Arabidopsis exchanger family members (AtCCX3 and AtCCX5) were previously studied and belong to CaCA  superfamily while none of the rice CCXs has been functionally characterized for their cation transport activities till date. Rice genome encode four CCXs and only OsCCX2 transcript showed differential expression under abiotic stresses and Ca2+ starvation conditions. The OsCCX2 localized to tonoplast and suppresses the Ca2+ sensitivity of K667 (low affinity Ca2+ uptake deficient) yeast mutant under excess CaCl2 conditions. In contrast to AtCCXs, OsCCX2 expressing K667 yeast cells show tolerance towards excess Na+, Li+, Fe2+, Zn2+ and Co2+ and suggest its ability to transport both mono as well as divalent cations in yeast. Additionally, in contrast to previously characterized AtCCXs, OsCCX2 is unable to complement yeast trk1trk2 double mutant suggesting inability to transport K+ in yeast system. These finding suggest that OsCCX2 having distinct metal transport properties than previously characterized plant CCXs. OsCCX2 can be used as potential candidate for enhancing the abiotic stress tolerance in plants as well as for phytoremediation of heavy metal polluted soil.In plant cell, cations gradient in cellular compartments is maintained by synergistic action of various exchangers, pumps and channels. The  Plants require various essential cations for numerous cellular metabolic activities, growth and development. Presence of excess essential ions in the cell leads to ion toxicity while concentration below optimal level leads to ion deficiency symptoms. Therefore, plants have employed wide range of mechanism to uptake mineral nutrients from soil by various transporters presents on the plasma membrane of epidermal root cells. Subsequently minerals from the roots are translocated to different plant organs and tissues for plant growth and development2+) is an essential mineral nutrient as well as a pivotal second messenger in plant cells2+]cyt leads to activation of various signal transduction pathways in the plant cells, which regulates the various cellular mechanisms such as opening and closing of stomatal aperture7811Calcium Ca+ is an e2+ absorbs in root by synergistic action of Ca2+-permeable transporters and translocated to shoot either by apoplastic or symplastic transport102+]cyt is required to regulate various signaling pathways14152+]cyt level is increased to several hundred folds which generates ‘calcium signature’2+]cyt burst is normalized by synergistic action of low capacity, high affinity (Km = 1–10 μM) calcium ATPases and high capacity, low affinity (Km = 10–15 μM) calcium exchangers (CAXs)1317182+-ATPases and CAXs maintain optimum [Ca2+]cyt either by apoplastic export or by sequestering excess Ca2+ to the lumen of vacuole against electrochemical gradient in the activated plant cells1520In soil, primarily Ca2+ (CaCA) superfamily exchangers are calcium transporters, which play an essential role in calcium signaling pathways in many organismsE. coli, a putative Na+/Ca2+ exchanger), NCX (K+-independent Na+/Ca2+ exchangers), NCKX (K+-dependent Na+/Ca2+ exchangers), CAX (H+/cation exchangers) and CCX (cation/Ca2+ exchangers)222+ across various membranes against its electrochemical gradient by utilizing the downhill gradient of other cation species such as H+, K+ or Na+2122242523The cation/CaArabidopsis calcium/cation exchangers (CCXs) were identified as CAX homologs but recently CAX7 to CAX11 were reclassified as CCX1 to CCX5 due to higher homology to mammalian NCKX (K+-dependent Na+/Ca2+ antiporters)+ antiporters that mediates H+-coupled antiport of Ca2+ and other metal ions and recognised as high capacity, low-affinity transporter resulting in the accumulation of these cations in vacuoles212930Arabidopsis) have been studied. AtCCX3 is an endomembrane localized H+ dependent K+ transporter, which suppresses yeast mutants defective in Na+, K+, and Mn2+ transport and therefore, it is a K+ transporter with apparent Na+ and Mn2+ transport properties+ uptake and Na+ transportArabidopsis CCX3 and CCX5 belong to the same family having a few similarities and distinction in cation transport properties322327Arabidopsis CAXs and CCXs.Previously, Arabidopsis CCXs in cation homeostasis while possible functions of rice CCXs are not characterized yet. To understand the functional role of rice CCXs, we have performed detailed expressional analysis of all four rice CCXs in abiotic stresses and calcium deficiency and found that only OsCCX2 showed differential expression under above mentioned conditions. We observed the subcellular localization of OsCCX2 to vacuolar membrane in plant epidermal cells. For functional characterization of OsCCX2, we did complementation analysis of OsCCX2 in heterologous yeast system to ascertain its role in transporting Ca2+, Na+, Li+ and distinct heavy metal ions  in appropriate yeast mutants.Studies in yeast support the role for CCX genes namely OsCCX1, OsCCX2, OsCCX3 and OsCCX4 were identified in rice genome. The probesets of OsCCX1 to OsCCX3 were available while probesets for OsCCX4 were not labelled on microarray chip (Agilent and Affimetrix). To determine the transcript expression profile of OsCCX1-3 under abiotic stresses , publically available rice microarray database, Rice DB:Oryza information portal (http://ricedb.plantenergy.uwa.edu.au/) was used. The OsCCX2 transcript was differentially upregulated under drought and salt stresses while no significant change was observed under cold stress  treated seedling, indicating role of OsCCX2 in calcium signaling and transport  conditions  while down regulated in shoot under P deficiency. OsCCX2 was also down regulated under potassium (K+) deficiency. The expression of other rice CCXs were not much affected in major nutrient deficient conditions. The expression of rice CAX1a, CAX1b and CAX1c were altered in low P and Fe (iron) conditions while transcripts of other rice CAXs were not much affected under different nutrient deficient conditions  was localized throughout the cytosol and the nucleus. GFP fluorescence of either OsCCX2::GFP or GFP::OsCCX2 was shown at vacuolar membrane in transient Agrobacterium mediated transformation of N. benthamiana epidermal peel cells , mitochondria, chloroplasts and cell wall217el cells . To conf2+/H+ exchangers (CAXs), four cation/Ca2+ exchangers, two EF-CAX, two MHX and single OsNCKX123OsCCX2 (LOC_Os03g45370) open reading frame contains 1,728 nucleotides, translating into a putative 60 kDa protein (576 amino acids) and its domain analysis reveals that it is a putative Na+/Ca2+ exchanger protein. Domain structure analysis of OsCCX2 reveals that it has typical 12 transmembrane domains, two Na+/Ca2+ exchanger domains and characteristic α-1 and α-2 motifs, which are considered necessary and sufficient for cation exchange activity. The presence of unique signature motifs GNG(A/S)PD in α-1 and (G/S)(N/D)SxGD motif in α-2 repeat regions232+ ion selectivity, binding, transport of OsCCX2 protein  and 41% protein sequence homology with Arabidopsis CCX1. In phylogenetic analysis, OsCCX2 groups with Arabidopsis and rice CCXs and its closest Arabidopsis orthologs are AtCCX1 and AtCCX2Arabidopsis and rice CCXs suggests possible involvement of OsCCX2 in similar calcium signaling pathways and prompted us to perform in-depth functional characterization to understand the cation/Ca2+ exchange properties of OsCCX2.It shows more than 60% protein sequence homology with cation/Ca2+ and other cations. K667, a low affinity Ca2+ uptake deficient triple S. cerevisiae mutant strain, lacking vacuolar ATPase, PMC1; vacuolar exchanger, VCX1; and cytosolic regulatory CNB1 subunit2+ concentration and is due to defective vacuolar Ca2+ transportPreviously, yeast heterologous expression system approach has been used successfully to decipher the functional role of plant exchanger proteins313233Arabidopsis CCX5 and CCX3 were unable to suppress the high Ca2+ sensitivity of yeast mutant K667, where the later was characterized as endomembrane localized H+ driven K+ transporter32Arabidopsis CCXs but OsCCX2 shares only 41% sequence homology with Arabidopsis CCX1 and lesser sequence similarity with previously characterized CCX3 and CCX5, indicating that OsCCX2 might have distinct transport activity. Most of the Arabidopsis and rice CAXs have Ca2+ exchange activity and are able to complement the Ca2+ sensitive phenotype of K667 mutant. Till date, plant’s CCXs have not been reported to complements yeast K667 mutant strain. To test whether rice OsCCX2 has the ability to suppress the Ca2+ sensitivity of K667, the complete ORF of OsCCX2 was cloned into galactose inducible pYES2 yeast expression vector and expressed in K667 mutant cells along with empty vector as a control. The K667 mutant cells expressing empty vector were unable to grow under excess CaCl2 either in presence of glucose or galactose (2 concentrations (2 (up to 150 mM)  .Arabidopsis CCXs with CAXs revealed that CCXs do not have N-terminal autoinhibitory domain in contrast to CAXs2+ exchange properties like other CAXs.Moreover, multiple sequence alignment of Arabidopsis CAXs and CCXs have broad substrate specificity and can transport a variety of monovalent  as well as divalent  cations. Although some CAXs such as AtCAX1 and Saccharomyces cerevisiae VCX1 (ScVCX1) are specific for Ca2729324142+/Ca2+ and Li+/Ca2+ exchange+ transport properties of OsCCX2, we have performed the complementation assay in K667 yeast mutant at higher concentration of exogenous NaCl. In contrast to empty vector control, OsCCX2 expressing mutant cells grew better on media supplemented with high concentration of NaCl (200–500 mM) (+ hypersensitivity of K667 in a manner similar to the suppression of Ca2+ sensitive phenotype of this mutant (–500 mM) . This res mutant . K667 