We applied sensitivity analysis to quantify the sensitivity of Shannon et al. model  to 5%–10% changes in currents conductance, channels distribution, and ion diffusion in rabbit ventricular cells. We found that action potential duration and Ca2+ peaks are highly sensitive to 10% increase in L-type Ca2+ current; moderately influenced by 10% increase in Na+-Ca2+ exchanger, Na+-K+ pump, rapid delayed and slow transient outward K+ currents, and Cl− background current; insensitive to 10% increases in all other ionic currents and sarcoplasmic reticulum Ca2+ fluxes. Cell electrical activity is strongly affected by 5% shift of L-type Ca2+ channels and Na+-Ca2+ exchanger in between junctional and submembrane spaces while Ca2+-activated Cl−-channel redistribution has the modest effect. Small changes in submembrane and cytosolic diffusion coefficients for Ca2+, but not in Na+ transfer, may alter notably myocyte contraction. Our studies highlight the need for more precise measurements and further extending and testing of the Shannon et al. model. Our results demonstrate usefulness of sensitivity analysis to identify specific knowledge gaps and controversies related to ventricular cell electrophysiology and Ca2+ signaling.Little is known about how small variations in ionic currents and Ca This model was the first to include (1) the subsarcolemmal Ca2+ compartment to the other two commonly formulated cytosolic Ca2+ compartments  Na+ buffering inside. Latest studies extended further the Shannon et al. ionic model in rabbit ventricular cells. Mahajan and colleagues modified L-type Ca2+ current and Ca2+ cycling formulations, based on new experimental patch-clamp data, and used the updated model to investigate the mechanisms regulating ventricular tachycardia and fibrillation (t)) in the compartment of interest only.Several ionic models have been developed to investigate the subcellular mechanisms regulating excitation-contraction coupling (ECC) in rabbit ventricular cardiomyocytes –7. In 20nd bulk, ), (2) thnts, and  Na+ buffillation . Morottitivation . Recentltivation  identifi include  the subsnts, and  Na+ buff2+ signals in rabbit ventricular myocytes with realistic t-tubule microanatomy have been investigated [2+ diffusion and buffering on ECC were not investigated.A few reaction-diffusion models that seek to incorporate the effects of idealistic or more realistic subcellular and whole-cell geometries and to compute the spatial distributions of [Ca] in cardiac, skeletal or smooth muscle cells have been published also –19. Recestigated . Limitat2+ dynamics and electrophysiological behavior in rabbit ventricular cells under control and certain pathological conditions. It remains, however, poorly understood how small variations in ion currents conductance and distribution and small changes in Ca2+ and Na+ transport between cellular subdomains affect important cellular biomarkers . In this study, we applied sensitivity analysis and used parameter estimation tools to investigate these questions. The sensitivity of each biomarker to 5%–10% changes in ionic currents properties and Ca2+ and Na+ diffusion coefficients were quantified using the Shannon et al. model. Validation of simulation results was performed by a comparison to experimental data in rabbit ventricular myocytes. This study identifies new experimental data required for better understanding of rabbit's electrophysiology and demonstrates the importance of sensitivity analysis as a powerful method for systematic and in-depth validation of AP models.Taken together the published computational models have provided important insights into the underlying mechanisms regulating intracellular Ca2+ fluxes included in the Shannon et al. electrophysiological model is shown in A diagram describing the arrangement of subcellular compartments, ionic currents and pumps, and intracellular Cahttp://somapp.ucdavis.edu/Pharmacology/bers/). The model behavior at 1 Hz pacing frequency is consistent with experimental measurements for AP-shape and global cytosolic Ca2+ concentrations ([Ca]i) changes under control conditions i remained unchanged while the variations in [Na]i were small (ranging from 20 μM to 100 μM) with insignificant effects on calculated AP and Ca2+ traces at 6–8 s. The APD60 and delta [Ca]j  quantities were used to analyze the model sensitivity. Δ[Ca]j is defined as the difference between peak Ca2+ concentration in the particular compartment and the diastolic Ca2+ concentration of 0.1 μM. The modified code was uploaded onto Nimrod/E toolset and was executed repeatedly using permutations of parameter sets that were ±10% or ±5% of default values )/std(x). Each z-score value was computed using the mean and standard deviations along each column of the matrices. The columns of matrices have mean zero and standard deviation one. The PLS regression coefficients, or sensitivity values  provide a qualitative overview of the inputs' relative effects on outputs and offer a comparison to the PLS-bar graphs. Changing the number of parameters studied slightly varied the magnitude of “regression coefficients” of bar plot and “estimate” of Lenth plot, but the relative effects and overall parameter-to-output relationships remained constant. The effects of sample sizes on model predictive efficacy by randomly picking subsets from sample pool, ranging from 15, 20, 50, 100, 200, and 500 to 1000 trials was examined. The adjusted R2 values  were calculated and averaged. At the low end of 15 samples, the regression model explained 90.8 ± 2.4% of the variance. At the high end of 1000 samples, the model explained 96.4 ± 0.3% of the variance. This relatively low decrease in prediction efficacy suggests that sample sizes do not significantly change the qualitative information obtained from the experiments. Considering this statistical analysis, the results were not included.To perform sensitivity analysis, the Shannon et al. MatLab code was modified to extract the desired model outputs. Under basal conditions or by varying one parameter at a time AP and Ca60, Δ[Ca]i, Δ[Ca]SL, Δ[Ca]jct).2+ channel permeability (PCaL) had the most pronounced effects on APD60 and Ca2+ signals, by increasing action potential duration and enhancing maximum Ca2+ peaks. The effects of GKr, Gto, and KNCX, while less pronounced than PCaL, were still significant. GKr and Gto decreased APD60 and Ca2+ peaks while 10% increase in KNCX decreased Ca2+ peaks but increased APD60. The increase in maximal NaK pump rate (KNaK) had still detectable but minor effect, by decreasing both APD60 and Ca2+ peaks. The changes in SR parameters  by 10% had negligible effects on APD60 and Δ[Ca]i whereas the submembrane and junctional Ca2+ peaks  were most affected. Krel and Kup increased Δ[Ca]SL and Δ[Ca]jct while Kleak had the opposite effect. Interestingly, 10% increase in background Cl− conductance (GClBk) showed similar magnitude as GKr and Gto and demonstrated the same effects in decreasing APD60 and Ca2+ peaks. Furthermore, Nimrod/E had the advantage in predicting how 10% increases in two-parameter group can affect the model outputs. Nimrod/E predicts here that PCaL and KNCX combined, as well all other two-parameter group combinations . The plots in PCaL, KNCX, GKr, Gto, KNaK, and GClBk on AP morphology and Ca2+ transients.The increase in L-type Ca2+ channels were assumed concentrated within the junctional membrane such that 90% of total number were located there while all other membrane transporters  were considered evenly distributed across the sarcolemma with 11% in the junctional cleft and 89% in the subsarcolemmal compartment i, Δ[Ca]SL, and Δ[Ca]jct), we performed systematic analysis, increasing by 5% the basic Fxj(jct) while decreasing Fxj(SL) to keep total number of transporter protein complexes within the sarcolemma unchanged; that is, Fxj(jct) + Fxj(SL) = 1  = 0.945) had the most pronounced effect on the model outputs, decreasing APD60 and Ca2+ peaks. The results also suggest that 5% increase in the junctional Na+-Ca2+ current (FxNCX(jct) = 0.1155) prolonged APD60 while slightly affected Ca2+ peaks in the junctional cleft and subsarcolemmal and bulk cytosol compartments. The 5% increase in the junctional ICl(Ca) (FxCl(Ca)(jct) = 0.1155) had similar effect on all model outputs as the increase in FxCaL(jct) but with much smaller magnitude. Results in Lenth plot (FxNCX(jct)FxCaL(jct) combination” as well all other two-parameter combinations had insignificant impact (not shown in Fxj(jct) by 5% and decreasing Fxj(SL) ) while keeping all other model parameters constant. The results in To examine how the changes in ion transporter distributions affect the selected cellular biomarkers  altered all model outputs, prolonging APD60 and increasing Δ[Ca]i and Δ[Ca]SL but decreasing Δ[Ca]jct. Moreover 10% increases in basal Di)Ca and Di)Na and Di)Ca and Di)Ca. The sensitivity of selected biomarkers  to the alterations in ionic currents' properties and intracellular ionic transport was quantified using the Shannon et al. ionic model in rabbit ventricular cells, the predictive power of which has been demonstrated previously i transient [i. Results demonstrate also that 10% increase in KNCX slightly decreased [Ca]jct and [Ca]SL peaks. Increase in GKr and Gto at 1 Hz resulted in APD shortening, which is consistent with experiments in rabbit ventricular myocytes i were smaller at 1 Hz (20–100 μM) versus 1–1.5 mM at 2.5 Hz, in this way affecting differently INaK and INCX activities. These results indicate that the stimulus frequency is important factor regulating the electrophysiological biomarkers. Accordingly, because the Shannon et al. model mimics rabbit electrophysiology more accurately at normal pacing rates of 1 Hz i, [Ca]SL, and [Ca]jct peaks. The reason for the predicted decrease in [Ca]jct was the strongly increased rate of Ca2+ transfer from the junctional cleft to the submembrane space evoked by the 5% increase of FxCaL(jct) and reduced ICa (~45%) into the SL compartment. The predicted decreases into [Ca]SL peak and APD60 were also consequence of FxCaL(SL) reduction to 45% though Ca2+ flux from the cleft was increased. The drop in [Ca]SL evoked decrease in the rate of Ca2+ transfer from the SL to cytosolic compartment thus causing drop in [Ca]i peak as well. Our studies also showed that the effects of INCX redistribution were quite different. The 5% increase in FxNCX(jct) (while decreasing by 40-41% INCX density in SL compartment to keep FxNCX(jct) + FxNCX(SL) = 1) prolonged APD60  to approximately the same magnitude as for ICa and slightly decreased the control Ca2+ peaks. Significant effects due to ICa redistribution and negligible effects due to INCX redistribution on the local and global Ca2+ transients' peaks were also found using our reaction-diffusion model in rabbit ventricular cells jct time to peak, (2) enhanced [Ca]SL and [Ca]i peaks, and (3) prolonged APD60. The effects of Di)CaCa(SL-) shortened APD60 and enhanced all Ca2+ transients' peaks. In addition, Di)CaCa in this way causing additional Ca2+ release from the SR and consequent elevations in [Ca]jct and [Ca]SL, respectively. The enhanced [Ca]SL transient produced faster Ca2+-dependent inactivation of ICa, thus leading to APD shortening. Nimrod/E showed also that simultaneous increases in control DCa(jct-SL) and Di)Ca. In summary, our results reveal that small variations in diffusion coefficients for Ca2+ may impact sensitively the cell electrical activity and Ca2+ homeostasis. New and more precise estimates of the effective Ca2+ and Na+ diffusion constants are needed to better understand ECC in rabbit ventricular myocytes under control and certain pathologies.In the Shannon et al. model the diffusion of Caed where  the juncdictable , 10–20. d[K]i/dt = 0 in Shannon's MatLab code) and whether or not this drawback was eliminated in the Romero's code or in recent modifications of the Shannon's code is unclear i appeared stable after ~200 s; (3) diastolic [K]i remained unchanged in the interval 0 ≤ t ≤ 8 min. Thus, models neglecting slow intracellular concentration changes in [K]i produced mainly by Na/K pump need to be revised further. Finally, the t-tubule compartment, the ion diffusion between the t-tubule lumen and bulk extracellular space, and the mitochondrial Ca2+ fluxes were omitted [2+ profiles as well.We need to acknowledge that although we demonstrated good correlation with experiment, the results of sensitivity analysis strongly depend on the model details and simplifications. For example, in the lumped Shannon et al. model the ionic diffusion from junctional cleft to cytosolic compartment goes solely through subsarcolemmal space which is questionable with regard to the contact areas between these compartments . In addi unclear –7, 9. Th omitted –50. Thes2+ and Na+ diffusion modulate ventricular myocyte electrophysiology and Ca2+ dynamics in rabbit ventricular myocytes at 1 Hz. We found that APD60 and Ca2+ peaks are (1) highly sensitive to 10% increase in ICa, while the effects of INCX, INaK, IKr, Ito, and IClBk were moderate, and (2) insensitive to 10% increases in all other Ca2+, Na+, and K+ transmembrane currents and SR Ca2+ fluxes. The myocyte electrical activity is highly sensitive to 5% variations in ICa and INCX and less sensitive to ICl(Ca) redistribution between junctional cleft and submembrane space. Small changes in submembrane and cytosolic diffusion coefficients for Ca2+, but not in Na+ diffusion coefficients, may impact notably the myocyte contraction. This study highlights the need for additional and more precise experimental data and further updating and testing of the Shannon et al. model and its recent modifications to fill specific knowledge gaps related to rabbit ventricular electrophysiology and intracellular Ca2+ signaling.Our studies demonstrate that parameter sensitivity approaches can be used to obtain new insights into relationships between model parameters, model outputs, and experimental data. More specifically, the simulation results provided information on how small variations in the ionic currents and intracellular Ca


