects of UFAs on the mitochondrial membrane potential. Effects were dependent on cell type and FA species. The ability of UFAs to induce a partial decrease in membrane potential was weaker in pachytene spermatocytes than in round spermatids. In the latter, the magnitude of the UFA-induced decrease in the mitochondrial membrane potential was consistent with what has been described as mild uncoupling , and theThe different uncoupling effects exerted by UFA on the mitochondria of each of these spermatogenic cell types is possibly linked to the observation that AA and DHA were able to decrease ROS production in round spermatids but not in pachytene spermatocytes. The ability to be partially uncoupled by several stimuli  is emerg2+ release. The fact that caspase 3 activation was more prominent in the absence of external Ca2+ strongly suggests that these cells had shifted to a pro-apoptotic state, likely due to the lowering of ICaS.The kinetics of AA-induced caspase 3 activation (occurring after 180 min of treatment) demonstrated that this was a late event as compared to AA-induced intracellular Ca2+]i and exhibit different effects on mitochondrial function in pachytene spermatocytes and round spermatids. These results strongly suggest that FAs originating in SC could be part of the cell-cell signalling mechanisms that control or modulate spermatogenic cell functions like metabolism, proliferation, differentiation or death in the seminiferous epithelium.Because AA can be released by SC in a FSH-dependent manner , and proS1 Fig(A) Pachytene spermatocytes or (B) round spermatids were exposed to 8 μM AA and subsequently to 25 μg/ml digitonin.(TIF)Click here for additional data file.


Inhibiting or silencing NCLX in A20 and DT40 B lymphocytes markedly increased random migration and suppressed the chemotactic response to CXCL12. In contrast to control cells, cytosolic Ca2+ was higher and was not increased further by CXCL12 in NCLX-knockdown A20 B lymphocytes. Chelating intracellular Ca2+ with BAPTA-AM disturbed CXCL12-induced chemotaxis, suggesting that modulation of cytosolic Ca2+ via NCLX, and thereby Rac1 activation and F-actin polymerization, is essential for B lymphocyte motility and chemotaxis. Mitochondrial polarization, which is necessary for directional movement, was unaltered in NCLX-knockdown cells, although CXCL12 application failed to induce enhancement of mitochondrial polarization, in contrast to control cells. Mouse spleen B lymphocytes were similar to the cell lines, in that pharmacological inhibition of NCLX by CGP-37157 diminished CXCL12-induced chemotaxis. Unexpectedly, spleen T lymphocyte chemotaxis was unaffected by CGP-37157 treatment, indicating that NCLX-mediated regulation of chemotaxis is B lymphocyte-specific, and mitochondria-endoplasmic reticulum Ca2+ dynamics are more important in B lymphocytes than in T lymphocytes. We conclude that NCLX is pivotal for B lymphocyte motility and chemotaxis.Lymphocyte chemotaxis plays important roles in immunological reactions, although the mechanism of its regulation is still unclear. We found that the cytosolic Na Migration of lymphocytes is one of the fundamental processes of the immune response. In the case of B lymphocytes, circulating naive lymphocytes enter lymph nodes through high endothelial venules, migrate to the B cell area where chemoattractants such as CXCL12 and CXCL13 are highly expressed, and are then activated and differentiated into plasma cells13452+ is an important second messenger which regulates chemotaxis. It is well known that various cellular processes involved in cell motility are Ca2+-sensitive. For example, a rise or oscillation in cytosolic Ca2+ activates cytoskeletal remodelling through the small GTPase Rac1, thereby modulating migration of T lymphocytes, tumour mast cells and podocytes782+ mobilization is mediated by Ca2+ influx via plasma membrane Ca2+ channels and/or Ca2+ release from intracellular stores such as the endoplasmic reticulum (ER). Various Ca2+ carriers localized at the plasma membrane and in the ER, such as transient receptor