yes mutant . Empty v2+, Mn2+, Zn2+, Co2+ and Cd2029314546+ and Na+ to divalent cations such as Mn2+ and Zn2+ 32On the basis of phylogenetic analysis of CAXs, type IB CAXs  are involved in transporting other metal ions with broader substrate specificity for divalent cations, such as Ca2+, Zn2+, Co2+ Mn2+ and Cd2+) mediated by OsCCX2, the sensitivity and tolerance of OsCCX2 expressing K667 cells were examined under excess concentrations of above mentioned heavy metals. We found that OsCCX2 expressing K667 yeast cells provide tolerance specifically against excess FeSO4, ZnCl2, and CoCl2 than empty vector control  while mid1 mutant shows impaired growth in presence of galactose at higher concentration of NaCl (upto 500 mM). The galactose inducible pYES2 empty vector expressed in mid1 mutant showed impaired growth at higher concentration of NaCl (500 mM), LiCl (25–100 mM) and FeSO4 (20 mM) in presence of galactose as carbon source and as an inducer of Gal1 promoter  is below 0.2 mg/g dry weight2+ and Na+ ions were in ppm range while total heavy metal Fe2+ and Zn2+ contents were in ppb range as reported previously2+, Na+, Fe2+ and Zn2+ ions than WT and OsCCX2 expressing K667 cells. The wild type strain has lowest Ca2+, Na+, Fe2+ and Zn2+ content than either OsCCX2 expressing K667 cells or empty vector expressing K667 cells under excess metal concentrations  under low Na+-K+ medium+ indicating its unique Li+ ion transport unlike reported for Arabidopsis CAXs and CCXs. As reported previously, Li+ could compete with Na+ transport in yeast cells54GmCAX1 expression is induced by Ca2+, Na+ and Li+ treatmentsArabidopsis plants overexpressing GmCAX1 were more tolerant to elevated Li+ and Na+ levels during germination and suggested that GmCAX1 may function as antiporter for Na+ and Li++ and Li+ transporter similar to GmCAX1. Interestingly, based on the ∆trk1trk2 yeast mutant complementation analysis, OsCCX2 does not have the ability to transport K+ ions like reported for other AtCCXs. Therefore, OsCCX2 have Na+ transport activity like AtCCX5 while in contrast to AtCCX3 and AtCCX5, unable to transport K+ in yeast mutant cells.The OsCCX2 protein complements the NaCl sensitivity of K667 mutant in presence of excess NaCl. It was reported that AtCCX5 showed Na2+, Zn2+ and Co2+ and provide tolerance to K667 mutant. Arabidopsis AtCCX3 and AtCCX5 also transport divalent heavy metal ions such as Mn2+, Zn2+ and Co2+, Cd2+, Li+ ions respectively by unknown mechanism32OsCCX2 shows similar properties like IB type of CAXs, which also transport heavy metals such as Feccc1, zrc1, cot1  under excess of respective metal ions  towards different metal ions transport and indicate its role in Fe2+, Zn2+ and Co2+ ions transport.In contrary to our expectation, OsCCX2 was unable to complement the single metal transport defective yeast mutants tal ions . In a prmid1 mutant for complementation analysis in this study. MID1 is N-glycosylated integral membrane Ca2+-permeable channel required for Ca2+ influx and stimulated by mating pheromone α-factormid1 mutant also show impaired growth in presence of excess NaCl and metal ions when galactose is used as a carbon source. We showed that mid1 mutant expressing OsCCX2 shows more tolerant phenotype in presence of galactose along with excess NaCl, LiCl and FeSO4. At present, we do not know the mechanism of tolerance provided to mid1 mutant by OsCCX2 in above-mentioned conditions but it may be speculated that OsCCX2 might be involved in excess metal ions transport and hence impart tolerance to mid1 mutant.To further validate the cation transport activity in other mutants than K667, we choose 2+, Na+, Fe2+ and Zn2+ ions accumulation is higher in K667 mutant than OsCCX2 expressing K667 mutant when grown in excess of these metal ions. We hypothesize that the higher accumulation of these cations leads to sensitive phenotype of K667 mutant but this notion require further experimental evidences. It was already reported that another yeast triple mutant, K616, where high affinity Ca2+ uptake is compromised, accumulates 8 times higher Ca2+ than WT when subjected to 20 nM external Ca2+5960PMC1, VCX1 and CNB1 genes. It is possible that in absence of functional calcineurin, expression of ENA1 system genes is compromised and therefore lesser efflux of excess cations like Na+, Li+ and other cation through ENA efflux system and hence higher accumulation of cations take place, especially Na+ in K667 mutant.Here, we have shown that total Ca2+, Na+, Fe2+ and Zn2+ in yeast cells. Hence, it leads to survival of OsCCX2 expressing K667 mutant under excess metal ions by some unknown mechanism, which requires further experimental work in near future.Overall, this study suggests that OsCCX2 is involved in providing tolerance to K667 mutant against over-accumulation of Ca2+/cation exchanger family. In contrast to other rice CCXs members, OsCCX2 transcript is highly inducible under abiotic stresses especially drought and salt and also under exogenous application of ABA. The expression of OsCCX2 is downregulated in seedlings grown under Ca2+ deficient conditions and the protein is localized to vacuolar membrane of N. benthamiana epidermal peel cells. The bioinformatic analysis reveals that OsCCX2 is a Ca2+/Na+ exchanger, with 12 transmembrane domains and peculiar cation exchange domains. OsCCX2 suppresses the Ca2+, Na+, Li+, Fe2+, Zn2+ and Co2+ sensitivity of low affinity Ca2+ uptake deficient yeast K667 mutant and Na+, Li+ and Fe2+ sensitivity of mid1 mutant (trk1trk2) yeast mutant suggesting inability to transport K+ ions under low K+ condition . Transcript profiling of rice OsCCXs in Ca2+ deficient conditions was analysed from the microarray expression data of rice seedlingOsCCX4 because probesets for OsCCX4 were not available in rice microarray chips (Agilent and Affimetrix).The microarray expression profiles of all the rice OsCCX2 under abiotic stress conditions and exogenous ABA application showed significant differential regulation, validated by quantitative PCR (qPCR) using two biological replicates. The microarray expression pattern of OsCCX2 under 5 days and 14 days of Ca2+ deficiency and 6 hrs of resupply were also validated by qPCR. The primers of OsCCX2 were designed from 3′ UTR region using PRIMER EXPRESS  with default parameters and analyzed using BLAST tool of NCBI and dissociation curve analysis after the PCR reaction for their specificity  was transformed with OsCCX2-GFP and GFP-OsCCX2 constructs. Overnight grown culture of transformed Agrobacterium (OD600 = 0.5) and helper strain p19 cells (OD600 = 0.3) were pelleted down at 5000g for 15 minutes. Pelleted cells were re-suspended in activation buffer  and kept for 2–4 hours at room tempreture. The re-suspended cells were then used to infiltrate the leaves of 4–6 weeks old N. benthamiana plants. After infiltration, plants were kept for incubation (3–4 days) under 12 h light/12 h dark photoperiodic cycle at 28 °C. Agrobacterium-infiltrated N. benthamiana epidermal peel cells were analyzed in TCS SP5 laser scanning confocal microscope  for sub-cellular localization and co-localization experiments. GFP fluorescence signals were detected at 500 to 535 nm after excitation at 488 nm while mCherry was excited at 543 nm and scanned at 600–630 nm. For co-localization experiments, sequential scanning was done for both the channels and then merged together to shows overlapping signals. All the images were further processed using Leica LAS AF Lite software.Agrobacterium OsCCX2 was amplified from cDNA of 7 day old IR-64 rice seedlings and was cloned into pJET1.2 vector (Fermentas Inc. USA). The cloned gene was confirmed by sequencing. OsCCX2 was subcloned into galactose inducible yeast expression vector, pYES2 (Clontech Inc. USA). For in planta subcellular localization, OsCCX2 ORF was cloned into pGPTVII-GFP.Kan vector to form fusion protein at the N-terminal of GFP (OsCCX2-GFP)Rice Saccharomyces cerevisiae yeast strains used in this study are listed in −1, 10−2 and 10−3 fold dilutions of 0.5 OD600 cultures. 5 μl of each serial dilution was dotted on the SC-Uracil/galactose media with mentioned different concentrations of cations. To observe the growth, plates were incubated at 30 °C for 3 days otherwise mentioned in figures.To test the different cations transport function of OsCCX2 in yeast, OsCCX2/pYES2 along with vector control, were individually transformed into different yeast mutant strains by LiAc/ss carrier DNA/PEG methodet al. (1984) is a Na+- K+ -free minimal synthetic medium frequently used to examine the effect of Na+ and K+ on ∆trk1∆trk2 mutant growth4, 0.2 mM CaCl2, 10 mM MES, 2% glucose, recommended vitamins, trace elements, amino acids and pH 6.5 was adjusted using L- arginine. 1% agarose was used to make solid AP medium plates.Arginine-phosphate (AP) medium, first described by Rodriguez-Navarro 2+, Na+, Fe2+ and Zn2+ were measured using Shimadzu Atomic Absorption Spectrophotometer 6300. Single colony of K601 having pYES2 vector and K667 mutant harbouring pYES2 and OsCCX2/pYES2 cultured in SC-uracil having different concentrations of metal ions  at a cell density of ~2 OD600/ml. The cultures of each set were harvested in pre- weighed microfuge tubes by centrifugation. The cells grown in CaCl2 were washed three times with 10 mM EGTA pH 5.5, while cells grown in other metal ions were washed with 10 mM EDTA and then finally with Milli Q water. After aspirating the supernatant, pelleted cells were dried in oven overnight at 60 °C. The dried samples were weighed again and re-suspended in HNO3 for atomic absorption spectrophotometry.Total CaHow to cite this article: Yadav, A. K. et al. A rice tonoplastic calcium exchanger, OsCCX2 mediates Ca2+/cation transport in yeast. Sci. Rep.5, 17117; doi: 10.1038/srep17117 (2015).