MLPH in two dilute and one black rabbit for polymorphism detection. In both color diluted rabbits, skipping of exons 3 and 4 was present resulting in altered amino acids at p.QGL[37-39]QWA and a premature stop codon at p.K40*. Sequencing of genomic DNA revealed a c.111-5C>A splice acceptor mutation within the polypyrimidine tract of intron 2 within MLPH. This mutation presumably causes skipping of exons 3 and 4. In 14/15 dilute rabbits, the c.111-5C>A mutation was homozygous and in a further dilute rabbit, heterozygous and in combination with a homozygous frame shift mutation within exon 6 (c.585delG). In conclusion, our results demonstrated a colour dilution associated MLPH splice variant causing a strongly truncated protein (p.Q37QfsX4). An involvement of further MLPH-associated mutations needs further investigations. Coat color dilution turns black coat color to blue and red color to cream and is a characteristic in many mammalian species. Matings among Netherland Dwarf, Loh, and Lionhead Dwarf rabbits over two generations gave evidence for a monogenic autosomal recessive inheritance of coat colour dilution. Histological analyses showed non-uniformly distributed, large, agglomerating melanin granules in the hair bulbs of coat color diluted rabbits. We sequenced the cDNA of  RAB27A , MLPH (melanophilin), and MYO5A (myosin VA) encoding for the proteins of the tripartite complex can cause Griscelli syndromes. However, dilution of the hair is commonly accompanied by further symptoms depending on the defective gene [MYO5A and hypomelanosis is accompanied by neurologic deficits [RAB27A mutations and patients show immune impairment in combination with the hair color dilution [MLPH or MYO5A [MLPH defects are usually not accompanied by severe clinical diseases [Coat color dilution in rabbits affects eumelanin as well as pheomelanin. A dilution of eumelanin in black and brown coat color leads to blue and cream-brown, respectively, while a dilution of pheomelanin in yellow coat color leads to cream-yellow . The rabive gene . Grisceldeficits , while Gdilution . Griscelor MYO5A  and onlydiseases . The humdiseases  and the diseases .MLPH was chosen as the most likely candidate gene due to histological analyses and phenotypic examination. We sequenced the cDNA of MLPH in black and dilute rabbits and performed mutation screening for the dilution phenotype.In this study, we analysed the dilute coat color in rabbits. In total 23 Netherland Dwarf, Loh, and Lionhead Dwarf rabbits and their crosses of our breeding trial, including six individuals showing coat color dilution, were clinically examined for defects accompanying the dilute color. No abnormal clinical findings were detected associated with this phenotype, neither serious clinical defects nor less severe signs like color dilution alopecia or comedo formations. The oldest rabbit showing the dilution phenotype was more than three years of age at examination. The phenotypes of a black and a blue colored rabbit are shown in Subsequently, histological analyses were performed using skin samples of black rabbits (n=6) and blue rabbits (n=4). Cells of the hair bulb matrix from black rabbits contained large numbers of fine melanin granules which are moderately and uniformly distributed in the cytoplasm. Nuclei in the horizontally sectioned hair bulb of black rabbits were free of melanin . The meloryctolagus cuniculus (OryCun2.0) map viewer of NCBI (http://www.ncbi.nlm.nih.gov/projects/mapview) locates rabbit MLPH on an unknown chromosome. MLPH is designated LOC100343360, which contains 35,141 bp genomic sequence. We ascertained the complete coding sequence of MLPH in one black (DD) and two dilute (dd) rabbits, including all exon-exon boundaries and 51 bp of the 5’ untranslated region (UTR) and 60 bp of the 3’ UTR  also seems to be located at a position conserved in mammals and marsupials  as well as the two polymorphisms located within conserved, non-coding regions  and four further polymorphisms located within exon 2 (c.1-1G>A), exon 3 (c.215AG) or exon 4  were analysed in all 23 rabbits of the breeding trial. The SNPs c.1-1G>A, c.111-5C>A, c.366CT, c.369AG, and c.585delG showed associations with the dilute phenotype and were subsequently genotyped in further 30 rabbits of several breeds, which were not related with those of the breeding trial. Of these rabbits, seven showed the dilution phenotype, two rabbits showed a coat color which was classified as dark-dilute and 21 rabbits were wild type colored. The polymorphisms c.1-1G>A, c.111-5C>A, c.366CT, and c.369AG were highly correlated with each other at r2>0.9 . -10 to 6.8x10-7) and c.585delG (1.3x10-10 to 1.8x10-7). The c.111-5C>A mutation was homozygous in 14/15 colour diluted rabbits and the c.585delG deletion in 13/15 colour diluted rabbits , while c.585delG is located further downstream. Both mutations are in most, but not all cases within the same linkage phase. In a single case of a dark-dilute rabbit showing a heterozygous genotype at c.111-5C>A and the homozygous deletion at c.585delG, the latter mutation might be of relevance, as it also leads to a frameshift and an altered amino acid sequence with a premature stop codon 123 or 166 amino acids downstream (p.L195LfsX123 or p.L195LfsX166), respectively (dependent on the polymorphism c.953TC). The c.585delG polymorphism therefore might have an effect on coat colour dilution when the individual has not the homozygous A/A genotype at c.111-5C>A. MLPH showed only low associations with the dilution phenotype. They may be assumed to have arisen subsequently to the causal mutations, due to missing selection pressure. Though c.366CT and c.369AG were in high linkage disequilibrium with c.111-5C>A, their wild type allele is not conserved among different species . TherefoMLPH two-exon skipping seen in dilute rabbits leads to a frameshift and results in a change in the amino acid sequence (p.QGL[The ce p.QGL-39QWA) fQWA fMLPHce NC, [GHR was recently described to cause exon skipping by disruption of the polypyrimidine tract [MLH1, and two mutations within intron 2 of ß globin at -8 NC, ) are rarne tract . Furtherne tract ) within  -7 (C>G ), respec -7  mutation within intron 13 of OXCT1. This splice donor mutation led to retention of intron 13, thus causing the retention of intron 11 and 12. This so called splicing paralysis resulted in skipping the introns 11-13 and exons 12 and 13 [MLPH-associated dilute phenotype in rabbits. A further characteristic in the present study is the skipping of not only the third, but also of the fourth exon. Missing of more than one exon can be due to an inversion or a deletion of genomic DNA . This wautations ,42-46. Autations . A splicMLPH, is the most probable cause for this exon skipping and therefore for the dilute phenotype in rabbits. The dilute phenotype seems to be further influenced by another frame-shift mutation (c.585delG), but only one individual was exclusively homozygous for this genotype. This deletion does not affect the RAB27A binding domain like the c.111-5C>A mutation. The mutations detected herein in rabbits expand the contingent of known dilution mutations in different species and provide an additional view on the splicing mechanism.In conclusion, the dilution phenotype in rabbits is likely caused by a two-exon skipping of exon 3 and 4 and a frameshift of the open reading frame causing a premature stop codon (p.Q37QfsX4). The c.111-5C>A mutation, located within intron 2 of  All animal work has been conducted according to the national and international guidelines for animal welfare. The Lower Saxony state veterinary office at the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany, was the responsible Institutional Animal Care and Use Committee (IACUC) for this specific study. The breeding experiment and EDTA-blood sampling of the rabbits for the present study had been approved by the IACUC of Lower Saxony, the state veterinary office Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany (registration number 33.9-42502-04-11/0563). Tissue samples were taken from rabbits, which had to be euthanized due to other medical reasons . For blood or hair sampling of living animals, no anesthesia was applied as this would have meant at least equivalent stress than taking a sample. Each rabbit was only sampled once. Samples from rabbits of the further breeds were taken in the context of diagnostic analyses by a veterinarian at the Clinic for Pets, Reptiles and Pet and Feral Birds, University of Veterinary Medicine Hannover. We obtained informed consent from all owners of the rabbits. We obtained blood or hair root samples of a total of 53 rabbits, whereof 23 animals were rabbits of the breeding trial . In thisAll rabbits were examined by veterinarians at the Clinic for Pets, Reptiles and Pet and Feral Birds and at the Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover Foundation for defects accompanying the dilute coat color like in dogs ,9,23 or The other 30 rabbits were from several breeds . Of these rabbits, 21 were wild type colored, seven showed dilute fur color and two rabbits showed a darker blue or blue-brownish coat color, which was classified as dark-dilute and might be a variant of the dilution phenotype. in situ for storing at -20°C. The RNA was extracted from tissue samples using the RNeasy Lipid Tissue Mini Kit (Qiagen) according to manufacturer's protocol and transcribed into cDNA using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR .Genomic DNA was extracted from EDTA-blood or hair samples using routine procedures. Tissue samples for the RNA extraction were skin tissue of one black and two dilute rabbits. These samples were stored in RNAlater RNA Stabilization Reagent  for stabilization and protection of cellular RNA Skin samples of six black and four blue rabbits were fixed in Bouin’s solution and Ca-formol and were embedded in paraffin wax  according to standard protocols . The par3 were prepared, carefully dehydrated with graded ethanol  and embedded in the water-soluble and rather shrinkage-free 2-hydroxy-methacrylate Technovit 7100 . TwoParaffin sections of all collected samples were stained with haematoxylin and eosin  and plastic sections were stained with Toluidine blue O .All skin samples  were investigated semi-quantitatively for their melanin granule distribution in hair matrix cells. A total of sixty longitudinal histological hair bulb sections per phenotype were separately evaluated by three scientists using a qualitative scoring system. The scoring system distinguished four categories including “-” = no melanin granules, “+” = few melanin granules, “++” = several melanin granules, “+++” = many melanin granules . Within each category, the presence of the respective score was then subjected to a statistical analysis using a one-way ANOVA test. P-values<0.05 were defined as significant with * = P-value<0.05 and ** = P-value<0.01.MLPH, we used cDNA of one black Loh and two dilute rabbits whereof one was a Netherland Dwarf and the other one was a cross of a Netherland Dwarf (75%) x Loh (25%). Four pairs of primers were designed based using the rabbit MLPH cDNA sequence obtained by Ensembl (www.ensembl.org) and the Primer3 software (http://frodo.wi.mit.edu/). These primers yielded products covering the complete MLPH open reading frame and all exon-exon boundaries, as well as 51 bp of the 5’ UTR and 60 bp of the 3’ UTR. The