potential channels (TRPs), the store-operated Ca2+ entry system (stim1/orai1), ER Ca2+ release channels (inositol trisphosphate receptors), and the ER Ca2+ pump (SERCA), have been implicated in the regulation of the migration of various kinds of cells101112132+ mobilization-mediated migration, though little information is available on B lymphocyte migration11Ca2+ store inside the cell. Mitochondrial Ca2+ dynamics is determined by influx via the Ca2+ uniporter and efflux via the Na+-Ca2+ exchanger and/or the H+-Ca2+ exchanger152+ uniporter, and MICU, which is a regulator of MCU, were reported to be involved in the migration of human endothelial cells and zebrafish blastomeres172+ dynamics and/or the production of mitochondrial reactive oxygen species were implicated in migration, though the exact mechanisms are still unresolved. Moreover, little is known as to whether, and if so, how mitochondrial Ca2+ efflux transporters participate in lymphocyte chemotaxis.Besides the ER, mitochondria provide an important Ca+-Ca2+ exchanger2+ to the endoplasmic/sarcoplasmic reticulum (ER/SR), and that NCLX-mediated Ca2+ recycling between mitochondria and the ER/SR modulates various cellular functions20212+ responses to antigens202+ dynamics participate in the migration of various kinds of cells, we hypothesized that NCLX may participate in lymphocyte migration and/or chemotaxis. In the present study, we studied the roles of NCLX in lymphocyte chemotaxis. We found that NCLX reduction/inhibition markedly suppressed chemotaxis while increasing the random migration of B lymphocytes. In addition, we found that the contributions of NCLX to motility were observed only in B lymphocytes and not in T lymphocytes.We recently demonstrated that NCLX, a gene responsible for the mitochondrial Na2+ carriers affected the chemotaxis of A20 B lymphocytes. A standard Transwell assay was employed to measure chemotaxis. When CXCL12 was present in the bottom chamber, there was a significant increase in the movement of A20 B lymphocytes toward the bottom chamber; that is, CXCL12 induced chemotaxis of A20 B lymphocytes (+-Ca2+ exchange by CGP-37157 (IC50 = 0.36 μM)2+ uniporter by Ru360 (IC50 = 0.2–2 nM)24We first investigated whether pharmacological intervention of mitochondrial Caphocytes . Perturb+-Ca2+ exchanger-mediated modulation of chemotaxis occurred in another B lymphocyte cell line, DT40. In heterozygous NCLX knockout DT40 B lymphocytes (NCLX+/−), in which NCLX protein expression was negligible+-Ca2+ exchanger is a key determinant for the chemotaxis of B lymphocytes.We then tested whether the mitochondrial Na+-Ca2+ exchange in A20 B lymphocytes (+/− DT40 B lymphocytes compared with that in wild-type (WT) cells  cells , in accoT) cells . Bath apphocytes ; this isphocytes . We confphocytes , suggest2+ sensitive101112132+ signalling during B cell receptor activation2+. In siNCLX cells, cytosolic Ca2+ concentration was higher than that in siControl cells in the absence of CXCL12 , and the ER Ca2+ binding protein calreticulin, were significantly higher in B lymphocytes than in T lymphocytes. It should be noted that the expression levels of mitochondrial adenine nucleotide translocator 2 (ANT2) and ATP synthase subunit ATP5b were comparable between B and T lymphocytes, suggesting that the cellular content of mitochondria is similar in the two cell types. In addition, the expression level of orai1, which mediates Ca2+ influx across the plasma membrane in response to ER Ca2+ depletion, was also higher in B lymphocytes than in T lymphocytes. On the other hand, the expression levels of other Ca2+ carriers in plasma membrane, such as the Na+-Ca2+ exchangers NCX1–3 and the Ca2+ pump PMCA1, were comparable between B and T lymphocytes. These results suggest that the contribution of organelle Ca2+ dynamics to cytosolic Ca2+ was larger in B lymphocytes than in T lymphocytes. In fact, the cytosolic Ca2+ response to antigen receptor stimulation was sensitive to the mitochondrial Na+-Ca2+ exchange blocker CGP-37157 in B lymphocytes but not in T lymphocytes , N = 3) and 