Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research.CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis , suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro.Differences were observed in the sequences and spatial structures of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.The sequences and spatial structures, molecular mass, and expression profile of The online version of this article (10.1186/s13071-017-2494-9) contains supplementary material, which is available to authorized users. Clonorchis sinensis, the causative agent of clonorchiasis, predominantly occurs in East Asia, including China, parts of Russia, South Korea, and northern Vietnam. Globally, it has been estimated that over 200 million people are at risk for clonorchiasis and that more than 15 million persons, of whom 13 million are in China, are infected  × 100%; worm reduction rate (%) = [ / average worm burden of control group] × 100%  × 100% .Clonorchis sinensis adults were recovered from infected rats, washed 4 times with sterilized PBS containing 1% antibiotics (streptomycin 100 U/ml and penicillin 100 μg/ml) and transferred to 12-well plates at 20 adults per well in 2 ml/well of low-glucose DMEM containing 1% antibiotics. Serum from rats that had been immunized with rCsPK or preimmune serum from the same animals was mixed with the medium at dilutions of 1:40–1:160. Low-glucose DMEM was used as the blank control. At 1–10, 15, 18, 20, 22, 24, 26 and 28 days of incubation, the adult worms were counted and monitored for 5 min using a microscope (Leica). Adults that failed to display pumping and muscle contraction after 5 continuous mechanical stimuli were classified as dead [ as dead .t-test. The survival rates of cultured adults were analysed using the Kaplan-Meier method, and differences between the groups were determined using the log-rank (Mantel-Cox) test. The results of the analyses are expressed as the mean ± standard deviation; significance was set at a P-value < 0.05.All experiments were performed at least 3 times. SPSS software  was employed for statistical analysis. Differences in IgG isotypes and immune protection between the groups were analysed using Student’s CsPK is 1458 bp in length and encodes 485 amino acids (aa). In its protein sequence, CsPK shows the identities to the PK of Opisthorchis viverrini (99%), followed Schistosoma japonicum (80%), Brugia malayi (59%), Hymenolepis microstoma (39%) and Echinococcus granulosus (33%). The protein sequence included the characteristic active site signature (IKLIAKIENHEGV)  residue that forms part of the catalytic site and that appears to be responsible for interconverting enolpyruvate and pyruvate. L205 was unique to CsPK, replacing the usual isoleucine (I). A207 of CsPK replaced the usual serine (S) found in the rat, mouse and human enzymes  domains described for PK are shown. The N-terminal domain, which only appears in eukaryotic PKs, is very long in  diverse . CsPK co cytosol .CsPK was simulated using SWISS-MODEL based on the molecular model of truncated TgPK1 , domain B (red) and domain C (green). The catalytic sites appear at the interface of domains A and B , which CsPK, the F16BP binding sites, which interact with the phosphate moieties, are also conserved with different conformations. The ribbon drawing shows a superposition between domain A of CsPK with the closed rabbit PK-M1 in complex with ATP and the inhibitor oxalate (PDB: 1A49 [In human PK-M2 (PDB: 3BJF ) and CsPDB: 1A49 ). The siCsPK and in that of LmPYK-suramin (PDB: 3PP7 [T. brucei glycolytic enzymes), the significant residues are highly conserved and exhibit the same conformation.In the active site of DB: 3PP7 ) complexCsPK grouped very closely with the PKs of trematodes. CsPK was closely related to PKs of vertebrates, followed by those of nematodes and cestodes, whereas it was distantly related to the PKs of protozoa. Phylogenetic, sequence and structural analysis revealed that CsPK (T52 and E56) is related to the group of monovalent cation-dependent isozymes (type I) [CsPKII  clusters with E. coli PKII (AAA24473.1), along with three isozymes from the protozoans T. gondii (KFH06835.1), P. falciparum (AAN35560) and T. parva (XP_764703.1). These results suggest a different evolutionary origin of the two isozymes in C. sinensis.In the phylogenetic tree of PKs Fig. , CsPK gr(type I) . In contCsPK is 6.69, and its predicted subunit Mr. is 53.1 kDa. Purified rCsPK containing a 6× His-tag displayed a single band of approximately 58.0 kDa, consistent with the calculated Mr. .The putative theoretical isoelectric point of Mr. Fig. , Lane 5.CsPK was between that of ferritin (440 kDa) and that of catalase (232 kDa). According to the equation describing the electrophoretic profile, the Mr. of rCsPK was approximately 290 kDa.In GFC, rCsPK eluted as a single peak between β-amylase (200 kDa) and ferritin (440 kDa)  was noted. This immunoreactive band did not appear on blots that had been probed with naive mouse sera. Additionally, CsESPs and total worm extracts probed with mouse anti-rCsPK sera, but not with preimmune sera, showed an immunoreactive band of approximately 53.1 kDa . The mRNA level of CsPK in the egg was higher than that in the adult  = 17.392, P = 0.003), metacercaria  = 15.844, P = 0.004) and excysted metacercaria  = 14.477, P < 0.001).The mRNA levels of sis Fig. . SignifiCsPK sera by Western blotting analysis. The protein level of CsPK was highest in the egg, followed by excysted metacercaria, metacercaria and adult  = 14.245, P < 0.001; t(14) = 7.299, P < 0.001). The egg reduction rate and worm reduction rates were 56.90% and 55.22% (compared with the PBS group), respectively. No significant difference in EPG or worm burden was observed among the PBS, adjuvant, and infection groups.The EPG values of the PBS group, the adjuvant group, the infection group, and the rC. sinensis adults in the blank control group, in preimmune sera groups , and in anti-rCsPK sera groups  were 15, 8, 8, 9, 2, 3 and 4 days, respectively . There were dominant differences of survival rates among all other groups (P < 0.01).The median survival times of CsPK with those of PKs from humans, rats, mice, and rabbits, all of which are definitive hosts of C. sinensis. CsPK is a 53.1-kDa homopentamer PK that lacks the characteristic N-terminal domain but possesses a characteristic active site signature (IKLIAKIENHEGV) and some unique sites. The mRNA levels, protein levels and immunolocalization of CsPK at various life stages of C. sinensis were characterized. CsPK was a component of CsESPs. Also, a Th1-biased immune response was elicited in rats immunized with rCsPK. Furthermore, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro.In the present research, we investigated and compared the sequence and spatial structure of CsPK possesses the typical characteristics of a PK . Based on SDS-PAGE, the Mr. of rCsPK was approximately 58 kDa (including a His-tag)  Fig. . A band ody Fig. , whereas enzymes , 21.E. coli, L. mexicana, yeast, rabbit muscle, human, and cat) are highly similar with respect to ordinary PK topology [S. japonicum but was absent in C. sinensis.The X-ray