Store-operated calcium channels were activated in a downstream process mediating entry of extracellular calcium. We provide the first detailed analysis of NPSR1 activity and the underlying intracellular pathways with respect to calcium mobilization in neurons.The neuropeptide S (NPS) system was discovered as a novel neurotransmitter system a decade ago and has since been identified as a key player in the modulation of fear and anxiety. Genetic variations of the human NPS receptor (NPSR1) have been associated with pathologies like panic disorders. However, details on the molecular fundamentals of NPSR1 activity in neurons remained elusive. We expressed NPSR1 in primary hippocampal cultures. Using single-cell calcium imaging we found that NPSR1 stimulation induced calcium mobilization from the endoplasmic reticulum via activation of IP In 2004, the 20 amino acid neuropeptide S (NPS) has been identified as the ligand for the formerly orphan g-protein coupled receptor GPR-154  .NPSR1 gene to diseases like asthma and allergy cyt upon NPSR1 stimulation, we used cyclopiaconic acid (CPA) to inhibit SERCA-ATPases  resulted in a transient increase in cytosolic calcium (1.6 ± 1.17), indicating calcium leakage from the ER upon blockade of SERCA  = 684.6, p < 0.0001).To identify the source of calcium leading to an increase in  r U73122  [10μM] t release . The mea2+ release was tested. In the presence of ryanodine [50 μM], the normalized mean fluorescence amplitude evoked by NPS was at 0.51 ± 0.37, a significant reduction compared to responses prior to ryanodine which were at 2.7 ± 1.48  = 337.0, p < 0.0001). Control recordings from the same batch of neurons without ryanodine treatment showed that the normalized mean fluorescence amplitude was reduced from 2.35 ± 1.9 for a first application of NPS to 1.74 ± 1.4 when NPS was applied again  = 111.4, p < 0.0001). Taken together, the amplitude of the response evoked by a second NPS application reached 0.77 ± 0.35 when normalized to the first application, while this value was reduced to 0.24 ± 0.25 when RyRs were blocked  to NPSR1-mediated Ca2+. The response amplitude was integrated over time for the slow component of the calcium signal in order to quantify the Ca2+ influx. The integral was significantly decreased from 36.6 ± 8.3 x 104 (arbitrary units) under control conditions to 23.4 ± 3.9 x 104 in Ca2+-free solution . In contrast, the duration and amplitude of the slow component was drastically reduced . This in2+. In most cells, calcium release from the ER and the according fall in ER-luminal Ca2+ concentration subsequently activates calcium channels in the cell membrane in a process called store-operated calcium entry (SOCE) [2+ to initiate SOCE (2+ (4 (arbitrary units) for a first application of NPS under calcium-free conditions to 2.1 ± 3.2 x 104 for a control application of Ca2+-free extracellular solution without NPS. After applying NPS under Ca2+-free conditions once again, the integral was significantly increased to 5.9 ± 4.9 x 104  = 67.16, p < 0.0001).These results suggested that calcium entry pathways located in the plasma membrane largely contributed to the second, slower component of NPSR1-evoked increases in cytosolic Cay (SOCE) . Next, wate SOCE . To quanSOCE (2+ . We foun4 (arbitrary units) for control conditions to 27.2 ± 5.7 x 104 in experiments with the drug  = 87.46, p < 0.0001). In presence of SKF96365, a reduction to 30.42 ± 3.8 x 104 could be observed  to the NPS-evoked calcium response was tested. Using a combination of Cav antagonists , we found the NPS-triggered calcium signal unaffected compared to control in 148 neurons (4 (arbitrary units) for control conditions and 37.4 ± 7.8 x 104 in presence of the Cav blockers, t-test: p = 0.2048).Finally, a possible contribution of voltage activated calcium channels (Ca neurons . The intin vitro in order to study intracellular mechanisms triggered by NPSR1 activation. We provide evidence that (i) NPSR1 can be functionally expressed in mouse neurons, (ii) application of NPS leads to calcium release from the endoplasmic reticulum via IP3 and ryanodine receptors in NPSR1 expressing neurons and (iii) these calcium release mechanisms in turn activate SOCE, thereby inducing influx of extracellular calcium.In this study, we used heterologous expression of the human NPS receptor in mouse neurons Human NPSR1-mCherry expressed after adenoviral transfection displayed punctual localization across the soma and dendrites of transfected neurons. A similar distribution has been shown for the G-protein coupled receptors (GPCRs) neurokinin1 and somatostatin receptor using immunohistochemistry ,41. In sin vitro studies, NPSR1 has been expressed in HEK293 or CHO cells [q and Gαs activity, i.e. their efficiency to induce intracellular calcium responses and cAMP production upon receptor stimulation, analyzing population responses from plate readers instead of confocal single-cell calcium imaging. The results of the present study extend these previous findings by providing a detailed characterization of the molecular signaling pathways and mechanisms underlying NPSR1-mediated Ca2+ mobilization in individual neurons with single-cell resolution. The cultured hippocampal neurons used represent a well-established and well-characterized expression system. They seem particularly suited for our study since untransfected hippocampal neurons proved to be non-responsive to NPS in our control experiments. Based upon our results, a model of NPSR1-coupled signaling pathways can be constructed  [3 receptor activation was amplified by subsequent activation of ryanodine receptors.In our experiments, blockade of RyRs by ryanodine significantly reduced the amplitude of the NPS-evoked calcium response, unmasking a second calcium release pathway from the endoplasmic reticulum. When IPe (CICR) . Taken tq-mediated release of calcium from the endoplasmic reticulum is followed by the activation of calcium channels at the plasma membrane, known as store-operated calcium entry (SOCE) [2+-free extracellular solution to isolate these calcium routes and found the fast component of the NPSR1-mediated calcium signal unaffected while the slow component was abolished under these conditions. Re-addition of Ca2+ restored the slow, extracellular component of the NPS-induced Ca2+ signal while re-addition of Ca2+ following application of Ca2+-free solution without co-application of NPS did not induce influx of extracellular Ca2+. These results show that Ca2+ mobilization from intracellular stores, i.e. a decrease of the ER calcium content, was a prerequisite for generation of the slow component of NPSR1-mediated Ca2+ signals, strongly suggesting that NPSR1 stimulation specifically activates SOCE in a downstream process.In many cell types, Gαy (SOCE) . We used2+ signals evoked by NPS application in the presence of ML-9 or SKF96365 were similar to those recorded under calcium-free conditions, abolishing calcium influx from the extracellular solution. An obvious explanation is that these drugs blocked signal transmission from the ER lumen to the plasma membrane via STIM, thereby inhibiting SOCE. In case of ML-9, washout of the drug increased the cytosolic calcium content to a level similar to control conditions, presumably because the ML-9-blocked signal transmission from the ER lumen to the plasma membrane via STIM was restored, resulting in SOC activation and Ca2+ influx from the extracellular solution. However, the question whether NPSR1-mediated SOCE comprises activation of ORAI or TRPC channels remains to be elucidated.Store-operated calcium channels (SOCs) responsible for SOCE are known to be composed by the calcium release-activated calcium modulator 1 (ORAI)  and membThe NPS system is known as a key player in the modulation of fear and anxiety in both humans and rodents ,21. The NPSR1 gene (rs324981) that codes for an amino acid exchange at position 107 (A107I) is more frequent in panic disorder patients [NPSR1 polymorphisms at the molecular level and, considering the proposed clinical background of NPSR1, might provide new insights in the intracellular mechanisms involved in the generation of anxiety and anxiety disorders.In human studies, it has been shown that a single-nucleotide polymorphism (A>T) of the patients –21. Morepatients . A more patients . The vir


In this way, global phenomena, such as propagating and abortive Ca2+ waves, as well as local events such as puffs, have been observed. Several experimental studies suggest that many features of global phenomena  depend on the interplay of biophysical processes such as diffusion, buffering, efflux and influx rates, which in turn depend on parameters such as buffer concentration, Ca2+ pump density, cytosolic IP3 level, and intercluster distance. Besides, it is known that cells are able to modify some of these parameters in order to regulate the Ca2+ signaling. By using a hybrid model, we analyzed different features of the hierarchy of calcium events as a function of two relevant parameters for the calcium signaling, the intercluster distance and the pump strength or intensity. In the space spanned by these two parameters, we found two modes of calcium dynamics, one dominated by abortive calcium waves and the other by propagating waves. Smaller distances between the release sites promote propagating calcium waves, while the increase of the efflux rate makes the transition from propagating to abortive waves occur at lower values of intercluster distance. We determined the frontier between these two modes, in the parameter space defined by the intercluster distance and the pump strength. Furthermore, we found that the velocity of simulated calcium waves accomplishes Luther’s law, and that an effective rate constant for autocatalytic calcium production decays linearly with both the intercluster distance and the pump strength.The functional properties of inositol-triphosphate (IP In order to control this variety of functions, calcium is precisely regulated in space and time. In cells that are not electrically excitable, calcium is stored in the endoplasmic reticulum (ER). Changes in the intracellular Ca2+ concentration are due fundamentally to the exchange between the cytosol and the ER. In this way, part of the calcium stored in the ER can be released by a variety of channels that form a set of sensory and release mechanisms. In particular, the inositol-triphophate receptor (IP3R) displays an autocatalytic amplification, since it is active when the IP3 and only one Ca2+ are bound to the receptor. This mechanism is called calcium-induced-calcium-release (CICR), since low Ca2+ levels in the cytosol favor channel opening. The receptor becomes inactive when a calcium ion is bound to the inhibitory binding site, rendering a highly nonlinear behavior. The cytosolic Ca2+ is removed by energy-dependent pumps, such as the sarco-endoplasmic reticulum ATPases (SERCAs), to be stored in the ER.Cytosolic-free calcium  of several Ca2+ channels in a cluster, an event named “puff”, as observed in early experiments  is the Ca2+ concentration in the cytosol, which can diffuse with diffusion constant D. channelJ is the calcium flux from the ER to the cytoplasm through the IP3R channels, leakJ is the calcium flux leakaged from the ER to the cytoplasm, and pumpJ is the calcium flux pumped by the SERCAs from the cytoplasm into the ER. The expressions for the fluxes are given bywhere ER is the Ca2+ concentration in the ER, γ1 is the maximal Ca2+ fluxes per channel, openN is a random variable that represents the number of open channels, and γ0 is the basal permeability of the ER membrane in the absence of IP3. The parameter 2+ efflux from the cytoplasm into the ER. Note that we do not make a distinction between the efficiency of the pump and the SERCA density; both features are represented by the parameter 2+ exchange with the extracellular medium since it is much smaller than the Ca2+ flux across the ER membrane [2+]ave = ([Ca2+] + rv [Ca2+]ER)/(1 + rv). The [Ca2+]ave is a control parameter whose value we can choose, but it is not a dynamical variable. We then define C = [Ca2+]/[Ca2+]ave and rewrite Eqs. where shold q^ . We neglmembrane . For thiolume, Ca+ER is tholume, Ca+ER is thx = 0.18 μm and Δt = 0.0005 s. The parameters of the model are given in p in the range 1.2 and 3.0 μM s-1.where 3 receptor is modeled by a stochastic version of the Othmer and Tang (OT) model  along the one-dimensional space  is represented in a gray scale.We assume that the Cad and the pump strength p in calcium dynamics, we perform 80 simulations varying these parameters and keeping the remaining one constant. The value of d, which varies between 1.6 and 3.7 μm  profile with eight-bit resolution for subsequent analysis with ImageJ program  μm/s, in the same manner as in [We also estimate the velocity of propagating waves by measuring the angle +2 release are limited to a region of the parameter space spanned by d and p. In this way, when the distance between clustered IP3Rs is shorter than 1.6 μm and the intensity of calcium uptake by SERCA is smaller than 1.2 μM/s, the high availability of Ca+2 observed in the cytosol prevents the identification of discrete Ca+2 release events. For distances longer than 3.7 μm and pump strength greater than 3.0 μM/s we observed a very small number of isolated events and the statistic is extremely poor. For this reason, we consider d ranging from 1.6 and 3.7 μm, and p between 1.2 and 3.0 μM/s.We observed that the events of Cad = 2.5 μm and two different values of p. From these profiles we can distinguish the presence of different kinds of release events: for a small value of p a variety of events, both local and global, can be observed , we compute the total number of waves and their durations, in order to obtain a more quantitative understanding of the events of calcium release presented by our model. We note that for the range of parameters studied here, abortive and propagating waves coexist in the same simulation, in agreement with experimental results [Since the number of detected puffs is in general one or two orders of magnitudes greater than the number of propagating or abortive waves , from which the number of events decreases. Also, the value of dmax decreases with increasing p, as can be seen in d (not shown). On the other hand, the intercluster distance d also affects the wave duration. In d depends strongly on the pump strength: for d ≥ 1.8 μm and small values of p (p ≤ 1.4 μM/s) the wave duration decreases monotonically with the intercluster distance, whereas for higher values of p this behavior is not monotonic with d.d, the propagating waves are more abundant than the abortive waves, whereas for higher values of d the situation is inverted. A similar situation was found with respect to the pump strength, since an increase in p favors the presence of abortive waves. In this way, we can identify a region of the parameter space dominated by propagating waves  and another region dominated by abortive waves . The points where wna = wn define a frontier between these regions, as can be seen in T criteria (solid line) in the characterization of the release events. Notice that the two different criteria used to characterize the type of calcium release events show good agreement to define the frontier. Also, we can see that by increasing p, the transition between the propagating region and the abortive region occurs at lower values of intercluster distance.When comparing the number of propagating and abortive waves, we observed that for small values of T, for the different events observed in our simulations as a function of d and p. From the comparison of 2+ released, almost independently of the parameter value. Also, whereas d and p, the CaT related to both puffs and abortive waves  as a function of the intercluster distance d, and the pump strength p. We found that the velocities are in the range 6.6 μm/s < wv < 9.0 μm/s. Also, we observed that the velocity of wave propagation decreases with the parameters d and p, in agreement with other theoretical studies [wv, the diffusion coefficient D, and the apparent first-order rate constant for autocatalytic calcium production k,Finally, we studied the effect of the parameters  studies , 11, 14. studies , 35. Fola is a dimensionless constant. Therefore, by adjusting a linear model to k obtained from the square calcium wave velocity k decreases with both the intercluster distance d and the pump strength p, according to: k = 4.30 - 0.44d - 0.49p, where we used a = 1 following [where city vw2 , we are ollowing .3R clusters by considering a partial differential equation for the Ca2+ dynamics in the cytoplasm and a Markov-stochastic kinetic model for the IP3R channels. The proposed model allows the simulation of global calcium release events such as abortive and propagating waves. Our main aim is to study how several features of the calcium events, such as: duration, wave propagation velocity, and number of events, are modulated by two relevant parameters for the calcium dynamics, the intercluster distance and the efflux rate through SERCA pumps.In the present work we propose a hybrid model of spatially distributed IPd and p. This fact indicates that an excessive amount of Ca2+ in the cytosol, due to small intercluster distances or low values of pump strength, prevents an orchestrated calcium release needed for the wave initiation and propagation. On the other hand, for high values of d and p, the initial trigger signal decreases before inducing the CICR mechanism, diminishing the probability of wave formation. In the region between these two extreme conditions, where global events are observed, we found a frontier between two regions: one where propagating waves are more abundant than abortive waves, and the other region where abortive waves prevail (et al. [The results of our simulations showed that waves are observed in a specific region of the parameter space spanned by  prevail . We foun (et al.  also stuμm/s < wv < 9.0 μm/s, in agreement with some experimental results in fertilizing eggs [d and p (Xenopus oocytes supporting that the velocity of calcium waves increases with the pump strength [et al. [2+ content in the ER. According to these authors [et al. [2+ in the intracellular Ca2+ oscillations points out that the fast recovery of Ca2+ in the ER lumen seems to be due to Ca2+ buffers rather than to the effect of Ca2+ pumps [From our model we found that the velocities of calcium waves are in the range 6.6 ing eggs . Also, w[d and p , accordi2+ pumps , 38.et al. [2+ coefficient diffusion in different deterministic models. For large intercluster distances they found that the speed of calcium waves is proportional to the diffusion constant, whereas for small distances it is proportional to 2+ release, whereas the latter is related to many sites that simultaneously release Ca2+. In the present work, we assumed that calcium wave velocity is proportional to 2+ simultaneously. Therefore, we are able to apply Luther’s law to the propagating calcium waves obtained from our model. Luther proposed a phenomenological equation that establish a relationship between the velocity of a traveling wave and both the diffusion coefficient of the propagator species and an apparent first-order rate constant for the autocatalytic production of the propagator [Keizer et al.  have stuopagator , 35. In opagator , 40, andopagator , 36. HerSumming up, we believe that the use of hybrid models, which take into account stochastic effects due to the random opening and closing of calcium channels, can offer new insights into the nature of calcium signaling, especially in the context where the hierarchy of different size events is the focus of the modeling.