0.002 in A20 B lymphocytes (N = 1)), suggesting that the contribution of MCU to chemotaxis in native lymphocytes, if any, is small.As far as we know, this is the first report which demonstrates the involvement of a mitochondrial Ca2+ dynamics between B and T lymphocytes, that is, the larger contribution of NCLX-mediated mitochondria-ER Ca2+ dynamics to cytosolic Ca2+ in B lymphocytes than in T lymphocytes  according to the protocol by Hara-Chikuma 3D (ibidi GmbH) according to the manufacturer’s instructions. Cells were conjugated with collagen I gel and applied to the observation area in the presence or absence of 100 ng/ml CXCL12 in one side of the reservoir. The chamber was placed in a CO2-incubator (Tokai hit) on the stage of a fluorescence microscope . Transmission images of cells were obtained every 2 min over 8 hrs using a digital CCD camera , and analyzed using a particle tracking tool of AQUACOSMOS software (Hamamatsu Photonics), a filtering tool of ImageJ (NIH) and a Chemotaxis and Migration tool (ibidi GmbH). Cells which moved directionally toward CXCL12 were evaluated by the position in the 90-degree area facing CXCL12 at the end of the experiment. The extent of chemotaxis was expressed as the percentage of directionally moved cells which moved over a distance equal to the average cell size . When pharmacological inhibitors were used, they were applied to both sides of the reservoir.Assays were performed with a μ-Slide Chemotaxis2+ was measured as described previouslyCytosolic CaCells were applied to cover glasses coated with 10 μg/ml fibronectin, starved for 1 hr at 37 °C in serum-free RPMI 1640 with 0.1% BSA, and incubated with or without 100 ng/ml CXCL12 for 2 hrs. After fixation with 3.7% formalin for 15 min at 37 °C, cells were permeabilised with 0.1% Triton-X/PBS for 10 min, blocked with 1% BSA/PBS for 30 min, and stained with Alexa Fluor 488 phalloidin (Invitrogen) and DAPI (Dojindo). Immunofluorescence images were obtained using a confocal microscope . A high intensity phalloidin signal was defined as a fluorescence intensity of more than 5000 a.i. and was expressed as % of cell area.Cells were applied to cover glasses coated with 10 μg/ml fibronectin, starved for 1 hr at 37 °C in serum-free RPMI 1640 with 0.1% BSA, and incubated with or without 100 ng/ml CXCL12 for 2 hrs. Then cells were stained with 1 μM MitoTracker Orange (Invitrogen), and images were obtained using a confocal microscope . Mitochondria-polarized cells were defined as cells in which the mitochondria were located within one-half of the cell area.+ T cell kit (#130–095–248); Miltenyi Biotec K.K.), according to the manufacturer’s instructions.Female 6–10-wk-old Balb/c mice were purchased from Japan SLC. All experimental procedures were conducted in accordance with the guidelines of the Research Center of Kyoto University Graduate School of Medicine and Regulations for Animal Research at University of Fukui. Procedures were approved by the Committee on Animal Research of Kyoto University Graduate School of Medicine and the Faculty of Medical Sciences, University of Fukui. Single splenocytes were obtained by standard proceduresQuantitative PCR analysis was performed as described previously20t test, respectively. P < 0.05 was considered significant.All data are presented as mean ± SEM of independent recordings. The number of independent experiments and the number of cells per recording are presented as N and n, respectively. In the microscopy measurements, the responses from n individual cells were averaged for each recording. Then the statistical evaluation was performed on averaged responses from N independent recordings. Each experiment was repeated independently more than three times. Statistical analyses were performed by one-way ANOVA multiple comparisons . Post hoc and two-group comparisons were performed using the Student–Newman–Keuls test and the unpaired Student’s How to cite this article: Kim, B. et al. Roles of the mitochondrial Na+-Ca2+ exchanger, NCLX, in B lymphocyte chemotaxis. Sci. Rep.6, 28378; doi: 10.1038/srep28378 (2016).