crystallographic structures of some PKs of diverse organisms , L205 and A207 replace the usual isoleucine (I) and serine (S) found in the rat, mouse and human enzymes. Phylogenetic analysis revealed marked differences between CsPK and PKs from the definitive hosts of C. sinensis, cat, rat, human and mouse  [CsPK as well as into its potential as a drug target for clonorchiasis.In human PK-M2 and moieties , are verenzymes) , the sigCsPK reacted with mouse anti-rCsPK sera by Western blotting, revealing its good immunoreactivity. The recognition of rCsPK by sera of mice infected with C. sinensis hints that CsPK might be a component of circulating antigens [CsPK reacted with mouse anti-CsESPs sera. Furthermore, CsESPs were recognized by mouse anti-rCsPK sera, showing a band of approximately 53.1 kDa. These results demonstrate that CsPK is also a component of ESPs.rantigens , 44. AddCsPK was widely distributed in the tissues of adult worm, suggesting that CsPK is significant for C. sinensis. The locations in which CsPK was found included the pharynx, the intestine, and the tegument, tissues from which ESPs are released [CsPK in ESPs might result from the shedding and recondition of the tegument [CsPK may be involved in obtaining a sustainable energy supply through the digestion and absorption of glucose from the host.As the key glycolytic enzyme, released . In tremtegument . The tretegument . With thCsPK within the musculature, including the pharynx, the oral sucker, and the ventral sucker, may be related to energy demands for adhesion behaviour as well as muscular contraction. Its localization in generative organs, including the ovary, vitellarium, uterus, testis, genital pore, and seminal receptacle, hints that the enzymatic activity of CsPK in glucose metabolism in the above organs may be needed to satisfy the energy requirements of C. sinensis for growth and reproduction. The vitellarium of the trematode makes a critical contribution to egg production by providing nutrients to the oosperm, providing eggshell material, and related catalytic activities [The localization of tivities .CsPK contains numerous predicted T cell as well as B cell epitopes. The high levels of specific antibodies induced by rCsPK may be attributed to its variety of B cell epitopes. It is well known that IgG1 and IgG2a are elicited by Th2 and Th1, respectively. These data suggest that rCsPK elicits Th1-biased immunoreaction.Bioinformatics analysis indicated that CsPK revealed its high immunogenicity and might account for its protective efficacy.During long-term infection, Th1 to Th2 shift has been shown to occur and to result in long-term persistence of adult worms as well as chronic clonorchiasis . Th1 celCsPK elicited a Th1-biased immune response. A majority of proteins that are regarded as vaccine candidates are ESPs or tegumental proteins. CsPK was identified as a component of ESPs and was shown to be deposited on the tegument of adult worms and metacercariae. Rat anti-rCsPK sera inhibited C. sinensis adult subsistence in vivo and in vitro. Our results indicate that CsPK may be a promising candidate for the development of vaccines for clonorchiasis.Nucleic acid-based, as well as protein-based vaccines for clonorchiasis, have been reported. Some DNA vaccines elicit Th1-biased immune responses, whereas other DNA vaccines and protein vaccines elicit combined Th1/Th2 immune responses [CsPK is a 53.1-kDa homopentamer PK without a characteristic N-terminal domain; it possesses a characteristic active site signature (IKLIAKIENHEGV) and some unique sites. CsPK was widely distributed in the tissues and organs of adult worms, including the oral sucker, ventral sucker, pharynx, genital pore, vitellarium, tegument, intestine, seminal receptacle, testicle, ovary, uterus and in eggs within the uterus. It was shown to be a component of CsESPs. rCsPK possesses strong immunogenicity as well as immunoreactivity. rCsPK provoked a Th1-biased immune response. In addition, rat anti-rCsPK sera suppressed C. sinensis adult subsistence in vivo and in vitro. This research reveals that CsPK might be a promising vaccine candidate and that it has potential as a drug target against C. sinensis infection. The selective development of specific inhibitors against CsPK and its immune protective mechanisms are worthy of further study.Additional file 1: Figure S1.a Ribbon drawing of superposed structure models of CsHK (darker tone) and truncated TgPK1 (lighter tone). The A, B, and C domains of CsHK are shown in blue, red and green, respectively. The catalytic site at the interface of domains A and B and the allosteric site in domain C are highlighted. b Ribbon representation of F16BP (red stick) binding sites of human PK-M2 (lighter tone). S434, S437 (yellow stick), W482 (magenta stick), and R489 (orange stick), which interact with the phosphate moieties, are indicated. The putative corresponding structure of CsPK (darker tone) is shown in panel c. In the active site signature of PK, I267 and S269 (dark red sticks) are replaced by L205 and A207 (blue-violet sticks) in CsHK. Oxalate is indicated as a ball model. d Ribbon representation of the K+-PK-MgIIoxalate-MgIIATP complex closed active site (rabbit PK-M1). ATP, oxalate, and significant residues are shown as red, magenta, and yellow sticks, respectively. e Ribbon drawing of the superposition between the A domains of CsPK with the closed rabbit PK-M1 in complex with ATP and oxalate . The corresponding significant residues of CsPK are shown in orange (stick). K+ and Mg2+ ions, ATP and oxalate, are shown for reference, with their positions derived from a superposition with 1A49. f Ribbon drawing of superposed structural models of CsPK (darker tone) and LmPYK-suramin (lighter tone) complexed with glycerol (magenta stick) and suramin . g Enlargement of the active site of the LmPYK-suramin structure. Significant residues are coloured yellow (stick). The putative corresponding structure of CsPK is shown in panel h. The corresponding significant residues of CsPK are coloured orange (stick). (TIFF 2964 kb)Putative tertiary modelling of CsPK. K+ and Mg2+ ions are shown as grey and black spheres, respectively. The N-terminal domain is shown in black. Additional file 2: Figure S2.CsPK. a Determination of the Mr. of rCsPK using 8% native PAGE according to Fig. CsPK. b Elution profile of rCsPK in GFC. c Mr. of rCsPK as detected with GFC. The calibration curve relating the elution volumes (Ve) and the log Mr. (kDa) of standard marker proteins were obtained with AKTA FPLC using a Sepharose 12 10/300 GL GFC column. Based on the deduced equation, the Mr. of rCsPK was calculated from the obtained Ve of rCsPK. (TIFF 252 kb)Apparent Mr. of r