exons 3 and 4 of MLPH, which were skipped in dilute rabbit cDNA, were sequenced on genomic DNA of the same three animals used for cDNA sequence analysis and in addition, a black Netherland Dwarf x Loh crossbred with the genotype Dd was sequenced. For this purpose, two pairs of primers were designed as described above. To exclude an inversion including exon 3 and 4, we further performed a long range PCR on genomic DNA of two black (DD and Dd) and two dilute (dd) rabbits, with the forward primer located within exon 3 and the reverse primer located within exon 5. Products of the appropriate size in all individuals in combination with the genomic sequences containing exons 3 and 4 would exclude the presence of an inversion. All primer pairs are shown in For mutation analysis of rabbit TaqDNA polymerase . Sequencing of the PCR products was performed using the ABI BigDye Terminator v3.1 sequencing kit . The products were analysed on an automated ABI 3500 capillary sequencer (Life Technologies). Mutation analysis was performed using Sequencher 4.8 . The open reading frame was determined using Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/). Translation into protein sequence was done using CLUSTALW . In the next step, all detected polymorphisms were analysed for their potentially damaging effect using PolyPhen2 [PCRs were carried out according to the standard protocol advised by the manufacturer of the olyPhen2 . DigestiolyPhen2 .Sequence data has been deposited at the GenBank Data Libraries . Figure S1Comparison of the different variants of truncated melanophilin proteins in dilute rabbits (variants 2-4) with the complete protein in wild type rabbits (1). Variant 2 is a result from skipping of exon 3 and 4 of MLPH (p.Q37QfsX4). Exon skipping is presumably caused by the c.111-5CγA transversion. Variants 3 and 4  are caused by the c.585delG mutation. Which one of these variants is generated depends on a further polymorphism affecting the premature stop codon of variant 3 (c.953T>C). The RAB27A domain is highlighted in green.(DOC)Click here for additional data file.Figure S2Linkage disequilibrium (LD) of the nine SNPs selected for genotyping in the 23 rabbits of the breeding trial  and in all 53 rabbits of different breeds . The latter five SNPs showed strong LD with the dilute phenotype. The pairwise r2-values are shown for each SNP pair. Red squares indicate complete linkage disequilibrium.(DOC)Click here for additional data file.Figure S3Pedigree of the rabbits from the breeding trial and the haplotypes of nine polymorphisms genotyped for these animals. Haplotypes include the polymorphisms c.1-10AγG, c.1-1GγA, c.111-5CγA, c.214A>G, c.215A>G, c.262A>G, c.366C>T, c.369A>G and c.585delG.(DOC)Click here for additional data file.Table S1MLPH) SNP positions detected in rabbits.Conservation of nucleotides in different species at the melanophilin Click here for additional data file.Table S2Case-control analysis for five melanophilin-associated polymorphisms with the dilution phenotype in 15 dilute and 37 colored non-dilute rabbits. The χ2- and P-values are shown for genotype, allele and trend tests.(DOC)Click here for additional data file.Table S3MLPH) c.111-5CγA, c.585delG and c.953T>C mutations in rabbits of different breeds.Distribution of the melanophilin .(DOC)Click here for additional data file.Table S4P) for amplification of sequences within melanophilin (MLPH) to be used for sequencing, long range PCR or genotyping as well as the targeted region and the appropriate template.Primer sequences with their product sizes Click here for additional data file.


ANF-RGC is the receptor and the signal transducer of the most hypotensive hormones, ANF– and B-type natriuretic peptide (BNP). After binding these hormones at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates the production of cyclic GMP. Cyclic GMP then serves the second messenger role in biological responses of ANF and BNP such as natriuresis, diuresis, vasorelaxation, and anti-proliferation. Very recently another modus operandus for ANF-RGC was revealed. Its crux is that ANF-RGC activity is also regulated by Ca2+. The Ca2+ sensor neurocalcin d mediates this signaling mechanism. Strikingly, the Ca2+ and ANF signaling mechanisms employ separate structural motifs of ANF-RGC in modulating its core catalytic domain in accelerating the production of cyclic GMP. In this review the biochemistry and physiology of these mechanisms with emphasis on cardiovascular regulation will be discussed.Atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), was the first discovered member of the mammalian membrane guanylate cyclase family. The hallmark feature of the family is that a single protein contains both the site for recognition of the regulatory signal and the ability to transduce it into the production of the second messenger, cyclic GMP. For over two decades, the family has been classified into two subfamilies, the hormone receptor subfamily with ANF-RGC being its paramount member, and the Ca Each of these two paradigms constituted an assemblage of three components – receptor, GTP binding protein, and the transducer catalyst – necessary for signal transduction. The astonishing discovery of a novel membrane protein that was simultaneously a receptor of a hormone and the transducer of its signal added a new dimension to our understanding of cellular signal transduction. The hormone was the newly described atrial natriuretic factor  sense the post-illumination fall in Ca2+ and stimulate ROS-GCs to resynthesize cyclic GMP at a faster rate  channels, and lowering the free Cahe GCAPs .2+ sensing modulators of ROS-GC activity. While increasing Ca2+ concentrations inhibit ROS-GCs activity through GCAPs, two other Ca2+ sensors, S100B, and neurocalcin δ (NCδ) stimulate ROS-GC1 in a Ca2+-dependent fashion , and the larger, C-terminal  tho503, L511, T513, T514, G580, S587, D590surround the ribose ring with L511 and T514 located within 2.5 Å radius from the ribose. The phosphate groups are surrounded by R504, G505, L511, K535, N633, D646, and K535 is the nearest residue (2.6 Å) forming a hydrogen bond with the phosphate and has been shown to be critical for ATP regulation of ANF-dependent ANF-RGC activity; D646 interacts with the triphosphate group of ATP through the formation of a coordinate bond with the metal ion Mg2+.GInteraction of ATP with the ARM domain. Kinetics of ATP binding to the ARM domain was determined through SPR spectroscopy (50) occurred when the concentration of AMP-PNP was ~0.2 mM and the calculated KD value was 0.21 mM. Similar results  were obtained when 8-azido-ATP was used.troscopy . AMP-PNPvide supra) was authenticated experimentally by cross-linking of the purified isolated ARM domain protein with 8-azido-ATP, trypsin digestion of the cross-linked product and sequencing of the resulting peptides  causing the side chains and the OH groups of T500, S502, S506, and T513 becoming directed toward the protein surface . The change in the positions of the side chains is most drastic for the S502 and S506 residues. Although upon ATP binding there is no toward the surface reorientation of the S497 and S510 OH groups, the entire residues are shifted toward the surface . This structural rearrangement permits the hypothetical protein kinase to access the side chains of the residues and transfer the phosphate group . These structural features define the fluorescence λmax of W601 at 332 nm. W669 residue is located at the end of a loop connecting β8 strand and EF helix . This environment causes red shifts in the fluorescence λmax of W669 to 345 nm.Another important result of ATP binding on the dynamics within the ARM domain is the translocation within the larger lobe of two helices, EF and F. Their movement was characterized by analyzing the ATP-dependent changes in the fluorescence intensity and wavelength of two tryptophan residues Wand W669 . Both retructure , 2005, iEF helix ; it is a669. The contraction results in shortening of the distance between W669 and the ATP binding pocket . ATP binding also causes reorientation of the W669 side chain. It turns and becomes more shielded by the surrounding amino acids. Turning of the W669 side chain pushes the remainder of the motif, 670TAPELL675, to the surface resulting in its exposure. In general, the movement of a hydrophobic motif toward the surface of the protein indicates its readiness for interaction. For the 669WTAPELL675 motif it was proposed that it interacts with subsequent transduction motif, possibly within the catalytic domain, propagates the ANF/ATP binding signal and activates the catalytic domain  interacts with the catalytic domain causing its partial activation; and (2) exposure and phosphorylation of six serine, threonine residues and full activation of ANF-RGC. Concomitantly, phosphorylation converts ATP binding site from the high to low affinity, ATP dissociates and ANF-RGC returns to its ground state  and C961(B) interact with guanine’s O6; second NH2 group of R959(B) interacts with guanine’s N1; the carboxy group of E889(B) interacts with R959(B) and through it with N1; G964(B) interacts with N7; T854(A), D893(A) and N968(B) form hydrogen bonds with ribose’s OH (3′) group; R972 binds β phosphate group; E974(A) through Mg2+ interacts with β phosphate group; and S971(A) binds γ phosphate group. Specificity toward GTP is determined by E889 and C961 of ANF-RGC. This conclusion is drawn based on studies showing that in ROS-GC1 mutation of the corresponding E to K and C to D converts guanylate cyclase activity into adenylate cyclase activity activity .669WTAPELL675 motif. It switches between the ATP-bound ARM domain and activates the catalytic domain. It was proposed that this motif interacts with and signals the antiparallel homo-dimer of the catalytic domain to undergo conformational changes and form a functional catalytic center  by Western blot to determine the proteins’ levels of expression; (2) for basal guanylate cyclase activity; (3) for ANF binding; and (4) for KM for the substrate GTP. The results demonstrated that (1) the wild type ANF-RGC and the 669WTAPELL675 deletion mutant were expressed to the same level as determined by Western blot; (2) the basal cyclase activity of the mutant, 20 pmol cGMP min-1 (mg protein)-1, was virtually identical to that of the wild type ANF-RGC, 21 pmol cGMP min-1 (mg protein) -1; (3) their receptor activities were equal with the respective specific binding values of 9.7 ± 0.6 and 10.1 ± 0.9 pmol [125I]ANF/mg protein and (4) the KM values for the substrate GTP, 614 μM for the wild type and 608 μM for the mutant protein were identical. Thus, the basal integrity of the protein remained intact despite the deletion  together with visinin-like proteins (VILIPs) and hippocalcin. They all are acylated at the N-terminus by myristic acid and undergo a classical calcium-myristoyl switch , e.g., tecoverin . Exposurne bound . Althoug2+-dependent affinity for S100B protein and tubulin β-chain , was chosen. And indeed, in a recombinant system, myristoylated NCδ stimulated ANF-RGC activity in a dose- and Ca2+-dependent manner; 0.5 μM Ca2+ and 0.5 μM NCδ triggered half-maximal activation of ANF-RGC . This activity increased when Ca2+ and NCδ were added, and the increase was Ca2+ and NCδ-dose dependent , heterozygous (ANF-RGC669WTAPELL675+/-) and homozygous (ANF-RGC669WTAPELL675-/-) mice  mice . Howevert is not . Thus, t669WTAPELL675 targeted mice and their isogenic controls implied that the expression of the mutant-cyclase must not differ from the expression of the wild type cyclase. This implication was confirmed by the results of immunocytochemical analyses. Side-by-side immunostaining with immunopurified anti ANF-RGC antibody resulted in identical images of the kidneys and adrenal glands of the wild type, heterozygous and homozygous 669WTAPELL675 targeted mice. Also, visual examination of the stained sections as well as of the respective differential interference contrast images indicated that the targeting of the 669WTAPELL675 motif does not change the integrity of these tissues  and fully (homozygous) suppressed ANF-RGC response to ANF, but the basal cyclase activity is unaffected the critical question was: does this suppression lead to increased blood pressure and other physiological alterations in the genetically modified mice?The main function of ANF/ANF-RGC signaling system is to regulate natriuresis, diuresis, vasodilation, and prevent cardiac and renal hypertrophy. It regulates blood volume homeostasis and blood pressure by off-setting the renin-angiotensin-aldosterone system (RAAS): inhibiting renal renin secretion and aldosterone synthesis in the adrenocortical zona glomerulosa . TherefoP value < 0.005  and by