Helicobacter pylori infection in acute and chronic spontaneous urticaria patients.Over the time, many infections were attributed to be the cause of urticaria. We proposed to assess the seroprevalence of hepatitis B virus (HBV), hepatitis C virus (HCV) and We conducted a prospective study which included 236 acute spontaneous urticaria patients, 168 chronic spontaneous urticaria and 220 volunteers without urticaria. The study was approved by the Ethics Committee of the Hospital and all the patients gave their consent for the inclusion into the research protocol.Helicobacter pylori were present in 139 acute spontaneous urticaria patients (58.89%), in 107 chronic spontaneous urticaria patients (63.69%) and in 124 (56.36%) controls.Serology for hepatitis B was found to be positive in 8 patients (3.38%) in the acute spontaneous urticaria group, in 9 patients (5.35%) in the chronic spontaneous urticaria group and in 5 subjects (2.27%) in the control group. Antibodies to HCV were identified in 3 patients (1.27%) with acute spontaneous urticaria, in 4 patients with chronic spontaneous urticaria (2.38%) and in one subject (0.45%) in the control group. Antibodies to Based on these results, we can conclude that the incidence of urticaria is higher in patients suffering from infections, but the analyzed pathogens in this study cannot be considered risk factors for the occurrence of urticaria, although their presence may exacerbate the symptomatology and the evolution of this disease. The therapy of these infections may ameliorate urticaria and the efficacy of the H1 antihistamine treatment.


Ca) produced action potential duration (APD) alternans seen clinically at slower pacing rates. Using single-cell clamps of voltage, fluxes, and state variables, we determined that alternans onset was Ca2+-driven rather than voltage-driven and occurred as a result of decreased RyR inactivation which led to increased steepness of the sarcoplasmic reticulum (SR) Ca2+ release slope. Iterated map analysis revealed that because SR Ca2+ uptake efficiency was much higher in control atrial cells than in cAF cells, drastic reductions in kiCa were required to produce alternans at comparable pacing rates in control atrial cells. These findings suggest that RyR kinetics may play a critical role in altered Ca2+ homeostasis which drives proarrhythmic APD alternans in patients with AF.Atrial fibrillation (AF) is the most common cardiac arrhythmia, but our knowledge of the arrhythmogenic substrate is incomplete. Alternans, the beat-to-beat alternation in the shape of cardiac electrical signals, typically occurs at fast heart rates and leads to arrhythmia. However, atrial alternans have been observed at slower pacing rates in AF patients than in controls, suggesting that increased vulnerability to arrhythmia in AF patients may be due to the proarrythmic influence of alternans at these slower rates. As such, alternans may present a useful therapeutic target for the treatment and prevention of AF, but the mechanism underlying alternans occurrence in AF patients at heart rates near rest is unknown. The goal of this study was to determine how cellular changes that occur in human AF affect the appearance of alternans at heart rates near rest. To achieve this, we developed a computational model of human atrial tissue incorporating electrophysiological remodeling associated with chronic AF (cAF) and performed parameter sensitivity analysis of ionic model parameters to determine which cellular changes led to alternans. Of the 20 parameters tested, only decreasing the ryanodine receptor (RyR) inactivation rate constant (ki Atrial fibrillation is an irregular heart rhythm affecting millions of people worldwide. Effective treatment of this cardiac disorder relies upon our detailed knowledge and understanding of the mechanisms that lead to arrhythmia. Recent clinical observations have suggested that alternans, a phenomenon where the shape of the electrical signal in the heart alternates from beat to beat, may play an important role in this process, but the underlying mechanisms remain unknown. In this study, we use computational models to conduct a detailed examination of the causes and contributors to