The optimum frequency of ultrasound was determined by 3--2,5-diphenyltetrazolium bromide assay. The apoptotic rate, reactive oxygen species concentration and decreased mitochondrial membrane potential (MMP) were analyzed by flow cytometry. Morphological changes were detected by a transmission electron microscope, and the concentration of intracellular Ca2+, [Ca2+]i, was detected by a confocal laser scanning microscope. In addition, the release of cytochrome c (cyt-c) was measured by western blot analysis. The results revealed that an increased apoptotic effect was induced by sonodynamic therapy (SDT), and this was found to correlate with the overloaded [Ca2+]i, derived from the intra- and extracellular sources in the early apoptotic process. The results also revealed an increased level of ROS production, a decreased MMP and an increased release of cyt-c. The present study indicated that low-level ultrasound in combination with HMME improved the apoptotic effect in C6 glioma cells. The overloaded [Ca2+]i was involved in the mechanism by which apoptosis was stimulated and enhanced by SDT.The present study investigated enhancement of apoptosis induction and the mechanisms underlying calcium overload on C6 glioma cells  Previous studies have indicated that ultrasound in combination with porphyrins may induce apoptosis in glioma cells , calcium overload and mitochondrial damage –12. The g others –16. Calcg others ,18. Honds  and the release of cytochrome c (cyt-c). The apoptotic rate and concentration of [Ca2+]i was determined following SDT-HMME treatment. The L-type Ca2+ channel antagonist nimodipine was added to cells prior to SDT to detect the source of overloaded [Ca2+]i.Accordingly, in this study we hypothesized that low-level ultrasound in combination with HMME may increase the apoptotic rate and the concentration of i was measured using a confocal laser scanning microscope  as described previously .The concentration of .The production of intracellular ROS was assayed by flow cytometry using DCFH-DA as previously described . After 2The loss of MMP was detected by flow cytometry using Rhodamine 123 as previously described . After 2c release, western blot analysis of cyt-c in the cytosolic fraction was performed as previously described . Actin expression was used as the loading control.To quantify cyt-escribed ,21. AfteData are expressed as the mean ± standard error of the mean. Comparisons between the different groups were performed via factorial design analysis of variance using SPSS, version 11.0 . P<0.05 was considered to indicate a statistically significant difference.2. The optimum experimental frequency was determined by MTT assay according to the cell viability. As shown in Cells were exposed to pulsed-wave ultrasound with an irradiation time of 60 sec and an average intensity of 1.0 W/cm2+ probe. The concentration of [Ca2+]i was increased significantly in the ultrasound group with PBS, HBSS, nimodipine and HBSS-nimodipine solution, and further increased in the SDT groups with the same medium , a higher elevation of [Ca2+]i was observed in the SDT-HBSS group . In addition, no significant difference was identified in the elevated concentration of [Ca2+]i between the SDT-HBSS-nimodipine (404±12.1 nM) and the SDT-HBSS group (408±11.6 nM) (P>0.05), which was the same as in the ultrasound-HBSS-nimodipine (181±16.2 nM) and the ultrasound-HBSS group (171±5.5 nM) (P>0.05).The concentration of intracellular free calcium was recorded by a confocal laser scanning microscopy for 30 min in a single cell with the fluorescent probe fluo-3/acetoxymethylester, a sensitive Ca2+]i, apoptotic rates were determined in groups with overloaded [Ca2+]i by flow cytometry. Cells were divided into control, HMME, ultrasound and SDT groups in PBS and HBSS solutions. The SDT and ultrasound groups in the two types of solution exhibited a significant increase in the apoptotic rate  and ultrasound-HBSS (29.6±1.3%) groups ; c was measured by western blot analysis. The release of cyt-c was upregulated in the SDT-PBS, SDT-HBSS, ultrasound-PBS and ultrasound-HBSS groups  group and further increased in the SDT-HBSS (59.9±3.2%) group following SDT, which was higher than that in previous studies whereby apoptotic rate was <40% in C6 glioma cells . This indicated that the [Ca2+]i overload was involved in the SDT treatment. In order to investigate the source of the overloaded [Ca2+]i, the cells were preincubated with HBSS buffer (containing 1.3 mM Ca2+). The results revealed a further elevation in [Ca2+]i concentration in the SDT-HBSS group. Therefore, the increased [Ca2+]i was resultant not only from internal store release, but also from extracellular medium influx during early apoptosis (2+]i was from the intracellular or extracellular environment (et al (2+]i was constant in the SDT-PBS/HBSS and ultrasound-PBS/HBSS groups when the L-type voltage-dependent calcium channel antagonist, nimodipine, was added. This indicated it was ineffective at regulating the concentration of [Ca2+]i by SDT treatment. Accordingly, the results indicated that the Ca2+ influx may be mediated via mechanisms other than voltage-dependent Ca2+ channels, including promotion of membrane permeability and membrane perforation, among others (2+]i concentration and exhibited the highest apoptotic rate. The higher the overload of [Ca2+]i, the higher the apoptotic rate following SDT treatment. Hence, the increased [Ca2+]i from intra- and extracellular sources contributed to the apoptosis induced by SDT.Calcium ions are important in living cells and are the key regulators of cell proliferation and death . A crucironments . Within chondria . Any chapoptosis –12. The poptosis ,21,24. Tironment ,18. Thisg others ; howeverin vitro and in vivo (c, apoptosis-inducing factor and Smac/Diablo were transmitted from the intermembrane space into the cytosol, which has been defined as an early stage of apoptosis, and then caspase-9 and-3 were activated, carrying out the irreversible apoptotic process (c. Therefore, mitochondrial damage was involved in the apoptotic process under the SDT parameter of the present study. In addition, the mitochondria acted as calcium buffers by sequestering excess calcium from the cytosol. Excessive calcium load to the mitochondria may induce an apoptotic program by stimulating the release of the apoptotic promoting factors from the mitochondrial intermembrane space to cytosol and by impairing their function. The results also revealed that the more ROS production and [Ca2+]i were increased, the more the MMP was decreased and the apoptotic rate was increased. Thus, the increased concentration of [Ca2+]i and the ROS production followed by the decreased MMP and the release of cyt-c may explain the higher apoptotic rate following SDT.Previous studies have revealed that ROS production and mitochondrial damage were associated with the apoptosis of C6 glioma cells following SDT ,17,18,24 in vivo ,13–16. W process ,21,23. W2+]i concentration from intracellular and extracellular sources, increased ROS production, a decreased MMP and the release of cyt-c. This effect may be applied to treat C6 glioma cells to cause an increased apoptotic effect. Low frequency and low intensity ultrasound with HMME improved the apoptotic effect in glioma cells. The overloaded [Ca2+]i was involved in the mechanism by which apoptosis was stimulated and enhanced by SDT.In conclusion, SDT under low-level ultrasound and HMME resulted in an increased [Ca


CCL28-induced growth modulation was used as marker of chemokine responsiveness, and 38 patients were then classified as chemokine-responsive. The effects of exogenous CCL28 (growth inhibition/enhancement/no effect) thus differed among patients and was also dependent on the presence of exogenous hematopoietic growth factors as well as constitutive AML cell cytokine release. The effect of CCR1 inhibition in the presence of chemokine-secreting mesenchymal stem cells also differed among patients. Chemokine-responsive AML cells showed altered expression of genes important for (i) epigenetic transcriptional regulation, particularly lysine acetylation; (ii) helicase activity, especially DExD/H RNA helicases; and (iii) angioregulatory proteins important for integrin binding. Thus, chemokine responsiveness is part of a complex AML cell phenotype with regard to extracellular communication and transcriptional regulation. Chemokine targeting in chemokine-responsive patients may thereby alter AML cell trafficking and increase their susceptibility toward antileukemic treatment, e.g., conventional chemotherapy or targeting of other phenotypic characteristics of the chemokine-responsive cells.Acute myeloid leukemia (AML) is an aggressive and heterogeneous bone marrow malignancy, the only curative treatment being intensive chemotherapy eventually in combination with allogeneic stem cell transplantation. Both the AML and their neighboring stromal cells show constitutive chemokine release, but chemokines seem to function as regulators of AML cell proliferation only for a subset of patients. Chemokine targeting is therefore considered not only for immunosuppression in allotransplanted patients but also as a possible antileukemic strategy in combination with intensive chemotherapy or as part of disease-stabilizing treatment at least for the subset of patients with chemokine-responsive AML cells. In this study, we characterized more in detail the leukemia cell phenotype of the chemokine-responsive patients. We investigated primary AML cells derived from 79 unselected patients. Standardized  Acute myeloid leukemia (AML) is an aggressive disease characterized by infiltration of malignant myeloblasts in the bone marrow; the only curative treatment is intensive chemotherapy potentially in combination with allogeneic stem cell transplantation . Howeverin vitro studies suggest that chemokines function as growth regulators in leukemic hematopoiesis only for a subset of AML patients, and a wide range of both CCL and CXCL chemokines can then modulate leukemia cell proliferation . Samples were collected after written informed consent. AML blasts were derived from 79 consecutive patients . Six patients had AML relapse Table .Prior to the study, we decided to include 80 unselected patients ; based on a previous study , we woul9/L) of leukemia cells among the peripheral blood leukocytes (The study included only patients with a high percentage (>80%) and a high absolute number (>15 × 10ukocytes . A cell ukocytes . Cells wukocytes .3H-thymidine incorporation (3H-thymidine incorporation) in transwell cocultures with bone marrow mesenchymal stromal cells  , and all recombinant cytokines were supplied by PeproTech . All exogenous cytokines were added at 20 ng/mL, i.e., corresponding to an excess of the added cytokine. Our methods for flow-cytometric characterization of AML cell viability . Amount and quality of the biotin-labeled cRNA were controlled both by NanoDrop spectrophotometer and Agilent 2100 Bioanalyzer, before 750 ng of biotin-labeled cRNA was hybridized to the HumanHT-12 V4 Expression BeadChip according to the manufacturer’s instructions. The chip targets 47,231 probes derived primarily from genes in the NCBI RefSeq database (Release 38).U-test was used to compare value distributions among different patient groups, and χ2 tests (Pearson’s χ2 test and the likelihood ratio) were used to determine correlations between different categories. Additionally, Kendall’s tau-b correlation tests were performed to determine the agreement between cytokine expression levels in cell supernatants and the mRNA levels of these cytokines. P-values <0.05 were regarded as statistically significant.Bioinformatical analyses were performed using the J-Express 2012 software . For hierarchical clustering of the cytokine secretion, all values were median-normalized and log(10)-transformed. Complete linkage and Pearson correlation were used as linkage method and distance measurement, respectively. The statistical analyses were performed with the IBM Statistical Package for the Social Sciences (SPSS) version 23 . The Mann–Whitney in vitro culture in medium alone . The