approximately 75% (up to 256 ± 22 pg/ml) in the plasma of the homozygous mice (P < 0.005)  and in their isogenic controls (wild type). The results demonstrated that, in comparison with the wild type mice, the plasma aldosterone concentration increased by approximately 40% (from 147 ± 12 to 204 ± 18 pg/ml) in the plasma of the heterozygous mice (< 0.005) .669WTAPELL675 targeted mice. The ratio of the heart weight (in mg) to whole body weight (in g) measured in 12 weeks old mice was 5.6 ± 0.3 and 6.1 ± 0.5 for the 669WTAPELL675(+/-) and 669WTAPELL675(-/-) mice, respectively, whereas it was 5 ± 0.3 for the wild type mice  exhibit significant cardiac hypertrophy. Which cyclic GMP-dependent signaling pathway involved in inhibition of myocardial proliferative responses is affected in these mice remains to be determined.Chronic pressure overload is almost always accompanied by cardiac hypertrophy  were tested for guanylate cyclase activity in the presence and absence of Ca2+. The activity in membranes isolated in the absence of Ca2+ was about 66 pmol cyclic GMP min-1 (mg protein) -1 for the control and NCδ+/- mice and the activity was not affected by the presence or absence of Ca2+ in the assay mixture. However, the activity in membranes isolated in the presence of Ca2+ was strongly dependent on the mice genotype and Ca2+ in the assay mixture. When assessed in the absence of Ca2+, the activity was ~70 pmol cyclic GMP min-1 (mg protein) -1 for the weight and NCδ+/- mice but when assessed in the presence of 1 μM Ca2+ the activity was 223 ± 20 pmol cyclic GMP min-1 (mg protein) -1 for the wild type mice and 135 ± 10 pmol cyclic GMP min-1 (mg protein) -1 for the NCδ+/- mice. Thus, the Ca2+-dependent NCδ-modulated ANF-RGC signaling pathway in the mice with one copy of NCδ gene deleted (NCδ+/-) is functionally half as active as in the wild type mice. To further authenticate that the lowering of the Ca2+-dependent cyclase activity in the adrenal gland membranes of NCδ+/- mice is the exclusive consequence of lower NCδ expression, 2 μM exogenous NCδ was added to the NCδ+/+ and NCδ+/- membranes (isolated in the presence of Ca2+) and the cyclase activity was determined in the presence of 1 μM Ca2+. The cyclase activity in the NCδ+/+ adrenal membranes increased only minimally, from 220 to 279 ± 21 pmol cyclic GMP min-1 (mg protein) -1 but in the NCδ+/- membranes, the increase was significantly larger, from 133 to 284 ± 24  mice. And the effect is exclusive for the aldosterone synthesizing glomerulosa cells, because corticosterone (synthesized in fasciculate cells) levels are unaffected by the absence of NCδ gene.What is the function of this pathway in the adrenal gland? In general, the primary role of ANF-RGC in the adrenal gland is to offset the renin-angiotensin system and inhibit aldosterone synthesis, and by doing this, to lower blood pressure . Therefo+/- mice correlates with the increase in blood pressure. Systolic blood pressure (measured by the non-invasive tail cuff method) was determined to be 92 ± 6 mm Hg for the wild type mice and 127 ± 9 mm Hg for the NCδ+/- mice. Therefore, the conclusion of these studies was that the ANF-RGC-Ca2+-NCδ transduction system is not only physically present but is of physiological significance at least in the adrenal gland.The increased plasma aldosterone level in the NCδ2+ is the additional signal of ANF-RGC. By entirely different mechanism, it regulates ANF-RGC catalytic activity and controls its physiological functions.In summary, this review has high-lighted studies which define the molecular and physiological mechanisms of the hormonal signal transduction of ANF-RGC. In addition, a new signal transduction mechanism and its present state of validation has been narrated. In this model Ca669WTAPELL675 motif “communicates” with the catalytic domain of ANF-RGC and signals its activation and (2) the mechanism of NCδ-Ca2+ signaling of ANF-RGC activity. The second venue relates to the physiology of both ANF-RGC signaling pathways (1) establishing whether the Ca2+ signaling mechanism is operative in other tissues in addition to the adrenal gland and (2) to determine the interrelationship of the hormonal and Ca2+ pathways. Finally, after deciphering the molecular and physiological details of the two signaling pathway the third venue will be translational, to design a molecule that can target directly the catalytic domain and bring ANF-RGC to its full activity and prevent the pandemic of hypertension, myocardial hypertrophy and obesity. The last objective is the most far-reaching, but hopefully can be achieved after the first two goals are accomplished.There are three venues through which, in these authors’ understanding, the future research will progress. The first venue is centered on the basic research to decipher (1) how the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation.Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish ( In meiotic maturation, the oocyte reduces the number of chromosomes to half through two successive meiotic cell divisions, in which the oocyte produces a normal ovum with high accuracy. Oocyte meiotic divisions are characterized by highly asymmetric division that occurs through dynamic surface actin remodeling In the starfish oocyte meiotic divisions, global and local actin remodeling has been observed in vivo cell shapes to a mathematical model in vivo cell shapes to a mathematical model is a powerful way to focus on the mechanical properties of a cell with high spatiotemporal resolution. One major consideration when applying the strategy to oocyte cell division, in which the changes in tensions on various spatial scales are all critical, is to acquire images having high spatial resolution and field of view Studies from mechanical perspectives on the morphology and tension of meiotic cell division have been performed on the starfish oocyte using image-based quantification in vivo cell shapes to a mathematical model. We adopted the Laplace formula that is a mathematical expression of the balance of forces of a thin elastic shell in vivo model have the advantage of simplifying the computation because of their spheroidal geometry. In order to detect the spatial change from a larger whole oocyte scale to a tiny polar body scale, rough and fine estimations of tensions at the surface were used in combination. We analyzed the images of oocytes during the meiotic cell division with an original computer program in which we approximated the surface shape data polynomially by means of a spline function In this study, we first clarified the tensions at the surface in detail during meiotic cell division, which is highly asymmetric, by fitting Individuals of the starfish, Asterina pectinifera, were collected from the Miura Peninsula, the Boso Peninsula, and the coast of Asamushi, Japan , and used as described previously The process of the rough estimation is shown in http://rsb.info.nih.gov/nih-image/).The principal radii of the oocyte surface curvature were calculated from the micrographs on the assumption that the oocyte was an ellipsoid of revolution whose axis running from the animal pole to the vegetal pole. In this rough estimation, as shown in T1 is the tension at the surface in the longitudinal direction, T2 is that in the latitudinal direction, and P is internal pressure, assumed to be 1. R1 and R2 are the principal radii of curvature of the cell surface  and 02T  are the tensions at the surface with a vertical distance of 20 µm from the animal pole before bulging, or base of the bulge after the bulging.The pseudocolor images near the animal pole were illustrated based on the tension obtained with fine estimation. The relative tension, Such a tension was represented with a pseudocolor on the three-dimensional (3D) cell surface whose image was restricted to the surface near the animal pole at the height of 20 µm from the animal pole, and the bulge of the polar body after the bulging. The fine estimation was done for four different oocytes.Spherical oocytes were selected for the analysis immediately after the 1-MeAde treatment. As shown in The oocyte recovered to a spherical shape at the onset of the bulge formation 0 min in . It collFollowing the method shown in rT, around the animal pole are shown. The animal pole of the image showed green 3 minutes before the onset of bulging, which meant that rT was about 1. One minute before the onset of bulging, the narrow region, which was supposed to become a polar body, changed to yellow, indicating that rT was more than 1. Then, rT at the animal pole decreased to less than 1, just before the bulge formation. One minute after the bulge formation, rT started to increase at the position where the furrow was supposed to form. As the bulging grew, rT at the top of the polar body in the animal pole decreased further, and rT at the furrow increased to more than 3 as shown by a change to red 2.5 minutes after the onset of bulging.To analyze the tension around the animal pole in detail, following the method shown in t = −3.0 in rT at the top of the animal pole and those in the maximum rT are assumed to be critical processes for oocyte in determining the site of the bulge before the onset of the polar body formation, and in the growth of the bulge after the onset. Thus, the temporal changes in the maximum rT, those in rT at the top of the animal pole, and the diameter of the ring with the maximum rT were estimated and shown in rT, at the top of the animal pole started to decrease before the onset of the bulge formation. One minute after the onset of the bulge formation, rT at the top of the animal pole was almost 0. In contrast to the rT at the animal pole, the maximum rT, as shown in rT increased after the bulge formation. The diameter decreased until 1.5 minutes before the onset of the bulge formation. Afterwards, the diameter increased until 0.5 minutes after the onset of the bulge formation. At this time, the position of the dividing furrow is assumed to be fixed. Then, the diameter decreased, indicating that the dividing furrow had started to ingress.At In this study, we first analyzed the spatiotemporal change in the tension at the surface during meiotic cell division. Our method successfully clarified the local change in the tension at the surface of a tiny polar body, simultaneously with the global change over the whole cell surface. In previous studies, spatial resolution of the tension at the surface has been limited and provided little information about the tension at the surface in the polar body To simultaneously obtain data on global deformation of the whole oocyte and that around the animal pole with a small bulge of the polar body, we analyzed these with coarse and fine estimation procedures respectively. In the coarse estimation procedure, the shape changes of the animal pole were ignored. On the other hand, in the fine estimation procedure, the tension around the animal pole with a small bulge of the polar body was obtained successfully and with high accuracy. Another feature of our analysis is to use the spline function to present a complex cell surface outline during the meiotic cell division by approximating the outline as a smooth curve. In this research, a fourth-dimensional B-spline function in which the second differential coefficient was continuous On a global whole oocyte scale, as seen in t = −1.0). As the bulge grew, the actin at the animal pole decreased (t = −0.5 to 2.5).On a local tiny polar body scale, before the onset of the bulge formation, the actin filaments around the animal pole are reorganized to form the bulge of the polar body. In ecreased . In contecreased . SimilarBy quantifying the global and local changes in the tension at the surface and by comparing the results with the distribution of actin filaments, as summarized in A detailed comparison between the actin quantity and the tension at the furrow indicates the increased efficiency of maintenance of the tension at the furrow, which should help to ensure normal meiotic cell division in the force generation system, as shown in Furthermore, the force generation system in Our approach successfully clarified the spatiotemporal regulation of force generation that drives the morphological change of the oocyte during highly asymmetric meiotic cell division. The results indicate that the force generation system involves machinery to increase the efficiency of tension maintenance at the furrow, and to achieve the movement of the meiotic apparatus for precise chromosome separation, which will help to accurately produce a normal ovum in the meiotic cell division.Video S1Shape change of a starfish oocyte during meiotic cell division. The top side is the animal pole.(AVI)Click here for additional data file.