alternans associated with human atrial fibrillation. We find that in atria remodeled by atrial fibrillation, alternans appears near resting heart rates because several aspects of calcium cycling are disrupted in the atrial cells. In particular, the release and uptake of calcium from the cellular storage compartment, the sarcoplasmic reticulum, becomes imbalanced, leading to alternation in calcium signals from beat to beat. These findings provide important insights into the mechanisms of proarrhythmic alternans in human atrial fibrillation which may be used to develop novel therapeutic targets and treatment strategies in the future. Atrial fibrillation (AF) is currently the most common cardiac rhythm disorder, posing a significant medical and economic challenge for the US health care system et al. reported differences in the rate dependence of action potential duration (APD) alternans in patients, with APD alternans occurring at pacing rates near rest in AF patients but only at fast pacing rates in controls et al. also found that APD alternans always preceded AF initiation, indicating that alternans may play an important role in establishing the arrhythmogenic substrate and creating vulnerability to AF. Thus, a better understanding of AF arrhythmogenesis will likely depend upon identification of the mechanism driving atrial alternans at heart rates near rest.Beat-to-beat alternation in the shape of cardiac electrical signals, a phenomenon called alternans, has been observed in the atria of AF patients, but the mechanism underlying these alternans is not known 2+ handling in atrial myocytes is known to play a crucial role in the generation of AF triggers and in AF maintenance 2+ cycling instabilities have been shown to underlie ventricular alternans in heart failure 2+ handling abnormalities, or other electrophysiological changes that occur in AF, lead to APD alternans. We identified a critical change in the kinetics of the ryanodine receptor (RyR) that was responsible for APD alternans onset at slower pacing rates, and subsequently aimed to elucidate the mechanistic relationship between this disruption in RyR kinetics and alternans onset. To this end, we employed single-cell clamping of ionic model parameters and iterated map analysis in order to dissect the mechanisms which drive alternans in atrial tissue, as well as to provide important insights into the pathophysiological changes that contribute to the development of alternans in AF patients.Interestingly, in AF patients the slope of the APD restitution curve was <1 during APD alternans onset at slow pacing rates. This suggests that a cellular mechanism other than voltage-driven instability underlies APD alternans at heart rates near rest et al. to induce APD alternans in AF patients In order to investigate ionic mechanisms in human AF that contribute to the generation of atrial APD alternans at the tissue level, we created a computer model of human atrial tissue incorporating ionic remodeling associated with chronic AF (cAF), as described in Ca) had the greatest effect on alternans onset CL in the human cAF-remodeled tissue  version of the cAF model Ca was decreased by 50% in the cAF model , APD alternans onset data from the human AF tissue model agreed well with data from persistent AF patients. Significant APD alternans began at 400-ms CL  at the onset CL  vs. the even (short) beat (2+ load before release (0.288 vs. 0.273 mM), higher peak RyR open probability (RyRo) (9.0e-4 vs. 4.7e-4), a larger intracellular Ca2+ transient (CaT) amplitude (Δ[Ca2+]i = 0.13 vs. 0.067 µM), similar L-type Ca2+ (LCC) current (integrated over one beat: 144 vs. 140 mC/F), and increased Na+/Ca2+ exchanger (NCX) current . The positive coupling between transmembrane potential (Vm) and Ca2+, with INCX as the primary electrogenic current, is consistent with experimental findings alt model provided the best agreement with clinical APD alternans data  was clamped to either the even or odd beat waveforms, alternans in both APD and CaT were eliminated (<−99%), demonstrating that the alternans were driven by SR Ca2+ instability (column 4 of i), RyR open probability (RyRo), junctional Ca2+ ([Ca2+]j), and SR Ca2+ release flux (JSRCarel)  . All fiv2+ ([Ca2+]sl) and sub-sarcolemmal Na+/Ca2+ exchanger current (INCXsl). Clamping [Ca2+]sl to the even beat eliminated all alternans; clamping to the odd beat greatly reduced APD and CaT alternans , although large alternation in SR load persisted , while clamping to the odd beat waveform resulted in elimination of all alternans  voltage clamp conditions so that changes in CaT alternans would be due solely to changes in Ca2+ homeostasis rather than bidirectional coupling between Vm and Ca2+. After clamping each ionic model at a CL of 400 ms until steady state was reached, we perturbed [Ca2+]SR and tracked SR load and SR Ca2+ release on the subsequent clamped beats  ionic models are depicted in alt model  at the beginning of a clamped beat and compared the unperturbed  and perturbed (dotted line) traces for each model (2+ regulation of the RyR (causing more opening), as well as to the increased concentration gradient between the SR and junctional compartments. In both the cAF and cAFalt models, these changes led to increased peak [Ca2+]j  and RyR opening  as a result of more Ca2+-induced Ca2+ release . However, the amount of positive feedback differed between the cAF and cAFalt ionic models. Positive feedback amplifies changes in release inputs, such as SR load; therefore, in the cAF model, where [Ca2+]j is higher and positive feedback is stronger, the increase in [Ca2+]SR produced a slightly greater change in release  during the rising phase of [Ca2+]j (t<48 ms) than in the cAFalt model  occurs. In the cAFalt model, negative feedback is decreased both directly, via reduction of kiCa, and indirectly, via reduction in [Ca2+]j that occurs as a result of decreased SR load. This causes prolongation of the Ca2+ release event and a larger peak [Ca2+]j  depends upon RyR inactivation by junctional Ca2+. However, recent work suggests that termination of release does not rely on direct Ca2+-dependent inactivation of the RyR but rather on local SR Ca2+ depletion 2+] decreases causing changes in RyR opening and closing rates.The results in column 1 of 34  is decreased from 100% to 50% , steady-state Ca2+ concentrations change modestly as compared to the original RyR formulation , peak [Ca2+]j is reduced by 10.5%  and delayed, and total release increases by 3.6% . When [Ca2+]NSR is perturbed in the Sato-Bers models by +20 µM, Ca2+ release increases more in the cAFalt model than in the cAF model  has the opposite effect as in Qu et al.2+ alternans.For each version of the human atrial cell model (cAF and control), we calculated the SR Caes , see , the thrCa are explored for the cAF model in Ca value (100%), as CL is decreased from 700 ms to 200 ms (−10 ms increments), 2+ cycling, coinciding with the iterated map parameter values residing very close to the theoretically predicted boundary given by Ca is set at 50% of the default cAF value , a similar trend is observed. However, the 50% kiCa cAF model reaches threshold at a lower pacing rate  fails to reach threshold and remains in the stable, no alternans region. This suggests that alternans in control patients, which occur at CL<250 ms 2+. As in the cAF model, the alternans threshold CL in the control model can be adjusted by modulating the value of kiCa (Ca must be decreased much more than in the cAF model in order to reach Ca reduced to 16% vs. 50%). The need for dramatic and possibly unrealistic reductions in kiCa to produce alternans at slow rates in control is consistent with the absence of alternans observed in control patients at CL≥250 ms We next performed the same iterated map analysis for the control atrial cell model with varying CL and kia values . When ki2+ cycling properties of the cAF and control models, we examined the effects of cAF cellular remodeling on iterated map parameters. Stochastic ionic model parameter variation and regression analysis CaL, gKur, koCa, IbarNCX, gto, gK1, and gNa, see 2+ handling . The effects of changing these three parameters from control to cAF values is depicted sequentially in CaL is decreased and then IbarNCX and koCa are increased to cAF values, resulting in an overall decrease in Ca is decreased to the cAFalt value (50%), the large increase in Ca value and