The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes , matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development. Prior to maturation, immature oocytes are arrested in prophase I of meiosis I, the germinal vesicle stage (GV). Oocytes remain arrested at this stage until puberty, when following the establishment of regular cycles and a surge of luteinizing hormone (LH), they resume meiosis (oocyte maturation), in preparation for fertilization. During maturation, oocytes undergo GV Breakdown, GVBD, complete meiosis I, and re-arrest at the metaphase II of meiosis II (MII stage), which is the stage of ovulation and fertilization2+ influx during maturation and fertilization in mammalian oocytes. Voltage-activated Ca2+ (CaV) channels have been reported to be expressed in oocytes of different species2+ store contents during oocyte maturationCacnah−/− female mice showed only a slight reduction in litter size8121415Several channels have been proposed to mediate Ca2+ influx in oocytes and eggs. TRP channels are a family of proteins that include ~30 members grouped in six subfamilies. TRP channels are in general cationic non-selective, weakly voltage sensitive, able to respond to different physiological stimuli and have a wide spectrum of physiological functions2+ influx in mouse eggs. Selective activation of TRPV3 channels provokes Ca2+ entry and parthenogenetic egg activation (parthenogenesis), although its physiological function in eggs remains undetermined, as Trpv3-deficient (Trpv3−/−) mice are fertile15The transient receptor potential channels, TRPs, are other possible mediators of Ca2+, pH, and phosphate inositol diphosphate (PIP2)+ channel blockerTrpm7 knock-out mice are embryonic lethal before E7.5 of embryogenesis24in-vitro embryo culture using TRPM7 antagonists and agonists, show functional expression of the channel in oocytes, eggs and 2-cell embryos, and suggest a role in pre-implantation development, as in the presence of TRPM7 specific inhibitors, development is curtailed or slowed.Most of the TRPs show tissue-specific expression and the generation of genetically modified mice has been helpful to understand their physiological function192+ promotes oscillations in both GV oocytes and MII eggs2+-induced oscillations and undergo egg activation, confirming the identity of the channel responsible for promoting Sr2+ influx in eggsTrpv3 KO mice are capable of displaying oscillations when exposed to Sr2+ is not known. This possibility should not be discounted given that during oocyte maturation new protein expression changes the protein profile of cytoplasmic proteins2+ influx in oocytes vs. eggs. To test the capacity of Trpv3−/− GV oocytes to mount Sr2+ oscillations, we exposed Wild Type (WT) and Trpv3−/− GV oocytes to Sr2+ (2+ induced the expected oscillations (left panel), which were comparable to those observed in Trpv3−/−oocytes (right panel). Importantly, the Sr2+ induced oscillations in WT and Trpv3−/− oocytes were unaffected by temperature, which also rules out TRPV3 as the active channel at GV, as TRPV3 is a temperature-sensitive channel29302+ influx in WT or Trpv3−/− oocytes is not TRPV3.Exposure to Sr to Sr2+ . In WT o2+) is a well-known inhibitor of some cationic non-selective channels members of the TRP family, including TRPV3 channels2+ also blocks TRP channels of the Melastatin sub-family, TRPM6 and TRPM720Trpv3−/− oocytes and eggs. In those gametes and, using internal and external solutions without Mg2+, a voltage ramp evoked outwardly rectifying currents with properties characteristics of TRPM7 and/or TRPM6 . Addition of 10 mM extracellular Mg2+ blocked the TRPM7 or TRPM6-like current in both cells , reducing its magnitude (current amplitudes are summarized in right panels). This data shows for the first time the presence of currents that are consistent with functional expression of TRM7- and/or TRPM6-like channels on the plasma membrane (PM) of mouse oocytes and eggs.Extracellular magnesium (Mg2+-activated K+ channels (SK)Ca 2.1–2.3 channels, was recently found to block with higher selectivity over other TRP channels, currents mediated by native and transfected TRPM7 channels2+. We also performed these studies in Trpv3−/− oocytes and eggs, as some modulators of TRPM7 channels can affect the activity of TRPV3 channels34To test the functional expression of TRPM7 in oocytes and eggs, we made use of a series of compounds with specificity for this channel and determined how they affected its function. TRPM7 current can be blocked by modulators targeting small conductance Ca2+ influx is required to fill the stores during maturation and to refill them following fertilization2+ influx in oocytes and eggs, we tested the [Ca2+]i responses induced by the TRPM7-specific activator Naltriben. WT oocytes responded to a wide range of Naltriben concentrations with brief [Ca2+]i peaks, which were preceded by a transient decrease in baseline [Ca2+]i (left panel). On the other hand, MII eggs required higher concentration of Naltriben and the response seems to be sustained (right panel). To confirm that [Ca2+]i increases induced by Naltriben were caused by Ca2+ influx and not due to intracellular off targets effects of the drug, we exposed the cells to Naltriben in media without Ca2+. Oocytes and eggs failed to show [Ca2+]i increase in response to the agonist in absence of Ca2+ in the media (left and right panels). These results suggest that Naltriben-induced [Ca2+]i increases in oocytes and eggs are caused by Ca2+ influx mediated by TRPM7 channels.Ca2+]i oscillations which are inhibited by high concentration of Nickel, an unspecific blocker of Ca2+ channels2+ influx supporting these oscillations in oocytes is unknown. As we have shown in this study, TRPM7-like channels expressed in oocytes are able to mediate Ca2+ influx. Therefore, to further evaluate the role of TRPM7 channels supporting Ca2+ oscillations in oocytes, we tested the efficacy of NS8593 in blocking these [Ca2+]i responses. As expected, WT oocytes displayed spontaneous [Ca2+]i oscillations in presence of 2 mM extracellular Ca2+ (left panel). The frequency of the oscillations rapidly increased by bringing the external Ca2+ concentration to 5 mM (see left panel), suggesting enhanced Ca2+ influx. The addition of NS8593 to the culture media prevented both spontaneous and Ca2+ (5 mM) enhanced oscillations (right panel). These results suggest that TRPM7-like channels may underlie [Ca2+]i oscillations in GV oocytes.GV oocytes display spontaneous i oscillations following release from antral follicles2+ release from intracellular [Ca2+]]i stores, but ultimately depend on extracellular Ca2+ and Ca2+ influx; these oscillations are not required for the GVBD to take place2+ with Sr2+. Exposure to Sr2+ is a well-known method of activation of rodent eggs, as Sr2+ induces persistent oscillations in MII eggs that promotes parthenogenesis26432+ in oocytes is not the same that permeates it in eggs, as whereas Trpv3−/− eggs are unable to mount Sr2+ oscillationsTrpv3−/− GV oocytes do so normally. TRPV3 channels, which are responsible for Sr2+ influx in mouse eggs, are temperature sensitive29302+ in oocytes can occur at RT. Accordingly, Sr2+ (and Ca2+) induced oscillations in GV oocytes may be mediated by the same channel(s). Consistent with this notion, NS8593 inhibited [Ca2+]i and Sr2+ oscillations in oocytes, but not in eggs. These results suggest that differential expression exists among oocytes and eggs regarding PM channels that mediate cation influx and efflux. Our results showing functional expression of TRPM7-like channels in GV oocytes suggest this