presence of 20 ng/mL exogenous CCL28 during culture did not significantly alter AML cell viability  when comparing the overall results (data not shown). Thus, exogenous CCL28 does not have any major effect on the regulation of spontaneous in vitro apoptosis for primary human AML cells.There was a wide variation among the 79 AML patients with regard to the viability of the leukemic cells after 40 h of 3H-thymidine incorporation assay, where the nuclide was added after 6 days and nuclear activity assayed 24 h later. A median value of at least 1,000 counts per minute (cpm) in triplicate cultures was defined as detectable proliferation, whereas a significant alteration of 3H-thymidine incorporation was defined as (i) an increase/decrease corresponding to at least 20% of the control culture, and the absolute value of this change being at least 2,000 cpm for patients with detectable proliferation or alternatively, (ii) a change from detectable to undetectable proliferation or vice versa , stem cell factor (SCF), or FMS-like tyrosine kinase 3 ligand (Flt3L). A detectable proliferative response for at least one of these three growth factors was detected for 42 of the 56 patients, and the results for these patients are summarized in the hierarchical cluster analysis presented in Figure CCL28 can bind to the CCR3 and CCR10 receptors –15. We u6 cells/mL, 48 h of in vitro culture). Detectable CCL28 release was observed for only 18 patients , whereas the other chemokines were released for most patients (Table in vitro apoptosis), differentiation , karyotype, and Flt3 or NPM1 mutations (data not shown). Finally, there was no significant association between constitutive CCL28 release and spontaneous/autocrine in vitro AML cell proliferation, and constitutive CCL28 release was seen both for patients with and without CCL28-associated growth modulation.The constitutive release of 11 chemokines was investigated for all 79 patients . The chemokine levels for each individual patient was median-normalized and log(10)-transformed prior to Pearson clustering in J-Express. The analysis is presented in Figure It can be seen from Figure Bone marrow failure with pancytopenia in peripheral blood is common at the time of diagnosis for AML patients . Furtherin vitro model, where primary human AML cells and normal bone marrow MSCs derived from a healthy donor were separated by a semipermeable membrane during coculture  of <0.01. The identified genes  as an immunosuppressive treatment. GVHD has antileukemic effects, and immunosuppression due to severe GVHD will reduce this effect. However, at least for patients with chemokine-responsive AML cells, the use of chemokine targeting may represent a therapeutic alternative that reduces the GVHD-associated antileukemic reactivity but at the same time has an additional antileukemic effect by itself against residual AML.Chemokine receptor blockers can be used as an immunosuppressive strategy, e.g., as GVHD therapy in AML patients receiving allogeneic stem cell transplantation . ChemokiAcute myeloid leukemia is a heterogeneous and aggressive disease, and in a previous study of another smaller patient population, we observed that exogenous CCL28 could modulate cytokine-dependent AML cell proliferation for a minority of patients. For this patient subset, CCL28 was one out of several chemokines with growth-modulating effect . SeveralCCL28 is a chemokine involved in normal hematopoiesis . We now We included 79 patients in the present study, and they represent a consecutive and thereby unselected group, except that all of them showed relatively high levels of circulating AML cells. This strategy was used to allow a standardized preparation of highly enriched AML cells by gradient separation alone, thereby avoiding the risk of inducing functional alterations in the cells by more extensive separation procedures . The usein vitro culture; (ii) an intermediate subset of clonogenic progenitors (often <1%); and (iii) a small minority of AML stem cells  a majority of more mature cells undergoing stress-induced apoptosis during em cells , 26. Stus often <%; and  in a more physiological model of AML/MSC cocultures; this model was used to study chemokine effects in a local cytokine network with a major influence of bone marrow stromal elements. Our results show that even in a cytokine environment formed by the constitutive cytokine release and the cytokine-mediated cross talk between primary AML and bone marrow MSCs, the CCR1/CCR3-mediated effects on AML cell proliferation are divergent, and the different effects of CCR1/CCR3 inhibition on AML cell viability and proliferation can be explained by the hierarchical organization of the AML cell population . The viain vitro, studies in chronic lymphocytic leukemia (CLL) have shown that altered regulation of in vivo cell trafficking can contribute significantly to the antileukemic effects of certain drugs  showed an altered expression of genes included in the GO-terms lysine acetylation/integrins/helicases. As discussed above, there is a functional interaction between CCL28 and integrins, but there is also a biological interaction between the integrin system and lysine acetylation. First, histone acetylation is important for regulation of gene expression , includiHelicases are also important for transcriptional and translational regulation and thereby seem to represent a functional overlap with lysine acetylation. First, a major part of the helicases with altered expression are members of the DEAD box family of RNA helicases, they belong to the helicase superfamily 2 together with the DEAH helicases and are often referred to as DExD/H box RNA helicases , 53. Thein vivo models  to see whether chemokine targeting alters AML cell trafficking/homing and thereby increases their chemosensitivity.Chemokines have growth-modulating effects on the AML cells only for a minority of patients; and these patients are then responsive to several chemokines. However, this chemokine responsiveness is only a part of a more extensive leukemia cell phenotype that includes several partly interacting cellular functions, i.e., altered extracellular communication (chemokines/integrins/acetylation) and transcriptional regulation  compared with the other non-responsive AML cells. Furthermore, the direct effects of chemokine targeting on primary human AML cells are difficult to predict because they depend both on differences among patients and on the overall local cytokine network; this is probably true both if chemokine targeting is tried as an antileukemic strategy or as an approach to treat immune-mediated complications in allotransplanted AML. A careful evaluation of the AML cells will possibly be important to avoid the risk of leukemia enhancement and thereby increased relapse risk; this would probably be important if the treatment is tried both as an antileukemic and an immunosuppressive strategy. A possible approach to avoid this risk may be combined targeting of other aspects of this complex phenotype. However, based on the lessons from recent studies in CLL where chemokines are important for the supportive function of neighboring stromal cells by keeping the CLL cells within their permissive microenvironment, one would emphasize that AML cells from chemokine-responsive patients should be further tested in relevant ØB conceived and designed the experiments; AB performed the experiments; AB and HR analyzed the data; and ØB and AB wrote the paper.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.


Corticosteroids may reduce the scavengers thatplay a crucial role for nerve regeneration, thus delay the recovery of thisdisease.Corticosteroids have been proved to be ineffective for Guillain-Barrésyndrome, but the mechanism remains unknown. In a rabbit model of axonalGuillain-Barré syndrome, treatment with corticosteroids significantlyreduced macrophage infiltration in the spinal ventral roots and the survival rate aswell as clinical improvement. On 30 Clinical trials have demonstrated that corticosteroids treatmentcannot benefit the recovery of patients with GBS13We hypothesized that corticosteroids inhibit the migration of macrophage into the injuredperipheral nerve and delay the recovery of GBS. In this study, we explored the effect ofmethylprednisolone on macrophage infiltration and clinical improvement of AMANrabbits.There was no significant difference in any of the baseline characteristics betweentwo groups of rabbits: inoculation times, days from first inoculation to onset,clinical score at onset and body weight .In experiment 1, macrophage infiltration was significantly decreased in themethylprednisolone group than the saline group ;whereasth day, one died on the 15th day and onedied on the 25th day. The autopsy findings showed gastrointestinalhaemorrhage in two of the rabbits who died on the 5th day. An obviouscause of death in the other rabbits could not be found. The life-table method showedsignificantly fewer survival in the methylprednisolone group than the saline group(p = 0.04)  (p < 0.001)  died within 30 days afterdisease onset. After the initiation of methylprednisolone treatment, three rabbitsdied on the 5 = 0.04) . On the = 0.01) . The fre< 0.001) .Corticosteroids are the most commonly used drugs worldwide for autoimmune diseasesbecause of its cost effectiveness. However, the application of corticosteroids inthe treatment of GBS remains to be disappointing for long time. Observationalstudies have demonstrated that steroids treatment has no beneficial effect on GBSduring the past decades1Anti-GM1 and anti-GD1a IgG antibodies cause complement-mediated disruption of Navchannel clusters at the nodes of Ranvier in the rabbit and mouse models of AMAN5Macrophages engulf and digest the cellular debris and foreign microbes, being dividedinto a killing/inhibitory type (M1 macrophage) and a heal/growth promoting type (M2macrophage)Previous clinical trials showed that a short course of high-dose steroids given alonein early stage of GBS was ineffective111121113In conclusion, corticosteroids inhibit the recruitment of scavengers, which arehelpful for the nerve regeneration, resulting in the delay of clinical improvementin GBS.AMAN rabbits were produced as previously describedth day, the rabbits were perfused transcardially and theventral roots of their lumbar cords were prepared as described elsewhereIn methylprednisolone group (n = 3), methylprednisolone wasinjected into the AMAN rabbits through the ear vein at a dose of 7 mg/kgper day for a total of five days. The dosage of methylprednisolone wascalculated according to the dose used in previous clinical trialsThe immunohistochemistry was performed as previously reportedU test wasused to compare the frequency of disrupted Nav channel clusters and C3depositions and the macrophage infiltration between different groups.Numbers of disrupted Nav channel clusters and C3 depositions were counted in twodifferent roots for each rabbit in at least 35 microscopic fields. Averageoptical density of staining area of macrophage infiltration per field wasmeasured for at least 40 images from each AMAN rabbit using Image-pro plus(version 6.0) . The quantification wasobserver-blinded. The results were shown asmean ± standard error. Mann-Whitney 2) in the ventral roots of the lumbar cord wascounted by a blinded observerU test wasused to compare the frequency of degenerative axons between different groups.Five normal rabbits without immunization or treatment constituted the histologiccontrols. Life-table method was used to evaluate the effect ofmethylprednisolone on AMAN rabbits surviving. A p valueof < 0.05 was considered significant. Analysis was performedwith the SPSS 19.0 analysis software by IBM .In both groups , AMAN rabbits were monitored daily until 30 days afterthe initiation of treatment. After the follow-up period, the rabbits wereperfused transcardially and the ventral roots of their lumbar cords were excisedfor the toluidine blue staining as previously describedHow to cite this article: Wang, Y.-Z. et al. Action mechanism ofcorticosteroids to aggravate Guillain-Barré syndrome. Sci. Rep.5, 13931; doi: 10.1038/srep13931 (2015).