pacing rate, SR uptake efficiency  could produce APD alternans at relatively slow pacing rates in a tissue model of persistent/chronic AF. In particular, decreasing kiCa by 50%  produced a good match to clinical data. We next aimed to provide mechanistic insight into why disruption of RyR kinetics, together with other electrophysiological changes occurring in AF, leads to alternans onset at pacing rates near rest. We established that alternans in the cAFalt model at the onset CL were Ca2+-driven rather than voltage-driven, and that they depended upon SR Ca2+ release. Furthermore, CaT alternans occurred in the cAFalt model at relatively long CLs because of steep SR Ca2+ release slope and decreased SR Ca2+ uptake efficiency. Lastly, we demonstrated that the ability to generate alternans at slower pacing rates by modulating kiCa depended upon the negative feedback properties of SR Ca2+ release.The first goal of this study was to identify the electrophysiological changes in human atrial cells that are responsible for the occurrence of APD alternans at heart rates near rest, as observed in AF patients. Using parameter sensitivity analysis, we found that of the 20 electrophysiological model variables tested, only changes in the RyR inactivation rate constant  enhanced alternans when clamped  is not consistent with ERP at the tissue level 2+ release, which was later confirmed through iterated map analysis.Our ionic model variable clamping protocol, which involved separately clamping the even or odd beat waveforms, was used to test for model variables which could robustly suppress alternans when clamped to either of two very different waveforms. An alternative approach would be to clamp model variables to the single unstable, non-alternating waveform obtained using a control algorithm 2+ depletion, rather than Ca2+-dependent RyR inactivation, as the main mechanism of SR release termination alt model relied on Ca2+-dependent RyR inactivation, other termination mechanisms which rely on SR Ca2+ (used in the Sato-Bers RyR model) may have similar effects on SR release slope and alternans susceptibility . APD alternans normalized magnitude (ANM), obtained by dividing the alternans magnitude by the mean APD over the last 10 beats, was used to compare alternans between cells of varying APD. Alternans onset CL was defined as the longest CL for which ANM was greater than 5% Voltage traces from the recording electrode  were ana2+ current (ICaL), rapidly activating potassium current (IKr), SR uptake, or SR release . Model variables were considered critical for alternans if clamping them to either the even or odd beat reduced both APD and CaT alternans magnitudes by >99% of baseline To determine which human atrial ionic model variables drive the occurrence of alternans, we clamped individual ion currents and state variables in a single-cell model paced at a CL exhibiting alternans 2+ cycling properties of the human atrial cell model under different pacing rates and parameter values, the following equation was used to clamp Vm to a generic atrial AP-like waveform so that comparisons between different conditions would not be influenced by variations in Vm:2+ cycling properties in ventricular myocyte models max = 10 mV, Vrest = −75 mV, and APD = 200 ms. CL ranged from 200 to 700 ms.To evaluate the Ca2+ cycling stability in the human atrial cell model via an iterated map analysis et al.2+ content of the cell are tracked from beat to beat. In our analysis, Ca2+ cycling stability depended upon three iterated map parameters: SR Ca2+ release slope . Lastly, To compute the iterated map parameters, a single atrial cell was repeatedly clamped to the AP waveform until model variables reached steady state. Following this, i (column 3), or [Ca2+]SR (column 4). Alternans persisted when Vm or [Ca2+]i was clamped, but clamping [Ca2+]SR eliminated alternans.(TIF)Click here for additional data file.S5 Figure2+ release parameter even beat clamps for the single-cell cAFalt model.SR Ca Traces of transmembrane potential , intracellular Ca2+ , and SR Ca2+  from two consecutive beats are superimposed to show alternans between even (red) and odd (blue) beats. Traces from the even beat at 400-ms CL pacing were used to clamp the relevant variable and are shown in row 4. Clamping RyR inactivated probability , RyR open probability , junctional Ca2+ , or SR Ca2+ release flux  eliminated alternans in Vm and Ca2+.