channel, which is permeable to divalent cations including Sr2+, is a candidate to mediate Ca2+ and Sr2+ influx in oocytes.GV oocytes undergo spontaneous i oscillations2+-transporters such as ZIP6 and ZIP10 have been shown to be responsible of the accumulation of Zn2+ 2+ efficiently2+ can regulate the binding of the α-kinase domain of TRPM7 to transcription factors containing zinc-fingers, it cannot be ruled out that TRPM7 contributes to gene expression in early embryos. Furthermore, the cleaved kinase domain of TRPM7 can bind components of the Polycomb Repressor Complex 1 (PRC1)2+ influx/efflux in oocytes and embryos, its activity regulating Zn2+ homeostasis might have an important role in epigenetic changes during embryo development.Zinc  medium and 100 μM isobutyl-1-methylxanthine (IBMX). Experimental protocols completed at Aarhus University were performed according to the Danish national and Institutional regulations and approved by the Animal Experiments Inspectorate under the Danish Ministry of Justice .Six-to-twelve-week-old females . Eggs were loaded with 1.25 μM Fura-2AM supplemented with 0.02% pluronic acid (Molecular Probes) for 20–30 min at room temperature. To estimate [Ca2+]i, oocytes/eggs were thoroughly washed and attached to glass-bottom chambers. For 2 were recorded in FCS-free (serum free) HEPES-buffered Chatot, Ziomek, or Bavister (HCZB) medium, where external Ca2+ was replaced by 10 mM SrCl2, and under mineral oil. Ca2+ measurements were performed on the stage of an Olympus IX70 microscope equipped with an Olympus LUCPlanFL N × 20 objective (N.A. 0.45) and an EasyRatioPro fluorescence imaging system . The oocytes were exited at 340 and 380 nm and emission was recorded at 510 nM. Intracellular Ca2+ ([Ca2+]i) was expressed from 340/380 fluorescence ratio. For 2+ (2.04 mM), and Mg2+ (0.492 mM) under mineral oil. For 2+ concentration described in the figure. For the Sr2+- induced oscillations measurements, Ca2+ was replaced by 10 mM SrCl2. Oocytes and eggs were monitored using inverted microscope (Nikon) fluorescence measurements. Fura 2-AM was excited between 340 nm and 380 nm by a filter wheel (Ludl Electronic Products Ltd.), and fluorescence was obtained every 20 s. After passing through a 510 nm, the emitted light was collected by a Cool Photometrics SenSys CCD camera  to calculate fluorescence ratios of 340/380 nm in the whole egg.[Ca2. Data were analyzed using Igor Pro and Origin 7.0 (OriginLab). Pipettes of 1–3 MΩ resistance were made from glass capillaries . The intracellular solution contained (in mM): 142 Cs-Methanesulfonate, 10 HEPES, 3 NaATP, 0.3 NaGTP, 5 EGTA, 3 mM CaCl2 (free 100 nM) pH: 7.3–7.4. Concentration of Calcium was calculated using WincMax Chelator. The external solution for giga seal formation contained (in mM): 125 NaCl, 6 KCl, 20 mM CaCl2, 1.2 MgCl2, 20 mM HEPES-NaOH, pH: 7.3–7.4. TRPM7 basal responses to agonists or blockers, were measured in an external solution containing (in mM): 140 mM NaMES, 10 mM HEPES, 10 mM glucose, 4 mM KCl, and 2 mM CaCl2. The solution of NaMES was used in order to avoid chloride currentsWhole-cell currents were measured at 22–24 °C using an HEKA 10 USB amplifier. Electrophysiology recordings were performed on the same day of oocyte, egg or embryo isolation up to 8 hours post-surgery. Eggs and embryo were maintained in human tubal fluid medium  and GV oocytes in CZB medium (EMD Millipore) at 37 °C and 5% CONS8593 (SIGMA), Naltriben (SIGMA).in vitro pre-implantation development even in control embryos. After collection, zygotes were cultured in KSOM media  with or without 10 μM NS8593 in four-well dishes . Assessment of embryo development was made every 12 or 24 h according to the experimental design until 96 h of culture, where blastocysts rates were assessed. At least two replicates were performed per strain and Chi square tests were performed for significance.Zygotes were collected from the oviducts of 6 to 8-weeks-old super ovulated CD1 and TRPV3KO females mated to CD1 males. CD1 zygotes were recovered 15 h post-hCG whereas TRPV3KO zygotes were collected at 20 h post-hCG, as earlier collection greatly reduced How to cite this article: Carvacho, I. et al. TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse. Sci. Rep.6, 34236; doi: 10.1038/srep34236 (2016).


Hair follicle stem cells exist in different sites. Most of the hair follicle stem cells are reside in niche called bulge. Bulge region is located between the opening of sebaceous gland and the attachment site of the arrector pili muscle.Data were collected using databases and resources of PubMed, Web of Science, Science Direct, Scopus, MEDLINE and their references from the earliest available published to identify English observational studies on hair follicle bulge region.Bulge stem cells are pluripotent with high proliferative capacity. Specific markers allow the bulge cells to be isolated from mouse or human hair follicle. Stem cells isolated from bulge region are label retaining and slow cycling hence these cells are defined as label-retaining cells. Bulge cell populations, due to their plasticity nature are able to differentiate into distinct linage and could contribute in tissue regeneration.The current review discuss about bulge stem cells characteristics and biology including their cycle, location, plasticity, specific markers and regenerative nature. Also the differences between mouse and human hair follicles are investigated. In recent years, the identification and characterization of adult stem cells was one of the biological and biomedical research interests . Adult sHair follicles reconstitute during the cycle that initiate with growing phase (anagen), followed by regression phase (catagen) and finally resting phase (telogen) .Since in recent years, adult stem cell therapy plays an important role in clinical application, thus more investigating about hair follicle stem cells may lead to treatments of injuries and diseases, therefor the aim of this study is to discuss about bulge stem cells characteristics and biology.Databases and resources of PubMed, Web of Science, Science Direct, Scopus, and MEDLIN from the earliest available online indexing year through 2016 were searched. The search keywords included bulge, hair follicle, stem cell, marker, regeneration, differentiation, pluripotent, and self-renewal. The reference lists of articles were read to obtain additional information. Only the English publications were studied. Lately published cohort and case-control studies on the interesting topic are cited. First, the titles and abstracts of all retrieved studies were screened, then the full texts of potentially eligible studies were reviewed before definitive inclusion and relevant data were extracted from studies.Hair as one of the skin appendages grows from hair follicle . Hair foHair growth rather than occurring continually is an alternating cycles of growth and quiescence . For genIn each growth cycle, for generating transiently amplified cells, stem cells resident in the niche proliferate, subsequently a differentiation process occurs and the new hair shaft is built . Indeed,Follicles generate hair shaft during