Urticaria, a heterogeneous group of diseases, often cannot be recognized by its morphology. Due to non-specific and non-affordable diagnosis, management of urticaria, especially chronic urticaria, is very challenging. This guideline includes definition, causes, classification and management of urticaria. Urticaria has a profound impact on the quality of life and causes immense distress to patients, necessitating effective treatment. One approach to manage urticaria is identification and elimination of the underlying cause(s) and/or eliciting trigger(s), while the second one is treatment aimed at providing symptomatic relief. This guideline recommends use of second-generation non-sedating H1 antihistamines as the first-line treatment. The dose can be increased up to four times to meet the expected results. In case patients still do not respond, appropriate treatment options can be selected depending on the cost.First-generation antihistamines can interfere with rapid eye movement (REM) sleep and impact on learning and performance. New GA²LEN/EDF/EAACI/WAO guidelines do not recommend the use of these sedating antihistamines for the routine management of CU as the first-line agents. Second-generation antihistamines should be considered as the first-line symptomatic treatment for urticaria because of their good safety and tolerability profile.Second-generation antihistamines in higher doses have been shown to be effective in control of chronic spontaneous urticaria. This has been verified in studies using even up to fourfold higher than recommended doses of desloratadine, fexofenadine, levocetirizineand rupatadine.Approach to chronic spontaneous urticaria.See Results Diagram in Figure Four-fold updosing of antihistamines is recommended in urticaria management by Indian consensus statement.


We have recently demonstrated that the Ca2+ signalling machinery driving the oscillatory Ca2+ response to this important growth factor is different in UCB-derived EPCs as compared to their peripheral counterparts. In particular, we focussed on the so-called endothelial colony forming cells (ECFCs), which are the only EPC population belonging to the endothelial lineage and able to form capillary-like structures in vitro and stably integrate with host vasculature in vivo. The present review provides a brief description of how exploiting the Ca2+ toolkit of juvenile EPCs to restore the repairative phenotype of senescent EPCs to enhance their regenerative outcome in therapeutic settings.Endothelial dysfunction or loss is the early event that leads to a host of severe cardiovascular diseases, such as atherosclerosis, hypertension, brain stroke, myocardial infarction, and peripheral artery disease. Ageing is regarded among the most detrimental risk factor for vascular endothelium and predisposes the subject to atheroscleorosis and inflammatory states even in absence of traditional comorbid conditions. Standard treatment to restore blood perfusion through stenotic arteries are surgical or endovascular revascularization. Unfortunately, ageing patients are not the most amenable candidates for such interventions, due to high operative risk or unfavourable vascular involvement. It has recently been suggested that the transplantation of autologous bone marrow-derived endothelial progenitor cells (EPCs) might constitute an alternative and viable therapeutic option for these individuals. Albeit pre-clinical studies demonstrated the feasibility of EPC-based therapy to recapitulate the diseased vasculature of young and healthy animals, clinical studies provided less impressive results in old ischemic human patients. One hurdle associated to this kind of approach is the senescence of autologous EPCs, which are less abundant in peripheral blood and display a reduced pro-angiogenic activity. Conversely, umbilical cord blood (UCB)-derived EPCs are more suitable for cellular therapeutics due to their higher frequency and sensitivity to growth factors, such as vascular endothelial growth factor (VEGF). An increase in intracellular Ca These Ca2+ signatures are detected, decoded and relayed to the target enzyme by a complex toolkit of Ca2+-binding proteins, pivotal among which are calmodulin (CaM), Ca2+/CaM-dependent kinases  and calcineurin, that act as Ca2+ sensors i may, indeed, be detrimental for the cell since a massive Ca2+ load may either induce the pro-apoptotic cascade or cause necrosis by stimulating both Ca2+-dependent proteases, such as calpain, and endonucleases [2+ oscillations that persist as long as the cells are presented with the growth factor [2+]i is the hallmark that features endothelial monolayers exposed to either VEGF or EGF, whose pro-angiogenic effect is suppressed when the intracellular Ca2+ signal is abolished i which arise even in the absence of extracellular Ca2+ influx i promote a phosphorylation cascade which is mediated by Ca2+/CaM-dependent protein kinases and leads IkB to site-specific ubiquitination and subsequent degradation. Consequently, NF-κB is released from inhibition and translocates into the nucleus, where it activates the transcriptional programme responsible for cell survival and proliferation i elevation ignited by VEGF in UCB-ECFCs. The higher proliferative response manifested by the latter respect to their peripheral counterparts prompted us to expect a difference in the kinetics of the Ca2+ signal. We were, therefore, surprise to observe a similar pattern of Ca2+ oscillations which were not synchronized between adjacent/neighbouring cells even from the same microscopic field of view (manuscript submitted). A major breakthrough in our studies was the discovery that the Ca2+ response to VEGF does not arise when UCB-ECFCs are stimulated in the absence of extracellular Ca2+. In other words, Ca2+ entry plays a primary role in these cells, by triggering their oscillatory activity in the presence of VEGF, while its role in their peripheral counterparts is limited to maintain the Ca2+ transients over time. Similar to PB-ECFCs, however, the Ca2+ machinery involved was placed downstream PLCγ activation, a feature implying InsP3 and/or DAG involvement (manuscript submitted). We utilised quantitative real time-polymerase chain reaction (qRT-PCR) analyses and immunoblotting to search for conductances other than SOCE in UCB-ECFCs and found that they express TRPC3 i in TRPC3-deficient cells (manuscript submitted). The signalling transduction pathway recruited by TRPC3-mediated Ca2+ entry was not different from that dissected in PB-ECFCs and involved the interaction between InsP3-dependent Ca2+ release and SOCE. Intriguingly, UCB-ECFCs are not endowed with InsP3R1, whereas they express the other two isoforms present in ECFCs harvested from PB (manuscript submitted). This feature is rather intriguing, InsP3R2 being the most suitable receptor subtype to drive repetitive Ca2+ spikes due to its peculiar InsP3 and Ca2+-sensitivity i is not sufficient to trigger a given Ca2+-dependent reaction, such as gene transcription or an increase in mitochondrial metabolism, if it does not involve the Ca2+ channel intimately associated to this process. Our finding that TRPC3 selectively initiates the pro-angiogenic Ca2+ signal in ECFCs isolated from umbilical cord blood, but not peripheral blood, might contribute to understand their higher sensitivity to VEGF. A large number of approaches are available to transfer angiogenic genes into endothelial committed progenitors [Cellular therapeutics is regarded as the most promising strategy for the future of cardiovascular diseases to overcome the limits intrinsic to surgical methods, catheter technologies and pharmacological treatments. Indeed, with the ageing of populations and improvement in survival, increasing numbers of patients are not amenable to revascularisation because of comorbid diseases, unsuitable anatomy or the risk of interventional procedures. The injection of autologous endothelial progenitor cells in clinical practice, however, is still far from coming of age due to the exceedingly number of concerns that have been raised by the lack of therapeutic consistency between pre-clinical studies and clinical trials. One of the issues that must be solved before progressing towards a safe application of stem and progenitor cells in the patients is to understand whether and how is possible to rejuvenate the phenotype of senescent EPCs, which are inadequate to meet the demands of repair and regeneration in diseased vessels. Whereas most of the efforts in the field have been carried out to understand which EPC subset utilize, routes and methods of cell delivery dosing, and timing of intervention, less attention has been paid to the biological mechanisms driving their angiogenic activity. In this regard, the discovery that ECFCs, which represent the most suitable candidates to recapitulate the vascular network genitors ,36,78. FThe authors declare that they have no competing interests.FM: conceived the study, analyzed and interpreted the data, drafted the manuscript. SD: conceived the study, critically revised the manuscript. MPC: critically revised the manuscript. SM: critically revised the manuscript. BA: critically revised the manuscript. GG: conceived the study, analyzed the data and critically revised the manuscript. VR: conceived the study and critically revised the manuscript. FT: conceived the study, analyzed and interpreted the data, critically revised the manuscript. All authors read and approved the final manuscript.FM: Assistant Professor of Physiology at University of PaviaSD: Ph.D Student at University of PaviaMPC: Assistant Professor of Anatomy at University of Naples "Federico II"SM: Full Professor of Anatomy at University of Naples "Federico II"BA: Associate Professor of Surgery at University of Naples "Federico II"GG: Assistant Professor of Anatomy at University of MoliseVR: Staff Physician at IRCCS Policlinico San Matteo, PaviaFT: Associate Professor of Physiology at University of Pavia