The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUGexp binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1–(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUGexp RNA in nuclear foci, owing to reduced turnover of the CUGexp transcript. By comparison, the monomer did not induce CUGexp accumulation in cells and was more effective at rescuing a CUGexp-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUG The undet al. ].n repeat in the 3’ untranslated region (UTR) of DM protein kinase (DMPK), a gene expressed in muscle, heart and brain  is retained in the nucleus in foci , respectively (In DM1 the expanded sequence is a (CTG)nd brain . When pa repeats . The DMP in foci ,14. SpliGexp RNA ,16. The Gexp RNA . For exaectively ,21.exp binding, thus restoring MBNL function in cells.As yet there are no disease-modifying treatments for DM1 or other RNA dominant disorders. However, several compounds or oligonucleotides have shown beneficial effects in cell or animal models . For sman binding to MBNL1, the predominant MBNL protein of skeletal muscle. Out of 279 433 compounds in the screen, the most potent inhibitor was lomofungin, a natural antimicrobial agent from Streptomyces lomondensis (n affinity and stronger MBNL-(CUG)12 binding inhibition in vitro, it was the monomer that showed greater correction of MBNL1-dependent splicing in cells. This discrepancy between the cell-free and intracellular activities appeared to result from a novel effect of dilomofungin on RNA turnover. Dilomofungin reduced the rate of CUGexp decay in cells, which led to a striking increase of CUGexp RNA in nuclear foci. These results are cautionary for development of high affinity RNA ligands to mitigate RNA toxicity, which in some cases may stabilize the target and promote the accumulation of mutant RNA in cells.We performed a high-throughput screen to identify compounds that inhibit r(CUG)ondensis ,36. We f12 binding was previously described (12 oligoribonucleotide and recombinant human MBNL1 protein. The (CUG)12 oligonucleotide was biotinylated at the 5′ end and contained a 4 bp GC clamp at 5′ and 3′ ends to stabilize the repeat in a hairpin conformation, with G·C and C·G base pairs separated by periodic U·U mismatch in the stem. The recombinant MBNL1 had a 105 amino acid deletion at the C-terminus, to reduce protein aggregation without loss of r(CUG)n binding, a GST tag at the N-terminus and a His6 tag at the C-terminus. The MBNL1-Δ105-His6 was expressed and purified from Escherichia coli as previously described (12 RNA and MBNL1-Δ105-His6 protein were mixed in equimolar concentrations (20 nM each) and dispensed in 1536 well plates at 2 μl per well. Compounds dissolved in DMSO were then added (23 nl per well) in a five point dilution series that ranged from 92 nM to 57.5 μM. All assay buffers contained 0.05% Tween-20 to reduce aggregation effects 12. The confirmatory assay employed a different detection system, consisting of AlphaScreen donor beads (conjugated to streptavidin) that emitted a singlet oxygen to activate AlphaScreen acceptor beads . Both systems were shown to detect MBNL1-Δ105-His6 when in close proximity to biotin-CUG12 in solution  assay for inhibitors of MBNL1-(CUG)escribed . In brieescribed . The bio effects . After 1solution . The ass1H NMR spectra were recorded at 25°C on a Bruker Avance 400 (400 MHz) or Bruker Avance 500 (500 MHz) instrument. Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane and referenced to the residual protium signal in the nuclear magnetic resonance (NMR) solvent . Data are reported as chemical shift, multiplicity , integration, and coupling constant (J) in Hertz (Hz). 13C NMR spectra were likewise recorded at 25°C on a Bruker Avance 400 (100 MHz) or Bruker Avance 500 (125 MHz). Chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane and referenced to carbon resonances in the NMR solvent. High-resolution mass spectra were acquired at the University of Buffalo mass spectrometry facility, Buffalo, NY, USA.Analysis of lomofungin  was performed on a Shimadzu LC2010 HPLC equipped with a C18 reverse-phase column. 10 RNA, also in the same buffer. After each addition the mixture was allowed to equilibrate for at least 10 min, or until no change in the fluorescence spectrum was observed. Changes in fluorescence emission at 565 nm (excitation at 550 nm) were measured. Raw data were corrected for dilution-dependent changes, plotted against compound concentration and fit to the one site binding equation equation :(1)\docin vitro transcription . The transcripts were purified on denaturing 10% polyacrylamide (19:1) gels. The RNA was visualized by UV shadowing, excised, eluted in 0.3 M NaCl and precipitated in three volumes of ethanol overnight at −80°C. The samples were resuspended in 10 mM phosphate buffer (pH 7.2). The RNA concentration was determined by absorbance at 260 nm using the corresponding extinction coefficients. Extinction coefficients were determined using HyTher version 1.0  were amplified by polymerase chain reaction (PCR) as previously described ,32. The sion 1.0 ,40 basedsion 1.0 .2PO4 and Na2HPO4 110 mM KCl, 2 mM MgCl2, 2 mM CaCl2 and 1 mM Na2EDTA at pH 7.0). A similar procedure was used to prepare a 10 mM stock solution of dilomofungin. Slide-A-Lyzer MINI dialysis units  were used for competition dialysis assays. Each unit was evaluated before use by dialyzing against water for 24 h. The volume in each unit was measured, and units with volumes exceeding the original 200 μl were discarded.A fresh 3.27 mM stock solution of lomofungin was prepared in 500 μl of DMSO and stored at −20°C. An aliquot of this stock solution was dissolved in 200 ml of phosphate buffer  sodium dodecyl sulfate (SDS) was added, in order to dissociate the lomofungin or dilomofungin from nucleic acid or MBNL1. The SDS step was completed to ensure accurate determination of the concentration of the bound ligand, as the spectroscopic properties of bound ligands could differ from unbound ligands.tC) within each dialysis unit was determined spectrophotometrically. Appropriate corrections were made for the small dilution resulting from the addition of the SDS stock solution. In parallel the free ligand concentration (fC) was determined from an aliquot of the dialysate solution, which typically did not vary appreciable from the initial 1 μM concentration. Absorbance and fluorescence measurements were made using a UV-Visible spectrophotometer . The bound ligand concentration (bC) was then determined by using Equation b). P-values for differential ligand binding were determined by unpaired t-tests, and α significance thresholds were false discovery rate (FDR) controlled for multiple testing.The total ligand concentration  that express firefly luciferase fused to the escribed . Assays escribed , 3 days in situ hybridization (FISH) for CUG repeat RNA and immunofluorescence for MBNL1 were performed as previously described  as endogenous control. Semi-quantitative RT-PCRs were also performed for Mapkapk5, Arid2, Nr3c1, Mapkap1 and rRNA 5′ external transcribed spacer (ETS) using primers listed in Supplementary Table S6. RNA was extracted 3 days after addition of vehicle or drug.The exp-MBNL1 binding we nucleofected cells with 2 μM of CAG25, a 25-mer morpholino oligonucleotide comprised of CAG repeats  was added to the media of fluc0 (no repeat) or fluc800 (800 CTG repeats) cells for up to 8 h in the presence or absence of 10 μM of dilomofungin. Levels of transgene mRNA were determined by qRT-PCR as previously described , normali12 binding was performed on 279 433 compounds in the MLSMR (Supplementary Table S1). The primary screen employed recombinant MBNL1 and biotinylated (CUG)12 in a solution-phase HTRF assay (PubChem AID 2675). Each compound was tested in a five-point 1:5 dilution series ranging from 92 nM to 57.5 μM, and concentration-response curve fitting was performed ; and (iii) a counter screen using an HTRF protein–peptide binding assay (Pubchem AID 651843). Ten compounds showed consistent inhibitory activity in the confirmatory and orthogonal assays but not in the counter assay, and were ordered as powder samples for retesting in the AlphaScreen . Among these, the most potent inhibitor was the antimicrobial agent lomofungin, which showed an IC50 more than 2-fold lower than other compounds in the screen erformed ,45. The 1H- and 13C-NMR spectra of the unknown compound confirmed this expectation. The structure of the dimer, which we designated dilomofungin, featured a carbon–carbon bond between the same aromatic carbon on two lomofungin monomers .HPLC analysis of lomofungin that was freshly prepared in DMSO showed a single peak with a retention time that was markedly different from a previously prepared solution . Mass spectrometry analysis of the unknown compound in the older solution indicated a mass of 626.2, two AMUs less than twice the molecular weight of lomofungin, suggesting dimerization with loss of two protons . Infrared and 10 RNA were determined by fluorescence titration . The change in emission at 565 nm was plotted against drug concentration, and then fitted to a one binding site equation. The calculated KDs were 998 ± 180 nM and 606 ± 300 nM for lomofungin and dilomofungin, respectively. To compare their efficiency for inhibiting MBNL1-(CUG)12 binding, we repeated the AlphaScreen assay using the monomer or fully converted dimer. Dilomofungin exhibited 17-fold stronger inhibition than lomofungin, with IC50s of 42 nM  and 717 nM , respectively 6 protein. Both lomofungin and dilomofungin showed a > 1.5-fold binding preference for RNA with a single internal 5′CUG/3′GUC loop, as compared to MBNL1 protein . The binding preference for CUG repeats versus MBNL1 protein was increased to > 4-fold for RNA with six internal 5′CUG/3′GUC loops   except A·A . The fluc cDNA was fused to a DMPK 3′ UTR that contained 0 or 800 CTG repeats . These results are consistent with previous in vivo observations that CAG25 injection produced partial release of CUGexp RNA and MBNL1 protein from nuclear foci, resulting in increased MBNL1 activity and increased nuclear export and translation of CUGexp mRNA (fluc-CUGexp transcripts in both systems ((exp RNA.Previously we found that a 25-mer morpholino antisense oligonucleotide comprised of CAG repeats (‘CAG25’) was able to compete with MBNL1 for binding to CUG repeat RNA, thus disrupting MBNL1-CUGomplexes . Injectin Figure , and nucn Figure . In contexp mRNA . Notablysystems  up-regulation at 10 μM  but not vehicle (DMSO) produced a striking increase in the number and intensity of nuclear foci in fluc800 cells   was applied to fluc800 or fluc0 cells, in the presence or absence of dilomofungin. The rate of fluc800 mRNA decay was markedly reduced by dilomofungin (10 μM), producing a 5-fold increase in the calculated half-life , an rRNA fragment that is generated and rapidly degraded in the nucleus . The alternative splicing of Serca1 exon 22 (Serca1ex22) is highly conserved and strongly promoted by MBNL1 . In recombined cells the level of Serca1ex22 inclusion was reduced to 54 ± 3% , consistent with partial release of sequestered MBNL1. Application of 10 μM lomofungin also promoted the inclusion of Serca1ex22 .Addition of lomofungin (10 μM) to the culture media led to increased diffuse nucleoplasmic staining for MBNL1 in fluc800 cells, consistent with partial alleviation of the MBNL1 sequestration Figure , F. Intes Figure . To testby MBNL1 . Among nnockdown ,18,31. T repeats . As the 12 cells . TransfeTargeting of RNA with small molecules has been advocated as a therapeutic approach, especially when viable protein targets are lacking . Source of open access funding: National Institutes of Health and institutional funds.Conflict of interest statement. None declared.


Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca2+-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2-/- . In UCP2-/- cardiomyocytes (Ca2+)m was decreased  while (Ca2+)c was unchanged  and transsarcolemmal Ca2+-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa2+-uptake in WT mitoplasts and (Ca2+)m of cardiomyocytes leading to an increase of (Ca2+)c while no ATP dependent effect was observed in UCP2-/-.Isolated mitochondria showed a Ru360 sensitive mCa2+-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.Our results indicate regulatory effects of UCP2 on mCa It has been shown to control the rate of mitochondrial energy  production [2+ signaling [Mitochondrial Caoduction , 2, moduignaling –5, regulignaling  and may ignaling , 7, 8.2+-uptake (mCa2+-uptake) is thought to be mostly mediated by the mitochondrial calcium uniporter (MCU) [2+-uptake mechanisms have been suggested [MiCa and ImCa1 have been reported for COS 7 cells and diverse tissues including human myocardium, respectively [Mitochondrial Caer (MCU) , 9, 10. uggested . Electroectively , 10, 12.ectively , 14. Morectively , 16. How2+-uptake [2+-uptake of isolated liver mitochondria from UCP2-/- mice was reported to be RuRed-insensitive and enhanced mCa2+-uptake was observed in an endothelial cell line overexpressing UCP2. Therefore, UCP2 was postulated to be fundamental for mCa2+-uptake [2+-homeostasis from UCP2-/- mice and wild-type controls.Uncoupling proteins (UCPs) are located in the inner mitochondrial membrane and belong to a superfamily of mitochondrial ion transporters . UCP1 is+-uptake –25, i. e+-uptake . However+-uptake . Thus, t2+ flux independent of the presence of UCP2, but that these Ca2+-currents are modulated by UCP2 in an ATP dependent manner. Our experiments also reveal a new compensational mechanism in UCP2-/- mice, which prevents possible cytosolic Ca2+ overload through inhibition of transsarcolemmal Ca2+-influx.Our data shows that the MCU may mediate Catm1Lowl/J  purchased from Charles River Laboratories, Research Models and Services, Germany [Animals were euthanized by cervical dislocation and hearts were obtained from the mouse strain B6.129S4-Ucp2 Germany . Hearts The animals used in this study were between 8 and 12 weeks of age. To verify genetic knockout of UCP2 mice tails were genotyped using primers UCP2f2 (CAGCCACTGTGAAGTTCCTGG) and UCP2r2 (CATTGTGACACACACTTAATG) for UCP2 as well as primers GAPDH-f (AGGCCGGTGCTGAGTATGTC) and GAPDH-r (TGCCTGCTTCACCACCTTCT) for GAPDH.-/- mice and single ventricular myocytes were isolated from murine hearts by enzymatic digestion, as previously described [Hearts were obtained from WT or UCP2escribed , 28. Forescribed . Isolate-/- mice by differential centrifugation, by a modified protocol, as previously reported [4 and 1 mM EGTA, pH 7.4 adjusted with KOH, atria were removed. The ventricular myocardium was minced in a KB buffer on ice containing: 143 mM KCl, 70 mM glucose, 20 mM taurine, 5 mM HEPES, 0.5 mM EGT, BSA 0.1%, pH 7.2 adjusted with KOH and incubated with 1500 U/ml collagenase B  at 37°C for 30 minutes. Subsequently the homogenate was centrifuged for 10 min at 750 g. The supernatant, containing the mitochondrial fraction, was further centrifuged at 7.000 g for 10 min, and the pellet was suspended in 1 ml of standard incubation buffer containing: 125 mM KCl, 20 mM HEPES, 15 mM NaCl2, 5 mM Na-succinate, 5 mM MgCl2, 2 mM K2HPO4, pH 7.0 adjusted with KOH and spun at 7.000 g for 10 min. Mitochondria were resuspended in standard incubation buffer. Protein concentration was determined using BCA Assays . Protein concentration was adjusted to 1 mg/ml. Mitochondria were kept on ice before experiments.Cardiac mitochondria were isolated from WT or UCP2reported –31. BrieIsolated intact cardiac subsarcolemmal mitoplasts were prepared from isolated myocytes by differential centrifugation, as previously reported , 10, 32.2HPO4, 20 mM HEPES, 5 mM Na-succinate, 5 mM MgCl2, pH 7.0 adjusted with KOH) and then transferred to a water-jacketed and magnetically stirred oxygraph chamber of an Oroboros Oxygraph-2k . Mitochondrial quality was assessed by measuring the respiratory control rate (RCR), using 1 mM ADP (state 3) or 1 mM ADP and 2.5 mg/ml oligomycin (pseudo state 4). Mitochondria consistently had RCR values between 4 and 7 with complex I substrates. The data were analyzed, as previously described [Isolated mitochondria were suspended in the standard incubation buffer  and incubated with 2 nM TMRM for 30 min to achieve transmembrane equilibrium of the dye [exc = 574 nm and emission was measured at λem = 590 nm. Only experiments which revealed a significant decrease in fluorescence signal after CCCP application indicating depolarization of mitochondria were included.Experiments were performed as previously described using a modified protocol –38. Mito the dye , 37. Mit2+-uptake, as previously described [2+ were added using an automated infusion system and rhod2-AM fluorescence was measured. Rhod2-AM was excited at λexc = 540 nm and emission was measured at λem = 605 nm. Data was acquired for 660 s.Isolated mitochondria were suspended in standard incubation buffer and loaded with rhod-2-acetoxymethyl ester  for 30 minutes at 4°C to determine mCaescribed . 1 μM ro2, 10 mM HEPES, pH 7.2 adjusted with Ca(OH)2 [3R; 10 μM xestospongin C, Sigma Aldrich, St. Louis, MO, USA), the mitochondrial Na+-Ca2+ exchanger  [All experiments were performed in the mitoplast-attached configuration of the patch-clamp technique . The bath solution contained 160 mM KCl, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA, pH 7.2 adjusted with KOH. Pipettes were filled with a solution containing 105 mM CaCl Ca(OH)2 , 10, 32.CA, USA) , 10, 32.CA, USA) , 40.Single-channel analysis was done using custom software only from single-channel patches, as previously reported , 32. Bri2+ measurements were performed, as previously described [2+)m. Using the patch-clamp technique, cytosolic traces of rhod2-AM were eliminated by whole-cell dialysis with a pipette solution containing 50 μM fluo4-pentapotassium-salt  to monitor (Ca2+)c [2, 5 mM Na-HEPES, 10 mM HEPES, 5 mM ATP, and 0.05 mM of the cell-impermeable fluo4-K5. Myocytes were equilibrated for >10 min. Thus, (Ca2+)m was reported by rhod2-AM and (Ca2+)c by fluo4-K5 [2, 10 mM Na-HEPES, 2 mM CaCl2, 10 mM glucose, 2 mM pyruvate, 0.3 mM ascorbic acid, pH 7.4 adjusted with NaOH. All myocytes were whole-cell voltage-clamped and Ca2+ transients were elicited by a rectangular pulse from -80 mV to 10 mV for 300 ms at 0.5 Hz, as described previously [2+ transients to eliminate cytosolic traces of rhod2-AM and normalize sarcoplasmatic reticulum (SR) Ca2+ load, as previously described [Caescribed . Briefly (Ca2+)c , 9, 41. fluo4-K5 , 9. Wherfluo4-K5 , or 5 mMeviously . Membraneviously  in wholeeviously . Series 2+)c, fluo4-K5 was excited at λexc = 470 nm, and emission was collected at λem = 520 nm, as described previously [2+)m, rhod2-AM was excited at λexc = 550 nm, and fluorescence (F) was recorded at λem = 605 nm.. Fluorescence data was normalized to F0 after completion of the stimulation protocol described above (F/F0). When necessary, (Ca2+)m and (Ca2+)c signals were corrected for rhod2-AM bleaching (~2% per minute) and fluo4-K5 bleaching (~10% per minute), respectively.To measure  [2+ by mCa2+-uptake. By using the stimulation protocol, fluo4-K5 and the detectors as described above we recorded (Ca2+)c. When necessary traces were filtered using a 30–50 Hz low pass Gaussian filter.To measure transsarcolemmal Caescribed . Briefly (SERCA) . Additio2+ load was registered as previously described [2, 0.33 mM NaH2PO4, 1 mM CaCl2, 10m M glucose, pH 7.4 adjusted with NaOH and an internal solution containing 110 mM CsCl, 30 mM TEA-Cl, 10 mM NaCl, 10 mM HEPES, 5 mM MgATP, pH 7 adjusted with CsOH. Miniature solenoid valves  controlled by PCLAMP digital outputs controlled the rapid solution exchange with an additional 5 mM caffeine in the bath solution. This enabled precise timing of solution exchanges in relation to the voltage clamp protocol. To quantify SR Ca2+ load in WT and UCP2-/- myocytes, we recorded INCX during an application of 5 mM caffeine for 1 s. We administered a train of five prepulses from -40 to 0 mV at 1 Hz lasting 100 ms immediately before caffeine application to ensure a steady-state SR Ca2+ load. A constant holding potential of -40 mV was maintained during exposure to caffeine. SR Ca2+ load was analyzed by calculating the integral of the INCX current recordings.SR Caescribed . BrieflyP<0.05 were regarded significant.In experiments obtained in isolated mitochondria n refers to the number of animals. In whole and single patch clamp experiments n refers to the number of patch-experiments obtained in mitoplasts or cardiomyocytes isolated from a minimum of three hearts. Pooled data are presented as mean ± SEM. Comparisons between two groups were performed with unpaired t-test and multiple comparisons with one-way ANOVA followed by Bonferroni test. Probability values of 2+-uptake in intact mitochondria we measured rhod2-AM fluorescence upon Ca2+ application (200 nM). Mitochondria had respiratory control rates from 4 to 7 with complex I substrates [-/- mitochondria revealed no difference in TMRM load  indicating a similar membrane potential in both groups. To rule out unspecific effects of other known mCa2+uptake mechanisms, mitochondria were incubated with various mitochondrial Ca2+-channel blockers (see -/- mitochondria showed a Ru360 (100 nM) sensitive Ca2+-uptake  without affecting single-channel amplitude (P<0.05)  , Table 1(P<0.05) , Table 12+-uptake and its potential regulative influence on mitochondrial Ca2+-uptake, we analyzed single-channel currents in cardiac mitoplasts of UCP2-/- mice. Notably, we recorded a Ca2+-current with a similar unitary conductance compared to mCa1 channels of WT mitoplasts . mCa1 occurred in 38% of total patches and compared to WT showed a trend towards an increase in the probability of occurrence  in the UCP2-/- group, however, without reaching statistical significance . The channel exhibited three different amplitude sublevels with -1.19±0.04 pA being the most common (P>0.05 vs. WT). However, the total open probability was significantly