anagen stage, subsequently within catagen and telogen follicles reset to be able to receive signals in order to onset the next cycle . BMP andMatrix keratinocytes located in the hair follicle bulb, rapidly proliferate and form the hair during anagen.The duration of anagen is variable in hairs of different sites . AccordiBy the end of anagen, epithelial cells below the bulge stop proliferation and undergo apoptosis; in the meanwhile the dermal papilla mesenchyme cells survive and after entering into catagen phase form the club hair . During LRC population localized at bulge area, it was though that, the hair follicle bulb area harbors stem cell and during anagen phase, the secondary germ ambulate to the follicle bulb in order to produce new hair, and in return during catagen, secondary germ move upward with dermal papilla  15)..15).Hair follicle stem cells (HFSCs) are major source of pluripotent adult stem cells . These cTo identify adult stem cells, the main method is using their slow cycling nature. Generally all the cells of a tissue are labeled with a DNA precursor, including bromodeoxyuridine (BrdU) or tritiated thymidine (3H-T). In this process, only the slow cycling cells retain their labels while rapidly dividing cells lose most of their labels . In thisTo identify and isolate stem cells, obtaining specific stem cell marker is essential. Likewis e, detecting bulge specific marker helps to identify the existent stem cells. Hence researchers utilized many experiments for the purification of bulge stem cells –21.Keratin 15 (K15) is an intracellular intermediate filament protein; this marker has high expression in the bulge region. Indeed the LRC reservoir is K15 positive .It is notable that K15 has low level expression in the lower follicle. Thus K15 is not limited to bulge cells and it is not a very specific marker of this region . HoweverFor the first time Lyle et al. found that human bulge cells are K15 positive . Ohyama CD34 is a surface protein, which is recognize as a specific marker of mouse hair follicle bulge stem cells  but not Ohyama et al. discovered that human bulge LRC expressed CD34 in low level . ConsequNobakht et al. limited their experiments on bulge area of rat vibrissa and found that stem cell marker including Nestin and CD34 were expressed in rat vibrissa bulge cells . On the Other parts of hair follicle could be identified by other specific markers . IsthmusThe bulge area is also reservoir of melanocyte stem cells (MSCs). Melanocytes proliferate to re-populate the melanocytes which raise melanin in order to accomplish hair pigmentation .Major researches for studying hair follicle stem cells are performed on mouse models. Significant differences exist between microanatomy of mouse and human hair follicle stem cells. Hence results from mouse models are necessary to be confirmed in human organism . Since tIn mouse pelage hair follicle, bulge is specified as a distinct swelling section of outer root sheath. Despite human bulge is also well identified in embryonic hair follicle, it is found as a petty distend in adult hair follicle .Human scalp follicles are 5 mm long, while the mouse pelage follicles are 1 mm in length . AnotherOhayama et al. found many identical and different genes that express in human and mouse bulge cells , and ideCD200, a membrane glycoprotein is a subset of immunoglobulin superfamily . Using mGene expressions control the noncyclic nature and quiescent of hair follicle stem cells, defining gene expression that distinct bulge and non-bulge cell populations provide intuitions of the maintenance of the stem cell . During A combination of bulge cell isolation and multiple gene expression microarray analysis allows distinguishing bulge stem cells form other differentiated cells. Mouse bulge cells molecular signatures by using different isolation methods were obtained .Bulge cells microarray results declared that genes involved in morphogenesis, organogenesis and development are up regulated, in contrast gene related to cycling and proliferation were down regulated, these results were compatible with quiescent property of bugle stem cells . These fImmunohistological staining declared that K19 and β1-integrin were highly expressed in bulge area . CD34 isOn the other side, human bulge cells surface markers were identified by microarray analyses, such as CD200 and CD59 which are up regulated in bulge and CD24, CD34, CD71 and CD147 are down regulated. Among these markers CD200 is the best human bulge cells marker. In addition, frizzled homolog 1 (FZD1), was up regulated in human bulge cells.Interestingly, some human bulge up regulated genes including DIO2 and ANGPTL2 were not expressed in mouse bulge cells. The mouse bulge cell marker CD34 were expressed in human bulge in low level .Hair follicle development and physiology are under control of signaling cascade. Signaling pathways regulate quiescent, differentiation and proliferation of hair follicle stem cells, including BMP, Wnt, Shh, Notch, FGF and TNF –4. For hHowever, Wnt and some other signals induce stem cell proliferation while BMP signals suppress cell activation. Major BMP signaling role is regulating terminal differentiation of hair shaft and IRS. BMP and FGF18 signals secreted from dermal papilla, the internal K6+ bulge area and bulge stem cells, these signals make bulge stem cells retain their slow-cycling nature. When Wnt signal activates, BMP signal suppresses and results in onset of hair germ proliferation .Another signaling pathway that control hair follicle stem cell function is sonic hedgehog (Shh) signaling. Shh is a second important signaling pathway that controls hair follicle bulge stem cells . Shh/GliStem cells due to their mulipotent nature are able to reorganize different lineages in the specific tissue. To study multipotnecy of bulge stem cells, various researches have been performed . Bulge sTaylor trace bulge stem cells of pelage follicle using double labeling, in lower follicle, faint labeling was detected in some cells. These results indicating that, these cells had originated from bulge region . ElsewheTo study bulge cells multipotency, Oshima et al. focused on transplantation. They worked on ROSA26 (transgenic mice) that express lacZ consistently. They dissected the bulge area from ROSA26 vibrissa follicles. Afterward, labeled bulge were transplanted into the vibrissae of non-ROSA26 mice. After downward migration of bulge cells through vibrissa follicle, expression of lacZ in all epithelial cell layers of some follicles were observed .Moriis et al. to study about bulge cells mulipotency used K15 promoter targeted with CrePR1. K15-CrePR1 transgenic mice were crossed with R26R (reporter mice) which could express lacZ under control of ROSA26 promoter. lacZ expression was observed in bulge cells and all their progeny. This experiment made it clear that bulge progeny could generate all epithelial cell layers located in the lower hair follicle .in vitro, repopulation of hair follicle, epidermis, sebaceous gland and bulge cells was observed, these results support the multipotency nature of bulge stem cells  have been completely observed by the authors.