(TIF)Click here for additional data file.S6 Figure2+ release parameter odd beat clamps for the single-cell cAFalt model.SR Ca Traces of transmembrane potential , intracellular Ca2+ , and SR Ca2+  from two consecutive beats are superimposed to show alternans between even (red) and odd (blue) beats. Traces from the odd beat at 400-ms CL pacing were used to clamp the relevant variable and are shown in row 4. Clamping RyR inactivated probability , RyR open probability , junctional Ca2+ , or SR Ca2+ release flux  eliminated alternans in Vm and Ca2+.(TIF)Click here for additional data file.S7 Figurealt model.Sub-sarcolemmal parameter clamps for the single-cell cAF Traces of transmembrane potential , intracellular Ca2+ , and SR Ca2+  from two consecutive beats are superimposed to show alternans between even (red) and odd (blue) beats. Traces from the even or odd beat at 400-ms CL pacing were used to clamp the relevant variable and are shown in row 4. Clamping sub-sarcolemmal Ca2+ ([Ca2+]sl) to the even beat (column 1) eliminated alternans in Vm and Ca2+, but clamping [Ca2+]sl to the odd beat (column 2) produced small alternans in Vm and [Ca2+]i and large alternans in [Ca2+]SR. Clamping sub-sarcolemmal Na+/Ca2+ exchanger current (INCXsl) to the even beat (column 3) eliminated alternans in APD but produced large alternans in [Ca2+]i and [Ca2+]SR. Clamping INCXsl to the odd beat (column 4) eliminated alternans in Vm and Ca2+.(TIF)Click here for additional data file.S8 FigureMultivariable regression between ionic model parameters and alternans threshold CL. (A) Bar graph of regression coefficient magnitudes. Twenty ionic model parameters were varied stochastically over 500 simulations to assess their effects on alternans cycle length (CL). Of the 500 simulations, 83 were excluded from the analysis because alternans threshold CL was below 100 ms or above 750 ms. Linear regression coefficients for each of the parameters are plotted in order of decreasing magnitude, with positive values plotted in red and negative values plotted in blue. Asterisks indicate p<0.05 for the t-statistic. (B) Bar graph of the predicted contribution of parameters to alternans threshold CL in the cAF-remodeled cell. Ten of the twenty parameters used in the regression analysis were altered from control values to represent cAF remodeling . Parameters whose changes were predicted to increase (decrease) the alternans CL are plotted in red (blue). Some unaltered parameters had nonzero predicted contributions to alternans threshold CL due to nonzero sample means from the regression analysis. The alternans threshold CL predicted by regression analysis (245 ms) was very close to the actual alternans threshold CL determined by simulation (244 ms).(TIF)Click here for additional data file.S9 FigureSingle-cell APD restitution in control model. With default model parameter values, APD alternans occurred at 200 ms CL (black). When the RyR inactivation rate constant (kiCa) was reduced to 95%, alternans occurred at slightly longer CLs (red). These results were comparable to alternans onset data from control patients (TIF)Click here for additional data file.S10 FigureAPD and CaT oscillations in single-cell and tissue models with Sato-Bers RyR formulation. Control (black), cAF (red), and cAFalt (dotted red line) versions of the model using the Sato-Bers RyR alt model, the calsequestrin-bound RyR closing rate (k34) was decreased by 50%. APD (A and C) and CaT (B and D) restitution data are plotted showing the mean±SD range . Oscillations in APD and CaT included but were not limited to alternans. Oscillations exhibited the reverse of the rate dependence observed in models using the original RyR formulation, with larger oscillations at longer CL. APD oscillations in these models were diminished as compared to the original models Click here for additional data file.S1 TextSupplemental methods.(PDF)Click here for additional data file.S2 TextSupplemental equations.(PDF)Click here for additional data file.