While the former is required for Ca2+ signalling linked to insulin secretion, the role of the latter in β cell function has not been well understood, primarily because the molecular identity of the mitochondrial Ca2+ transporters were elusive and the selectivity of their inhibitors was questionable. This study focuses on NCLX, the recently discovered mitochondrial Na+/Ca2+ exchanger that is linked to Ca2+ signalling in MIN6 and primary β cells. Suppression either of NCLX expression, using a siRNA construct (siNCLX) or of its activity, by a dominant negative construct (dnNCLX), enhanced mitochondrial Ca2+ influx and blocked efflux induced by glucose or by cell depolarization. In addition, NCLX regulated basal, but not glucose-dependent changes, in metabolic rate, mitochondrial membrane potential and mitochondrial resting Ca2+. Importantly, NCLX controlled the rate and amplitude of cytosolic Ca2+ changes induced by depolarization or high glucose, indicating that NCLX is a critical and rate limiting component in the cross talk between mitochondrial and plasma membrane Ca2+ signalling. Finally, knockdown of NCLX expression was followed by a delay in glucose-dependent insulin secretion. These findings suggest that the mitochondrial Na+/Ca2+ exchanger, NCLX, shapes glucose-dependent mitochondrial and cytosolic Ca2+ signals thereby regulating the temporal pattern of insulin secretion in β cells.Mitochondria mediate dual metabolic and Ca Cellular uptake and metabolism of glucose by pancreatic β cells stimulates ATP production 2+ transport that is powered by their steep membrane potential. Cytosolic increase in Ca2+ is followed by Ca2+ influx into the mitochondria via the mitochondrial uniporter recently shown to be linked to MCU  2+ is buffered by calcium phosphate and subsequently extruded by an electrogenic 3Na+/Ca2+ exchanger that uses the mitochondrial membrane potential and Na+ gradient to pump Ca2+ out of the mitochondria back into the cytoplasm 2+ shuttling has several roles: first, Ca2+ activates at least three key intra-mitochondrial dehydrogenases 2+ store and the Ca2+ efflux by the mitochondrial exchanger can control the amplitude and duration of cytosolic Ca2+ transients, for example, in neurons + dependent efflux of Ca2+ has been described also in isolated mitochondria from β cells 2+ shuttling, and in particular, the mitochondrial Na+/Ca2+ exchanger in glucose dependent Ca2+ signalling in β cells are not well understood. A major complication is that the molecular identity of the mitochondrial Na+/Ca2+ exchanger has been elusive until recently and the concern that the inhibitor of the exchanger, CGP-37157 may interact with other major Ca2+ transport pathways in β cells. For example, CGP-37157 was suggested to trigger mitochondrial Ca2+ rise by blocking the exchanger thereby leading to enhanced mitochondrial oxidative metabolism and insulin secretion 2+ signals by blocking the LTCC in β cells 2+ transporters among them: sarcoma(-endo) plasmic reticulum Ca2+-ATPase, SERCA, and ryanodine receptors, RyR Mitochondria however, are also a major hub for cellular Ca+/Ca2+ exchanger super family member NCLX is localized in the mitochondria where it mediates Ca2+ efflux and is therefore likely the mitochondrial Na+/Ca2+ exchanger 2+ signalling linked to insulin secretion. Our results indicate that NCLX is not only critical for mitochondrial Ca2+ efflux but also affects cytoplasmic Ca2+ responses. A major conclusion of this study is that mitochondrial Ca2+ shuttling, catalyzed by NCLX, plays a dominant role in shaping glucose-dependent cytosolic Ca2+ transients and thereby regulates the temporal pattern of insulin secretion.We and subsequently others, found that the NaSix-eight week old female DBA/2J mice were purchased from Jackson laboratories, Bar Harbor, ME, USA. Mice were kept in a pathogen-free environment at the Ben-Gurion University of the Negev Research Animal Facility and were cared for according to the Ben-Gurion University of the Negev Care and Use of Animals committee guidelines.Animals were anesthetized prior to islet harvest by standard ketamine/xylazine and islets were isolated by collagenase digestion Isolated islets were cultured in RPMI 1640  for 2–3 days and MIN6 cells in DMEM . Both media were supplemented with 10% fetal calf serum , 1% Pencillin/Streptomycin , 1% L-Glutamine , 5 mM Glucose . Islets were hand-picked under a stereoscope and dispersed into single cells by Trypsin-EDTA . Dispersed primary islet cells and MIN6 cells were seeded onto coverslips for imaging experiments Pancreatic primary β cells and MIN6 cells cultured on glass coverslips were transfected with siNCLX vs. siControl using DharmaFECT siRNA Transfection Reagents . siRNA NCLX or siControl was diluted in DharmaFECT siRNA transfection reagent, incubated approximately 20 min at room temperature and then added in the antibiotics free media. The efficiency of transfection was assessed by visualizing Dharmacon siGLO Red  transfection particles according to the protocol provided by manufacturer. The transfection efficiency, for siNCLX delivery as determined by siGlo fluorescent marker was high, ∼80%. Pancreatic primary β cells and MIN6 cells were co-transfected with a dominant negative active form of NCLX 2+ imaging was performed in pancreatic primary β cells and MIN6 cells attached onto coverslips and superfused with Ringer solution containing (in mM): 126 NaCl , 5.4 KCl , 0.8 MgCl2 , 20 HEPES , 1.8 CaCl2 , 15 Glucose , pH adjusted to 7.4 with NaOH  or with high K+ (50 mM) Ringer solution when indicated. In glucose dependent experiments the pancreatic primary β cells or MIN6 cells were pre-washed for 30 min with low glucose (3 mM) Ringer solution followed by high glucose (20 mM) Ringer solution.The imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator  and a SensiCam cooled charge-coupled device . Fluorescent images were acquired with Imaging WorkBench 4.0 . Ca2+ measurements were performed in pancreatic primary β cells infected with lenti-pericam and in MIN6 cells expressing ratiometric mitochondrial pericam, which is targeted solely to the inner membrane of mitochondria Pancreatic primary β cells and MIN6 cells were loaded with Fura 2AM, excited with 340/380 nm wavelength light and imaged using a 510 nm long pass filter 2+ imagingFluorescent Ca), as previously reported Mitochondrial membrane potential was monitored in MIN6 cells loaded and superfused with TMRM , excited at 545 nm and imaged with a 570 nm emission filter as previously described Cell fractionation was performed as previously described AACGGCCACUCAACUGUCU) or siNCLX (AACGCGCAUCCAACUGUCU) using TRIZol reagent  following the manufacturer's instructions. The pancreatic primary islets were homogenized in TRIZol, followed by phase separation with chloroform and centrifuged at 12000×g for 15 min at 2°C. RNA was then precipitated from the aqueous phase by mixing with an equal volume of isopropyl alcohol and centrifuged at 12000×g for 10 min at 2°C. Finally, the RNA pellet was dried and dissolved in RNA-ase free water. The cDNA was generated using First Strand cDNA Synthesis Kit . Thermal cycling (40 cycles) conditions were 50°C for 15 min, followed by 95°C for 15 min, 95°C for 15 sec and 60°C for 60 sec. As reference gene, we employed glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR reactions were conducted using a SDS7500 Real Time PCR machine and TaqMan probes (ThermoScientific).RNA was isolated from pancreatic primary islet cells transfected with either siControl (Pancreatic primary islets cells were kept in low glucose (3 mM) Ringer solution for 30 min that was then replaced with high glucose (20 mM) containing Ringer solution. Cells were lysed at the indicated time intervals and ATP content was determined using the luciferin/luciferase luminescence assay with Bioluminescent Cell Titer-Glo Assay Kit  according to the manufacturer's instructions.Insulin secretion was monitored using a commercial ELISA kit . Pancreatic islet cells were incubated for 30 min in low glucose (3 mM) Ringer solution and then stimulated with high glucose (20 mM) Ringer solution. Aliquots were collected at the indicated time intervals and the amount of insulin secreted from pancreatic primary islet cells was determined according to the manufacturer's protocol (Mercordia mouse insulin ELISA kit).2+ into the cytosol or mitochondria consistently began after stimulation of pancreatic β cells with High K+, ATP or high glucose. Similarly, Ca2+ efflux out of the mitochondria followed the influx phase as previously described +, ATP or high glucose) for Ca2+ influx or Ca2+ efflux as previously described 2+ response or their amplitude (ΔCa2+ cytosolic rates or peak amplitude) were calculated by averaging either rates of fluorescence Ca2+ change or the amplitude of the fluorescence Ca2+ response of number of experiments, n, indicated at the figure legends The traces of all fluorescent imaging experiments were plotted using KaleidaGraph 4.0. Influx of CaThe results of the experiments are the mean ± S.E.M. (standard error of the mean) of at least 3 independent experiments (n), using 20–30 cells in each. Statistical significance for all experiments was determined using a one-way ANOVA test followed by Bonfferoni post-hoc analysis. * Significantly different (P<0.05) compared to control.2+ transport. Immunoblot analysis of NCLX in total lysates and isolated mitochondria from MIN6 cells demonstrated that NCLX was enriched in mitochondrial fractions  Ringer's solution, mitochondrial Ca2+ influx was observed in the siControl cells and was followed by a robust efflux phase. In contrast, in cells transfected with siNCLX the mitochondrial Ca2+ influx rate increased by 50±11% and a dramatic 80±13% reduction of mitochondrial Ca2+ efflux was measured  and a decrease in efflux (65±4%) in the cells transfected with the dnNCLX construct  followed by application of high glucose (20 mM) Ringer solution. Application of high glucose triggered a robust mitochondrial Ca2+ transient (2+ influx (50±4%) and a decrease of Ca2+ efflux (56±7%) .Considering the major effect of NCLX on mitochondrial Caransient . Knockdo (56±7%) . To dete (56±7%) . Compari (56±7%) . We next2+ efflux and also regulates the rate of Ca2+ influx transient. Notably, we find that NCLX controls mitochondrial properties, also under resting conditions as the knockdown of NCLX is followed by a mild mitochondrial depolarization and a rise in resting mitochondrial Ca2+.Altogether, the results of this part indicate that NCLX participates in glucose-dependent mitochondrial Ca2+ influx is a major step in initiating the secretory process in β cells. We have therefore sought to determine the role of NCLX in regulating this process. Cytosolic Ca2+ entry was initially triggered in pancreatic MIN6 cells by depolarization with high K+ (50 mM) Ringer while monitoring Fura 2 AM fluorescence. We then asked if knockdown of NCLX expression will modulate cytosolic Ca2+ signals. Comparison of cytosolic Ca2+ rise of siNCLX vs. siControl transfected MIN6 cells showed a marked decrease in the rate of cytosolic Ca2+ changes of 45±10% and a decrease of 10±5% in the amplitude of the cytosolic Ca2+ signals transfected with siNCLX H/NAD+. The mitochondrial Ca2+ transient signal was monitored in primary β cells co-transfected with a lenti viral construct of mito-pericam H/NADenhanced  suggestipression  was suff2+ transients induced by high glucose and it is also affecting the basal NAD(P)H production rates.Altogether, the results of this part suggest that NCLX is regulating the rates and amplitude of influx and efflux phases of mitochondrial CaWe next determined the time course of glucose-dependent ATP production by monitoring luciferase luminescence in primary β cells lysates 2+ response, we asked if it also participates in regulating the rate of insulin secretion. We used primary β cells transfected with siNCLX vs. siControl while monitoring insulin secretion before and after application of high glucose Ringer's solution. The supernatant