Decreased Ca2+ signaling is observed in aged cells, probably secondary to the formation of disulfide bonds among Ca2+ signaling-related proteins. Skin aging processes are modeled by treating keratinocytes with H2O2. In the present study, H2O2 dose-dependently impaired the adenosine triphosphate (ATP)-induced Ca2+ response, which was partially protected via co-treatment with β-mercaptoethanol, resulting in reduced disulfide bond formation in inositol 1, 4, 5-trisphosphate receptors (IP3Rs). Molecular hydrogen (H2) was found to be more effectively protected H2O2-induced IP3R1 dysfunction by reducing disulfide bonds, rather than quenching ROS. In conclusion, skin aging processes may involve ROS-induced protein dysfunction due to disulfide bond formation, and H2 can protect oxidation of this process.Based on the oxidative stress theory, aging derives from the accumulation of oxidized proteins induced by reactive oxygen species (ROS) in the cytoplasm. Hydrogen peroxide (H This theory began as a proposal that oxygen was poisonous2, followed by the notion that reactive oxygen species (ROS) are a cause of aging, and was eventually modified as the oxidative stress theory in 19723. Among the large number of aging models proposed to date, the oxidative stress hypothesis is considered the most likely, because ROS are continuously produced in aerobic cells. Stepwise reduction of O2 produces several ROS, such as superoxide radicals 2O2), and hydroxyl radicals 4. Despite the enormous amount of data, however, the molecular mechanisms of aging are not clearly elucidated.The oxidative stress theory of aging suggests that aging results from the accumulation of aberrant proteins in the cytosol, chemical damage to macromolecules, and mitochondrial DNA changes2O2 is generally used as an instrumental ROS species despite some limitations, such as the complex effects of H2O2 on catalase. Further, H2O2 may be transported across the membrane by aquaporin channels5 and act as superoxide anions, major ROS released from the mitochondria that are converted to H2O2 by superoxide dismutase6, and the increased release of H2O2 mimics the aging process7. Interestingly, H2O2 selectively allows for the oxidization of cysteine or methionine sulfhydryl groups to sulfenic acid or disulfide bonds9, inducing cytoplasm protein dysfunction with the formation of disulfide bonds10. Therefore, H2O2 impairs various physiologic processes via the oxidation of thiols, especially those in proteins. In addition, over the last 20 years, Ca2+ signaling has been identified as crucial for normal physiologic processes13. The effect of protein oxidation (or effect of aging) on Ca2+ signaling is therefore an important topic. Based on several published papers, most aged cells have decreased Ca2+ responses in the endoplasmic reticulum and decreased Ca2+ release16. These findings suggest that inositol 1, 4, 5-trisphosphate receptors (IP3Rs)-mediated Ca2+ release from ER must also be decreased14. Accordingly, the aging-related reduction of Ca2+ signaling may be mimicked by H2O2-induced disulfide bond formation.H2 gas selectively reduces ROS, especially hydroxyl radicals, and can strongly slow the rate of aging processes or the progression of aging-related diseases, such as ischemia, reperfusion brain injury, and Parkinson’s Disease18. Since the lifetime of ROS in the tissue is very short19, the effect of H2 on quenching of ROS is through to be limited. If H2 could be made to more effectively reduce the formation of disulfide bonds between SH groups, H2 could more efficiently reduce ROS-induced damage. Among human tissues, the skin is very prone to damage, thus the present study focused on the effect of treating human skin cells, keratinocytes (KC), with H2 to protect against ROS-induced damage. Specifically, the protective effect of H2 treatment against ROS-induced dysfunctional disulfide bond formation and recovery Ca2+ signaling was examined. The data demonstrated that aging processes in KC was found to be selectively oxidized IP3Rs, especially IP3R1-mediated Ca2+ signaling by inducing the formation of H2O2-mediated disulfide bonds in the skin. In addition, a major protective effect of H2 was to reduce disulfide bond formation in the protein caused by oxidative stress, and not by eliminating the generation of ROS in the skin.Recently, a large number of studies have demonstrated that H2O2 were used to mimic the aging process induced by ROS accumulation. Approximately 105 KC were plated onto 24-mm coverslips in 3.5-cm2 dishes, incubated for 2 days, pre-stained with 20 μM 2′,7′-dichlorofluorescein-diacetate (DCFH-DA), then treated with 5 μM, 50 μM, or 500 μM H2O2. Imaging of the DCFH-DA staining showed ROS generation in the cells, as evidenced by an increase in fluorescence intensity, and dose-dependent effects of H2O2 exposure were observed with time-lapse recordings -induced Ca2+ elevation and store-operated Ca2+ (SOC) influx in a dose-dependent manner in KC . ATP-induced Ca2+ elevation was completely inhibited by 500 μM H2O2  to 248  relative to vehicle, when 2-ME was applied with H2O2  or 79 ± 12% (500 μM H2O2 + H2) relative to that of the vehicle group  or 407 ± 73 nM (500 μM H2O2 + H2)  that of vehicle and the Ca2+ signal of H2-treated cells was 80 ± 3%  Fig. . There wone Fig. . To furt2-BSS had different effects on 500 μM H2O2-induced damage of Ca2+ release with IP3Rs in KC, we performed additional experiments to make the difference between the effects of H2 and 2-ME. Briefly, cells were pretreated with 2-ME or H2 gas for 30 min and compared  was 185 ± 19% that following pretreatment with vehicle   and Western blot analysis were performed as described previously10. We focused on examining the formation of disulfide bonds of IP3Rs with three subtypes: IP3R1, IP3R2 and IP3R3 via treating the specific antibodies23. All IP3Rs display the molecular weight over 250 kDa. As shown in Fig. 3R1 with disulfide bonds, as detected by IP3R1 antibody and migration in a broad band secondary to oxidized S-S formation. However, IP3R2 and IP3R3 did not present clear disulfide bonds formation based on the similar molecular weight of broad band  SDS-PAGE and reducing SDS-PAGE (with 2-ME in protein loading buffer)10. As expected, KC exposed to H2O2 had IP3R1 containing disulfide bonds, as evidenced by the lower broad band when electrophoresed under non-reducing conditions, but KC treated with a reducing agent  had a higher band and lightly stained bands similar to the vehicle-treated KC  Fig. . The enoand Fig. . The for KC Fig. . Western KC Fig. . This fi KC Fig. . The fin2O2, is labeled with maleimide-PEO2-biotin (MPB)29. We examined whether MPB was applicable to assay H2O2-induced intermolecular disulfide bond formation in receptor protein-tyrosine phosphatase α (RPTPα). MPB-containing peptides signal located the intermolecular disulfide bond to the Cys-723 of RPTPα, and the counts of H2O2-induced MPB-containing peptides was higher than the counts in the group of vehicle or H2O2 with 2-ME  the counts of MPB-containing peptides . The sulfhydryl residue Cys or Met, through which disulfide bridges can be produced by Hins Fig. ; Met-5, ins Fig. . H2 trea2+ signal dysfunction via the formation of H2O2-induced disulfide bonds10 in IP3Rs that lead to selective damage of the IP3Rs. Co-treatment with 2-ME and 500 μM H2O2 decreased the disulfide bond formation, evidenced by the protection of ATP-induced Ca2+ release in KC with 2-ME. This provides clear evidence