lower  in UCP2-/- mitoplasts compared to mCa1 obtained from WT mitoplasts, indicating mCa1 to be present but less active in UCP2-/- mitochondria  mediated cytosolic and rhod2-AM mediated mitochondrial Cas report  using veansients . Furtherpartment . Ru360 i control . Inhibit2+-uptake and EC coupling under physiological conditions we recorded dual Ca2+ transients from intact UCP2-/- cardiomyocytes. Consistent with the single-channel data UCP2-/- cardiomyocytes showed a significantly lower amplitude and slower rate of rise of mitochondrial Ca2+ transients vs. WT. The amplitude of (Ca2+)c was unchanged in UCP2-/- vs. WT while rate of rise was reduced suggesting a counter-regulatory mechanism against cytosolic Ca2+ overload in UCP2-/- myocytes  and SR Ca2+-ATPase (SERCA) using 0.2 μM ryanodine and 0.01 mM thapsigargin, respectively, as previously described [2+ by mCa2+uptake via the MCU. UCP2-/- cardiomyocytes presented a significantly down-regulated transsarcolemmal Ca2+-influx with a significantly diminished slope factor in comparison to WT  or ATP depleted pipette solution. Mitochondrial Ca2+ transient amplitude and rate of rise were significantly decreased by additional delivery of high amounts of ATP (15 mM) via the pipette solution  and ATP free solution. Consistently neither mitochondrial Ca2+ transients nor cytosolic Ca2+ transients were significantly altered by either ATP concentration in intact cardiomyocytes from UCP2-/- mice  measurement of Ca2+-uptake in intact isolated mitochondria by using rhod-2 AM, 2.) single-channel recordings of subsarcolemmal mitoplasts prepared from isolated cardiomyocytes, and 3.) analysis of dual Ca2+ transients elicited by voltage-clamp depolarizations from the mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in whole-cell patch-clamped, isolated ventricular cardiomyocytes.To define the role of UCP2 we used three complementary approaches in WT and UCP22+-uptake in both WT and UCP2-/- mitochondria. However, compared to WT mitochondria UCP2-/- mitochondria showed a significantly lower Ca2+-uptake.The experiments performed in isolated mitochondria demonstrated a Ru360 sensitive Ca-/- mitoplasts, we detected a murine cardiac Ca2+-channel which presented burst like open states, a mean amplitude of -1.1–1.2 pA at -100 mV and a conductance of 12–13 pS. These characteristics are not equal to the IMiCa which was proven to underlie the MCU [MiCa activity was reported to differ between various tissues and species showing the lowest activity in the heart [2+-channels in mitoplasts isolated from HeLa cells  [i-MCC matches the human cardiac mCa1 [i-MCC described in HeLa cells. Therefore, this channel might display activity characteristics probably suggestive for the MCU-established current in the murine heart [Using single-channel recordings in WT and UCP2 the MCU , 47 and d b-MCC) . The autiac mCa1 . Since, iac mCa1 . Thus, wne heart .-/- mitoplasts. Finally, our detection of a reduced Ru360 sensitive (Ca2+)m in intact cardiomyocytes from UCP2-/- mice further confirmed this observation. Since none of our experiments neither showed a complete inhibition of mitochondrial Ca2+ uptake in isolated mitochondria, a fully inhibited mCa1 activity in the single channel experiments nor a completely reduced (Ca2+)m under basal conditions, we conclude that UCP2 is not essential for mitochondrial Ca2+ uptake as previously suspected [2+ uniporter as a highly Ca2+-selective protein complex that consists of the pore-forming mitochondrial Ca2+ uniporter protein (MCU) [2+ handling. Our data suggests that in the heart, through interaction with mCa1, UCP2 could operate cooperatively or sequentially for the modulation of transporting Ca2+ across the inner mitochondrial membrane as another part of this complex system. However, regulatory effects on additional mitochondrial Ca2+ channels or Ca2+ uptake mechanisms in the heart should also be considered. Of note, in a recent study in HeLa cells UCP2 was reported to regulate the extra-large mitochondria calcium channel (xl-MCC), that essentially contains MCU and EMRE. Notably, UCP2 was shown to have a much higher affinity to this particular channel than to other MCU-dependent calcium currents  [Consistent with intact mitochondria we found a mCa1 current with a high sensitivity to Ru360 but decreased single-channel activity in UCP2uspected . This coin (MCU) , 51, thein (MCU) –55, whicl i-MCC) .2+-uptake [2+ transients from the mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in whole-cell patch-clamped, isolated, intact cardiomyocytes confirmed MCU inhibition by ATP. However, no ATP effect was observed in UCP2-/- mice, supporting the notion that the inhibition of mCa2+-uptake by ATP is mediated via UCP2. Since our whole-cell experiments revealed decreased (Ca2+)m using high concentrations of the nucleotide (15 mM) and increased (Ca2+)m in the absence of ATP as compared to physiological ATP concentrations (5 mM) we suggest that under physiological conditions MCU activity is diversely dependent on the intracellular ATP concentration. In addition, previous data reported the MCU to be involved in the regulation of mitochondrial energy (ATP) production [2+-uptake indicating a physiological feedback mechanism in situations with high energy demands.Previous studies also described an inhibitory effect of adenine nucleotides especially ATP on mCa+-uptake  and on U+-uptake , 18, 22.2+ uptake could be the modulation of (Ca2+)m by increased ATP and increased SERCA activity leading to reduced depletion of the internal Ca2+ stores as observed by De Marchi et al. in intact HeLa cells under UCP3 depletion [2+ load was not different between WT vs. UCP2-/- we did not specifically monitor SERCA activity. This needs to be further investigated in future studies.An alternative explanation as to how UCP´s and ATP might regulate mitochondrial Caepletion . In our 2+)m and subsequent upregulation of (Ca2+)c by Ru360 at concentration of 10 nM, as well as the inhibitory effect on both (Ca2+)m and (Ca2+)c in the presence of 100 nM Ru360 in WT cardiomyocytes were consistent with a previous report [2+)c in the presence of 10 nM Ru360 is that moderate increases in ROS production activate RyRs and therefore (Ca2+)c. This suggest a close interplay of mitochondrial and cytosolic redox state/ROS production and elementary Ca2+ release events as previously described. We found no evidence for an unspecific Ru360 effect at 10 nM, e.g. by downregulation of cytosolic Ca2+ release which subsequently affects mitochondrial Ca2+ uptake, Additional control experiments analyzing cytosolic Ca2+ uptake using only Fluo4-K5 in the presence or absence of 10 nM Ru360 also confirmed this observation (data not shown).The inhibitory effect on (Cas report . A possi2+ in WT cardiomyocytes caused by inhibited mCa2+-uptake could lead to an increased (Ca2+)c transient, possibly due to the spatial vicinity of these structures in an area of limited diffusion like the dyadic cleft. This hypothesis is further being supported by our data using an alternative mode of inhibiting mCa1/MCU, i.e. elevation of (Ca2+)c with high ATP concentrations in isolated cardiomyocytes, which should not influence RyRs in WT mice but also inhibited (Ca2+)m.We provide an additional explanation for the observations at 10 nM Ru360 since a shift of available dyadic cleft Ca2+)m and subsequent downregulation of (Ca2+)c supports the notion of modulated local Ca2+ distribution based upon the ATP dependent modulation of mCa1/MCU as mentioned above.Furthermore, our finding that the absence of ATP in WT cardiomyocytes led to an increase of (Ca2+)c in the presence of 100 nM Ru360 [-/-. Strikingly, the amplitude of (Ca2+)c in UCP2-/- mice was not significantly altered vs. WT (p>0.05) while (Ca2+)m was reduced. Since cytosolic Ca2+ overload is considered as unfavorable and is observed in the onset of heart failure [-/- mice are likely. We hypothesize that the decreased transsarcolemmal Ca2+-influx in UCP2-/- vs. WT is such a mechanism. A similar modulation where reduced Ca2+ influx was observed as a potential compensatory mechanism against Ca2+ overload has been shown in NCX knockout mice [Higher Ru360 concentrations are known to inhibit the RyR, which could explain the diminished m in HeLA cells [2+-uptake apparently uses alternative, Letm1-dependent routes in the same cell model [2+-influx should occur in UCP2-/- cardiomyocytes only while using identical amounts of thapsigargin in both groups. Additionally, we analyzed murine cardiomyocytes and it is unclear whether the observations by De Marchi et al. and Waldeck-Weiermair et al. in HeLA cells can be transferred to our results. This should be addressed in future studies with focus on SERCA-activity and (Ca2+)m in intact murine cardiomyocytes.With regard to our experiments analyzing transsarcolemmal CaLA cells . A subsell model . This me2+ transient in UCP2-/- vs. WT, a reduction of mitochondrial Ca2+-transients in UCP2-/- vs. WT is possibly directly attributable to this observation. This suggests either a diminished SR Ca2+-load, altered RyR kinetics or differences in transsarcolemmal Ca2+ influx in UCP2-/- vs. WT as alternative explanations.Since we observed a trend towards a diminished peak cytosolic Ca2+ load but detected a significant alteration in (Ca2+)c rate of rise suggesting modulated RyR kinetics in UCP2-/- vs. WT. We did not further analyze this aspect, which limits the conclusions in our paper. It should also be further investigated in future studies.However, we found no difference in SR Ca2+-uptake, which most likely has a direct impact on Ca2+ handling of the entire murine cardiomyocyte under steady state conditions and is modulated by ATP. The quantities of mitochondrial Ca2+ uptake on a beat-to-beat basis, however, remain an area of debate, and should be rather low according to recent data by other groups [In summary our study supports an essential modulating role for UCP2 in mCar groups , 62.-/- mice from these otherwise disadvantageous Ca2+ regulations.Our data also indicate that UCP2 inhibition by ATP could serve as a potential regulatory mechanism in controlling energy production under physiological conditions via the MCU. Compensational mechanisms seem to protect UCP2