of treated cells was collected at the indicated time intervals and amounts of secreted insulin levels were determined. Partial inhibition by 40±8% - 1 min, 40±14% - 2 min, 60±16% – 5 min, in the rate of insulin secretion was monitored in siNCLX primary β cells during the first 5 minutes  NCLX is localized in the mitochondria of β cells, 2) silencing of NCLX expression leads to inhibition of mitochondrial Ca2+ efflux, 3) transfection of β cells with a dominant negative NCLX construct has a similar inhibitory effect on mitochondrial Ca2+ efflux activity, 4) both knock down of NCLX expression or activity inhibit mitochondrial calcium efflux following a glucose-dependent Ca2+ rise. However, our results indicate that NCLX in β cells has an additional role that has not been documented in previous studies 2+ influx in β cells. This suggests that the Ca2+ efflux mediated by NCLX is strongly activated already at the early phase of mitochondrial Ca2+ influx and thus, NCLX has the capacity to shape not only the mitochondrial Ca2+ efflux phase but also indirectly the Ca2+ rise in this organelle. This effect is particularly remarkable considering that the influx phase, that was previously monitored in many cell types is about 2 orders of magnitude faster than the efflux phase 2+ response, we observed that NCLX determines the resting levels of mitochondrial Ca2+ in β cells, a finding that may explain the role of NCLX in metabolic processes (discussed below). These major effects of NCLX are consistent with the high pancreatic expression of NCLX in β cells Towards this goal, we first asked if NCLX, recently identified by our group as a candidate for the mitochondrial Na2+ influx. The effects of NCLX elimination on mitochondrial calcium changes prompted by physiological stimulation with glucose were not explored, nor were the kinetics of mitochondrial transport, mitochondrial membrane potential or glucose-regulated insulin secretion. In the detailed studies described here we now show that NCLX plays a key role in regulating the glucose-dependent Ca2+ response in the cytosol and the mitochondria. Our results indicate that NCLX activity is critical for clearance of mitochondrial Ca2+ and is therefore a rate limiting player in the mitochondrial Ca2+ response induced by glucose. In addition, we demonstrated that by catalysing the mitochondrial efflux, NCLX also shapes the cytosolic glucose-dependent Ca2+ response and thereby, regulates the rate of insulin secretion.In our recent report 2+ in distinct tissues. For example, in cardiac tissue they occupy ∼30% of the total volume 2+ responses 2+ uptake. The estimated occupancy of mitochondria in β cells is even lower at about 4% 2+ signalling of β cells. We find that silencing either the expression or the activity of NCLX, decreases the rate of cytosolic Ca2+ changes by glucose by approx. 40% and the amplitude of the Ca2+ signals by 30%. Considering the small volume occupied by the mitochondria and the large change that it triggers in cytosolic Ca2+, our results indicate that it outpaces by several fold, the transport rate mediated by the plasma membrane and ER Ca2+ transporters. Remarkably, despite the major cytosolic Ca2+ changes triggered by NCLX, the mitochondrial changes are relatively modest. Several studies have underscored the powerful Ca2+ buffering capacity of mitochondria, in particular the formation of calcium phosphate, that is at least 10 fold stronger than the buffering capacity of the cytosolic Ca2+2+ buffering, dependent on matrix pH, allows a rapid dissociation of Ca2+ phosphate, thus readily providing the exchanger with Ca2+2+ provide sufficient amounts of Ca2+ required to change its cytosolic concentrations.Mitochondria are occupying vastly different relative volumes in different cell types and play a highly heterologous role in regulating cytosolic Ca2+ in the vicinity of mitochondria followed by enhanced mitochondrial Ca2+ uptake 2+ clearing mechanism thereby minimizing the potential toxic effect of a strong cytosolic Ca2+ surge. On the other hand, a rapid decline in cytosolic Ca2+ may interfere with the first phase of insulin secretion, which requires a sufficient rise in cytosolic Ca2+ because of the low Ca2+ affinity of the secretory machinery 2+ rise during the first phase of insulin secretion 2+ channels and requires a more moderate Ca2+ rise 2+ levels in β cells by mediating a continuous and robust mitochondrial Ca2+ efflux, thereby preventing rapid cytosolic Ca2+ decline and augmenting sustained elevated cytosolic Ca2+ levels required for this secretory phase. This role in secretion is supported by our finding demonstrating a delay in glucose-dependent insulin secretion triggered by silencing of NCLX. Thus, we suggest a more dominant role for strong Ca2+ rise during the first phase of insulin secretion. An important finding of this study is that the role of the mitochondrial exchanger NCLX in regulating the cytosolic Ca2+ determines the rate of insulin secretion during the initial phase of secretion. Previous studies using the mitochondrial exchanger inhibitor CGP-37157 suggested that the inhibition of the exchanger led to enhanced ATP production and insulin secretion 2+ in β cells. Our results indicate that the silencing of NCLX leads to a small but significant rise in basal mitochondrial resting Ca2+. Because the affinity of the Ca2+ sensitive Kreb's cycle enzymes is relatively high, even such a modest rise in Ca2+ may be already sufficient to induce their activation at low levels of glucose 2+. Consistent with such a mechanism, we observed that the acceleration of the basal metabolic rate was induced following knockdown of NCLX expression. In addition, knockdown of NCLX is followed by partial mitochondrial depolarization that may further decrease the efficiency of ATP production. Alternatively, the rise in resting mitochondrial Ca2+ could trigger the formation of oxygen radicals of NO species that may have an inhibitory effect on metabolic processes in β cells 2+ signalling linked to insulin secretion as previously suggested based on the inhibition of the exchanger by CGP-37157. The latter effect may have also been related to the modulatory effect of this compound on other major Ca2+ pathways such as the L-type Ca2+ channel or SERCA, that can affect ATP consumption and thereby could indirectly change the energy balance in β cells 2+ transport activity mediated by NCLX and its strong effect on increasing cytosolic Ca2+ responses, are the primary roles of NCLX that are linked to insulin secretion. Further in vivo studies, employing for example transgenic NCLX knockout mice, will be required to study the contribution of mitochondrial Ca2+ shuttling on these aspects of islet physiology.Recent studies have highlighted the physical proximity and functional cross-talk between the L-type calcium channels, LTCC and the mitochondrial network. This leads to a robust LTCC-dependent rise of local Ca


TARA) under Cd stress. Plants grown in the presence of Cd exhibited reduced growth traits  and concentration of Ca2+, K+, Chlorophyll (Chl) a and Chl b content, except content of MDA, Cd and (Pro). The antioxidant enzymes [peroxidase (POD) and superoxide dismutase (SOD)] slightly increased as compared to control under Cd stress. However, a significant improvement was observed in all growth traits and content of Ca2+, K+, Chl a, Chl b, Pro and activity of antioxidant enzymes catalase (CAT), POD and SOD in plants subjected to Ca2+ and/or K+. The maximum alleviating effect was recorded in the plants grown in medium containing Ca2+ and K+ together. This study indicates that the application of Ca2+ and/or K+ had a significant and synergistic effect on plant growth. Also, application of Ca2+ and/or K+ was highly effective against the toxicity of Cd by improving activity of antioxidant enzymes and solute that led to the enhanced plant growth of faba bean plants.Cadmium (Cd) in soil poses a major threat to plant growth and productivity. In the present experiment, we studied the effect of calcium (Ca Increasing cadmium (Cd) in soil is an enormous constraint for agriculture worldwide. Cd is a highly toxic metal that causes deleterious effects on plants, and limits crop productivity . Cd is c+) has an outstanding role in plant growth and development. K+ is the most abundant cation in plants (up to 10% on dry weight basis) , as compared with the controls. However, combined application of Ca2+ and K+ showed more enhancing effect on all growth traits than individual treatment. Application of Cd inhibited all growth attributes of faba bean. However, the subsequent treatment of Cd-stressed plants with Ca2+ or K+ alone as well as in combination alleviated the adverse effect of Cd on faba bean and caused a considerable improvement of RL, SL, SFW, RFW, SDW, and RDW. The combined application of Ca2+ and K+ had maximum alleviating effect of Cd toxicity on all growth characteristics when compared with Ca2+ and K+ supplied alone. In contrast, combined application of Ca2+ and K+ showed parity with the application of Ca2+ alone for SFW and RFW.The significant changes of growth parameters in faba bean plants subjected to Ca2+ and K+ alone as well as together significantly increased the chlorophyll (Chl) a and Chl b as compared to the control under normal conditions. The combined application of Ca2+ and K+ exhibited maximum value for both Chl a and Chl b when compared with individual application of Ca2+ and K+, under normal conditions. However, presence of Cd in growth medium suppressed both the photosynthetic pigments. Application of Ca2+ and/or K+ significantly enhanced Chl a and Chl b under stress. Moreover, application of Ca2+ and K+ together was found to be more effective in alleviating the adverse effect of Cd stress on Chl a and Chl b.2+ and/or K+ inhibited accumulation as compared to the control. However, application of Ca2+ and K+ alone as well as in combination arrested the ill effect of Cd up to a considerable limit by decreasing the accumulation of MDA in the leaf of faba bean plants. Under stress, the lowest accumulation of MDA was recorded in the plants that received Ca2+ and K+ together.2+ and K+ alone was found to be effective in suppressing leaf-Cd concentration, but maximum inhibition of Cd content was recorded in plants fed with the combined Ca2+ and K+ application. The presence of Cd in growth medium suppressed both the content of both nutrients in the leaf. However, application of Ca2+ and K+ alone as well as in combination increased leaf-Ca2+ and K+ content under normal as well as stress conditions. Application of Ca2+ and K+ individually was found to be effective in improving the content of leaf-Ca2+ and K+, but degree of efficiency of combined application of Ca2+ and K+ in alleviating the adverse effect of Cd stress on leaf-Ca2+ and K+ content was found to be maximum , peroxidase (POD), and superoxide (SOD) in faba bean plants exposed to Ca2+ and/or K+ under both stress and non-stress conditions are shown in 2+ and K+ individually decreased the activity of CAT and POD except SOD, but they gave a maximum value for these enzymes’ activity when they were applied together. Under Cd stress, application of Ca2+ individually gave maximum value for POD and SOD, but its effect was at par with K+ alone for CAT activity. However, the subsequent application of Cd-stressed plants with the combination of Ca2+ and K+ neutralized the adverse effect of Cd and resulted in considerable improvement in the activities of these three enzymes compared to their individual treatment H oxidases and maintaining photosynthetic electron transport .To meet the objectives mentioned in the introduction, the response of fabe bean  and Cd was determined with the help of Inductively Coupled Plasma Optical Emission Spectroscope .To determine Ca, K and Cd concentrations, we followed the digestion approach of Zheljazkov and Nielson  as modifet al. [Proline concentration was determined spectrophotometrically by adopting the ninhydrin method of Bates et al. . We firsg for 10 min. The absorbance of the supernatant was read at 532 nm and 600 nm against a reagent blank.Malondialdehyde (MDA) content was determined according to the method of Heath and Packer . Leaves g for 20 min at 4 °C. The supernatant was used for the enzymatic assays described below. All enzyme activities were expressed as units mg−1 protein min−1.To determine the enzymatic activities of the antioxidant proteins, a crude enzyme extract was prepared by homogenizing 500 mg of leaf tissue in extraction buffer containing 0.5% Triton X-100 and 1% polyvinylpyrrolidone in 100 mM potassium phosphate buffer (pH 7.0) using a chilled mortar and pestle. The homogenate was centrifuged at 15,000 × 2O2, and 1 mL of the enzyme extract diluted 20×. This was incubated for 5 min at 25 °C, after which the reaction was stopped by adding 0.5 mL of 5% (v/v) H2SO4. The amount of purpurogallin formed was determined by measuring absorbance at 420 nm. A unit of peroxidase activity was the amount of purpurogallin formed per mg protein per minute.We used the method of Chance and Maehly  to deter2O2 was monitored by the decrease in absorbance at 240 nm. For the assay, a 50 mM phosphate buffer (pH 7.8) and 10 mM H2O2 were used.Aebi  method w−2·s−1 for 15 min. The absorbance at 560 nm was read against a blank (non-irradiated reaction solution). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction.The activity of SOD (EC 1.15.1.1) was determined by measuring its ability to inhibit the photoreduction of nitro blue tetrazolium (NBT) according to the methods of Giannopolitis and Ries . The reap < 0.05% level.Each pot was treated as one replicate and all the treatments were repeated four times. The data were analyzed statistically with SPSS-17 statistical software . Means were statistically compared by Duncan’s Multiple Range Test (DMRT) at the 2+ and/or K application in the detoxification of Cd toxicity. These results clearly show that application of Ca2+ and/or K+ decreased in MDA and Cd contents, and increased in photosynthetic pigment, Pro, antioxidant enzymes activity  and accumulation of nutrients (Ca2+ and K+) may be responsible for better growth in terms of RL, SL, RFW, SFW that were responsible for enhanced dry matter production under Cd stress. Therefore, inclusion of Ca2+ and/or K+ in growth medium could be an adequate strategy to alleviate the harmful effects of heavy metal stress and to enhance plant metabolism to perform better under normal as well as different environmental conditions.In summary, results presented above confirm the importance of Ca


The coordination dynamics and coordination mechanism of Et2Cit with Ca2+ ions and the effect of pH on the complex were examined. The result was compared with that for the conventional anticoagulant sodium citrate (Na3Cit). The reaction order (n) of Et2Cit and Na3Cit with Ca2+ was 2.46 and 2.44, respectively. The reaction rate constant (k) was 120 and 289 L·mol−1·s−1. The reverse reaction rate constant (kre) was 0.52 and 0.15 L·mol−1·s−1, respectively. It is indicated that the coordination ability of Et2Cit with Ca2+ was weaker than that of Na3Cit. However, the dissociation rate of the calcium complex of Et2Cit was faster than that of Na3Cit. Increased pH accelerated the dissociation rate of the complex and improved its anticoagulant effect. The Et2Cit complex with calcium was synthesized and characterized by elemental analysis, XRD, FT-IR, 1H NMR, and ICP. These characteristics indicated that O in –COOH and C–O–C of Et2Cit was coordinated with Ca2+ in a bidentate manner with 1 : 1 coordination proportion; that is, complex CaEt2Cit was formed. Given that CaEt2Cit released Ca2+ more easily than Na3Cit, a calcium solution was not needed in intravenous infusions using Et2Cit as anticoagulant unlike using Na3Cit. Consequently, hypocalcemia and hypercalcemia were avoided.Diethyl citrate (Et Sodium citrate (Na3Cit) is an important anticoagulant used in clinical settings  at t time was also calculated. Given that CaCl2 was mixed with Na3Cit or Et2Cit (1 : 1) and that the reaction of Ca2+ with Na3Cit or Et2Cit was equal in solution  with t in reaction system of Et2Cit and Na3Cit with CaCl2 is shown in c(Ca2+) was observed with prolonged t from 0 s to 30 s. This finding indicated that Et2Cit or Na3Cit was rapidly coordinated with Ca2+. At t = 30 s, c(Ca2+) decreased from 1.0 mmol/L to 0.49 mmol/L in the Na3Cit system and from 1.0 mmol/L to 0.87 mmol/L in the Et2Cit system. c(Ca2+) slowly decreased when t > 120 s, indicating that the system was in a dynamic equilibrium of complexation dissociation.The change in concentration of free Cadx/dt) of points on the curve, that is, the reaction rate r of each point formula (r  versus  logc(Ca2+)  , the slo(formula ). The in2Cit and Na3Cit with Ca2+ were as follows:The reaction rate equations of Etk can directly reflect the reaction rate,  in serum, as well as the reduced concentration of free Ca2+ in plasma that disturbed the blood clotting process from reaching the anticoagulation effect in vitro [3Cit, particularly as an anticoagulant, can coordinate a large number of Ca2+ ions in the blood. This phenomenon can lead to the low serum concentration of calcium in patients, as well as to hypocalcemia and all kinds of complications [3Cit [2+ after entering the body in the dialysis process. Additionally, hypercalcemia easily ensued in patients with presupplementary Ca2+. Therefore, the incidence of hypocalcemia and hypercalcemia can be reduced if we can reduce the coordination ability of anticoagulant.The anticoagulant mechanism of Nain vitro –11. Howeications –15. Therns [3Cit . MeanwhiThe reaction rate was equal to the inverse reaction rate when the reaction reached equilibrium, as shown in the following:k is the reaction rate constant, kre is the inverse reaction rate constant, and Ks is the complex stability constant.The above equation can be written as follows :(7)xn, wherein Na3Cit was alkaline. Therefore, when Na3Cit was used as an anticoagulant, the blood pH decreased and metabolic alkalosis likely ensued.In the dialysis process, the pH values of different dialysates varied. The acidities of different anticoagulants also differed. Therefore, the pH of blood in the dialysis process also changed. Considering that Na2Cit molecule only had one –COO−, the possibility of causing alkalosis was significantly reduced when Et2Cit was used as anticoagulant. With increased pH from 6.0 to 8.0, free c(Ca2+) decreased faster in the system , indicating a hydrogen bond.The peak at 3430 cm2Cit split into two peaks, which were 1709 and 1624 cm−1, respectively, indicating two different coordination environments in carbonyl. The position of both peaks red-shifted by approximately more than 30 and 110 cm−1 compared with the carbonyl absorption peaks of Et2Cit at 1736 cm−1. This finding indicated that the carbonyl of Et2Cit was coordinated with the calcium ions and was consistent with the change in the carbonyl characteristic absorption peak before and after coordination, as reported in [The carbonyl absorption peak (C=O) of CaEtorted in .2Cit was at 1100 cm−1. However, the peak split into two in the complex, that is, at 1081 and 1041 cm−1, respectively. This phenomenon was ascribed to one of the three C–O–C groups of the Et2Cit molecular complex with Ca2+, in which C–O–C absorption was bimodal and red shifted.The absorption peak of the symmetric stretching vibrations of (C–O–C) in C–O–C of Et−1 was the absorption peak of the methyl hydrocarbon of CaEt2Cit. It did not significantly change compared with the absorption peak of Et2Cit methyl hydrocarbon (2986 cm−1).The peak at 2982 cmThe FT-IR spectra of Et1H NMR spectra of Et2Cit and CaEt2Cit were studied using CDCl3 as a solvent, and the results are shown in 1H NMR are shown in δ = 7.26 and 6.28 ppm disappeared, indicating that –COOH participated in the coordination reaction. Meanwhile, the hydrogen in –OH group is very active; it can be easily dissociated and be partially or entirely substituted by deuterium in CDCl3 solution.The proton peaks of the ligand at 2Cit quartets were –CH2C=O , this peak of the complex (δ = 4.12 ppm to  4.17 ppm) shifted to a high field. This finding indicated the weakening of the induction effect of attracting electrons of O in –OCH2 from H after the O atom in –OCH2 coordinated with Ca. Thus, the total electron density of H increased, and the absorption peaks moved to a high field.The peak at OOCH2CH3 . CompareThe 1H NMR revealed that Et2Cit formed a 1 : 1 complex with Ca2+, that is, CaEt2Cit.Elemental analysis, ICP, XRD, FT-IR, and 2+ was coordinated with Et2Cit. O in –COO and C–O–C of Et2Cit was coordinated with Ca2+ in bidentate ligand. Two kinds of –OCH2CH3 had different chemical environments in the crystals, that is, 1,3-CaEt2Cit and 1,5-CaEt2Cit. However, their proportions were still difficult to ascertain because of their similar physical and chemical properties. Based on the above characterization results, two kinds of coordination of Et2Cit with Ca2+ are shown in The above results showed that Ca2Cit based on the reason that the FT-IR  of coordination with CaCl2 were 0.52 and 0.15 L·mol−1·s−1 for Et2Cit and Na3Cit, respectively. The dissociation rate of Ca2+ of CaEt2Cit was faster than that of CaCit. The increased pH accelerated the dissociation of the complex. With increased pH from 6.0 to 8.0, kre of Et2Cit-CaCl2 increased from 0.04 to 19.80, which was beneficial in improving the anticoagulant effect. Et2Cit and Ca2+ were coordinated to form a 1 : 1 complex, and O atoms in –COOH and C–O–C of Et2Cit were coordinated with Ca2+ in bidentate ligand. Et2Cit was able to coordinate with Ca2+, and its release capacity of Ca2+ was stronger than that of Et2Cit. Thus, it did not require an intravenous infusion of calcium when used as an anticoagulant, thereby avoiding hypocalcemia and hypercalcemia that can be caused by Na3Cit. Overall, Et2Cit was a better anticoagulant than Na3Cit.The coordination dynamics and effect of Et