that oxidative stress-related skin damage is associated with Ca2+ signaling defects, similar to 2-ME, H2 gas in BSS decreased IP3R1 disulfide bond formation due to ROS exposure but not IP3R2 and IP3R3. The major function of H2 in BSS in skin was the protection of ATP-induced Ca2+ signaling, but not by direct quenching of ROS. ATP is thought to be an external 1st cell-to-cell communication around the other cells equipped with GPCR. Because BSS media has no Cu and Fe to make hydroxyl radical, contribution of hydroxyl radical will be smaller than usual when the cells are treated by H2O2. Previous reports indicate that the presence of reducing agents decreased number of disulfide bonds, resulting in a loss of cross-link-induced stability produced by the chemical microenvironment32. It is reasonable to assume that the skin aging process is associated with ROS accumulation in the skin, which leads to IP3Rs dysfunction by inducing conformational changes due to disulfide bond formation.The findings of the present study reveal the potential association of the skin aging process with Ca2 in acute erythematous skin diseases, which are associated with large ROS accumulation in KC33. H2 improved significantly in erythema of these patients and did not affect physiological parameters and deterioration of the blood chemistry33. Although elimination of ROS produced by H2O2 is also performed in H2, our study firstly indicates H2 also reduces disulfide bond formation and restores protein function. However, our finding revealed IP3R1 but not IP3R2 and IP3R3 is recovered the function by H2 treatment. It may be possible due to the different distribution of S-S bond formation in each subtype of IP3Rs. H2O2 and H2 independently oxidize and/or reduce each subtype of IP3Rs; therefore, the exposed site (Cys or Met) of each subtype of IP3Rs could be affected by H2O2 and H2 independently or correlatively. This is reasonable to explain why IP3R1 displays more obvious in disulfide bond formation and is more efficient for H2 reduction.Recent study has pointed out the treatment of H2 acts also as a reducing agent at a lower concentration, but the amount of H2 was sufficient to disrupt metabolic oxidation reducing reactions or to interrupt ROS-induced disruption of cell signaling17. H2 induces superoxide dismutases (SODs) and heat shock proteins (HSPs) activity to quench ROS production6 and may thereby completely protect the IP3Rs Ca2+ signal. Nevertheless, pretreatment with H2 failed to decrease H2O2-induced ROS production. Another possible mechanism underlying the H2-induced elimination of ROS damage of IP3Rs is the activation of glutathione/thioredoxin systems, which reduces H2O2-induced disulfide bond formation. To date, however, it is not clear whether H2 facilitates glutathione/thioredoxin system activity, although studies have reported that glutathione/thioredoxin systems are involved in the modulation of disulfide bond formation during oxidative stress34.Besides, H35. Superoxide radicals (∙O2−), hydroxyl radicals (∙HO), and peroxyl radicals (LOO∙), singlet oxygen (1O2), and hydrogen peroxide (H2O2) are involved in UV-stimulated oxidative stress6. According to a previous report17, however, H2 selectively reduces the hydroxyl radical, yet it is not involved in the efficient elimination of oxidative-stress-induced damage due to UV irradiation. The results of the present study indicate that, rather, a major mechanism underlying H2 effects is to protect IP3Rs-mediated Ca2+ signaling by reducing ROS-induced disulfide bond formation. IP3Rs-mediated Ca2+ signaling has a central role in several physiologic processes, such as fertilization, proliferation, muscle contraction, cell metabolism, vesicle and fluid secretion, and information processing11. As aging-induced dysfunction of Ca2+ signaling leads to cell death or genome instability, H2 can protect skin cells from destruction by maintaining the Ca2+ signal and thus preserving normal physiologic conditions. The results of the present study clearly demonstrate the role of H2 in protecting against the aging process in skin and provide evidence to support further investigation of the clinical applicability of H2.In the skin aging process, UV irradiation increases ROS production36. The skin specimens were washed with phosphate-buffered saline (PBS), cut into small pieces, and cultured in medium containing 0.25% trypsin (Gibco BRL) overnight at 4 °C. The epidermal sheet was lifted from the dermis utilizing fine forceps. The epidermal cells were then pelleted using centrifugation  and separated into individual cells by repeated aspiration. The KC were incubated in serum-free KC growth medium, supplemented with human recombinant epidermal growth factor and bovine pituitary extract (5 μg/mL each), insulin (5 μM/mL), and hydrocortisone  at 37 °C in humidified 5% CO2, and supplemented with KC growth medium for five generations37.Human primary KC were isolated from foreskins obtained via routine circumcision as described previously2+ response was induced by application of ATP (Sigma-Aldrich), according to methods previously described20. Before the experiments, cells were stained with 1 μM Fluo-4-AM (Molecular Probes) at 37 °C for 20 min and then washed with BSS buffer . Intracellular Ca2+ concentrations were estimated based on the ratio of fluorescence intensities emitted upon excitation with consecutive 3-s pulses of 488-nm light at a resolution of 1376 × 1038 pixels using an Olympus Cell^R IX81 fluorescence microscope (Olympus) equipped with an MT 20 illumination system (Olympus) and UPLanApo 10 × objective lens. The intracellular Ca2+ concentration was estimated based on calibration curves as follows. A Ca2+ calibration curve was created using a Ca2+ Calibration Buffer kit (Molecular Probes). Intracellular Ca2+ ([Ca2+]i) was calculated from Fluo-4 excited at 488 nm and imaged using an Olympus Cell^R IX81 fluorescence microscope and UPLanApo 10 × objective lens at 20 °C. Fluo-4 signals were calibrated by measuring the fluorescence intensity from microcuvettes containing 10 mM K2-EGTA (pH 7.20) buffered to various [Ca2+] levels. Ca2+ concentration was calculated using the following formula: [Ca2+]i = KD *(F − Fmin/Fmax − F). Plotting the fluorescence intensity versus [Ca2+] yielded the calibration curve with the formula of: [Ca2+]i = KD *(F − Fmin/Fmax − F), where KD = 345 nM, F = Fluo-4 intensity, Fmax = 640, and Fmin = 21.7 for Fluo-4.The intracellular Ca3Rs by photolysis using UV light (300–400 nm) as described previously20. For photolytic uncaging, an Olympus FV1000 MPE multiphoton laser scanning microscope equipped with an argon laser was used to produce a collimated light beam as the principal uncaging laser line at λ = 408 nm. To detect the effect of H2O2 (Sigma-Aldrich) on IP3Rs in Ca2+ pathways, 105 primary KC were plated on 2.4-mm coverslips in a 4-cm dish. For H2O2 treatment, cells were pretreated with 1 μM caged IP3 (Molecular Probes) for 2 h38 and stained with the Ca2+ dye Fluo-4 to analyze changes in intracellular Ca2+ levels. Ca2+-induced fluorescence was observed using an Olympus FV1000 laser-scanning microscope. Caged IP3 was uncaged by illumination with UV light (λ = 408 nm) and the released IP3 molecule was immediately able to bind to the IP3Rs.Caged compounds were uncaged to investigate modifications of IP3R1 , phospho-IP3R1 , IP3R2 (Merck Millipore), IP3R3 (BD Biosciences) and α-Tublin (Santa Cruz Biotech), and then with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence reagents (Amersham Biosciences).Western blot analyses were performed utilizing whole-cell lysates. Briefly, cells were lysed by incubating for 30 min on ice in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), containing proteinase and phosphatase inhibitors. Cell debris was removed via centrifugation at 10,000 g for 10 min at 4 °C. The protein concentration of cell lysates was determined using the Bradford method (Bio-Rad). Proteins (100 μg) in cell lysates were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 7% gel with or without 2-ME and then transferred to a Hybond-P polyvinylidene fluoride membrane (Amersham Biosciences). The membrane was first incubated with primary antibodies against IP2O2, reducing agent 2-ME, or H2 gas-containing BSS (H2-BSS) at 37 °C for 30 min. ROS production was determined using flow cytometry  with fluorescence emission at 488 nm.DCFH-MA (Sigma-Aldrich) staining was used to quantify ROS generation from the cells. Briefly, cells were stained with 20 μM DCFH-DA at 37 °C for 20 min and then washed with BSS buffer. The cells were collected after application of Het al. study29. Briefly, the cells were treated 2.5 mM Methyl-PEO12-maleimide  with 0.05% TritonX-100 (Sigma-Aldrich) in PBS containing 1% bovine serum albumin  at 4 °C for 30 min; MPM can entry the cell and bind the intracellular free sulfhydryl groups with TritonX-100 treatment. After washing the cells with PBS containing 1% BSA at 25 °C for three times (1 min/time), the cells were reduced with 2.5 mM tris(2-carboxyethyl)phosphine  and 10 mM dithiothreitol  at 25 °C for 30 min. After washing (1% BSA in PBS) three times, cells were labelled with 2.5 mM Maleimide-PEO2-biotin  in PBS containing 1% BSA at 4 °C for 30 min, and then cells were washed for three times. Total protein lysates were collected for in solution digestion after adding M-PER protein extraction reagent (Thermo Fisher Scientific).To investigate the intracellular disulfide bonds formation, we modified the labeling protocol from Clive Metcalfe E).We utilized nitrogen gas to blow-dry the total protein lysates to protein pellet, and suspended protein pellet with 0.1% RapiGest SF solution (Waters). Protein sample was treated with 20 mM iodoacetamide  in the dark for 30 min and then desalted by using Amicon® Ultra-0.5 Centrifugal Filter Device (Merck Millipore). After incubating with trypsin for peptides digestion overnight, protein sample treated with 1% of formic acid  and confirmed pH <2 with a pH paper. The tryptic peptide sample was analyzed disulfide bonds labeling by use of quantitative proteomics techniques utilizing serially coupled lipid chromatography data-independent parallel fragmentation mass spectrometry (LC/MSE). Parallel ion fragmentation will be programmed to switch between low (4 eV) and high (15–45 eV) energies in the collision cell, and data will be collected from 300 to 2000 m/z utilizing glu-fibrinopeptide B  as the separate data channel lock mass calibrant. Data will be processed with ProteinLynx GlobalServer v3.0 (waters) for qualification and Progenesis QI for proteomics (Waters) for relative quantification, respectively. Deisotoped results will be searched for protein association and modification from the Uniprot (www.uniprot.org) human protein database.Quantitative analysis will be performed essentially on a Waters Xevo G2 qTof mass spectrometer (Waters). In brief, the tryptic peptide sample will be chromatographically separated on M-class UPLC separations module (Waters) incorporating 50 femtomole tryptic digested BSA as the internally spiked protein quantification standard. Peptide elution will be executed through a 75 μm × 25 cm BEH C-18 column (Waters) under gradient conditions at a flow rate of 300 nL/min over 70 min at 40 °C. The mobile phase will be composed of acetonitrile as the organic modifier and formic acid (0.1% v/v) for molecule protonation. Mass spectrometry was performed on Xevo G2 qTof (waters) instrument equipped with a nanoflow electrospray ionization (ESI) interface and operated in the data-independent collection mode (MS2 water) was produced using an Aurora H2 water-making machine . To generate H2-containing BSS, First, H2 water with one-tenth concentration of BSS was made from sterile water using the Aurora machine. Then, H2 water was mixed with BSS stock solution to make final H2-containing BSS (pH = 7.4). The H2 content was measured using a hydrogen electrode .Hydrogen-containing water  in Kaohsiung Medical University Chung-Ho Memorial Hospital, number KMUH-IRB-960119. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent of all participants was obtained. A total number of 32 samples (foreskins) were collected from 2007 to 2008 of which were further analyzed by isolating KC from skin.GraphPad Prism  was used to generate bar charts; error bars indicate standard deviations. A one-way, two-tailed analysis of variance (ANOVA) was also utilized to compare means of each group. A P-value of less than 0.05 for differences between groups was considered statistically significant.supplementary information


These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca2+ sensors is also presented.Ca In plants, continuous exposition to changing and potentially harsh conditions induces diverse spatial and temporal patterns of Ca2+ levels [2+ signatures”, these changes provide plants with information about external stimuli that are decoded using highly-specific protein sensors which trigger the appropriate physiological responses. Ca2+ sensors affect the activity of downstream effectors that synchronize changes in metabolism, gene expression, and turnover of proteins.As second messengers, Carocesses . The pro2+ sensors contain a highly-conserved helix-loop-helix motif, called the EF-hand, which is composed of 29 amino acids; the central 12 residues form a loop structure that coordinates one Ca2+ ion (Most CaCa2+ ion .2+ sensors in growth and development of plants is highlighted by the diversity and large number of proteins identified to date with Ca2+ binding domains. There are three families of Ca2+ sensor proteins in plants: (i) calmodulin (CaM) and CaM-like (CML); (ii) calcineurin-B-like (CBL); and (iii) Ca2+-dependent protein kinases  [The importance of Caidopsis) ,5,6. Of 2+ sensor; it is highly conserved from an evolutionary standpoint and is present in all eukaryotic cells [2+ with positive cooperativity. In addition to multiple CaM isoforms, plant genomes encode a remarkable number of CMLs whose primary sequences have ≥16% overall identity with the canonical CaM sequence ; functional motifs other than EF-hands are notably absent [Arabidopsis, seven genes are present that code for four CaM isoforms  as well as 50 genes that code for CML proteins [Oryza sativa [Lotus japonicus [2+ sensors in the green lineage [CaM is undoubtedly the best characterized Caic cells ,10,11,12y absent ,13. In Aproteins ,13. The a sativa , 52 in ta sativa , 36 in waponicus , and 79 aponicus ), revealArabidopsis CMLs in development and response to both biotic and abiotic stimuli have been summarized in several recent reviews, and convincing evidence has been provided that these proteins are not likely to have redundant functions, but rather play central and highly specific roles in coordinating environmental responses of plants. In addition, many CMLs are now known to recognize a specific target [The functions of c target ,21,22,232+ to CMLs and the Ca2+-induced structural rearrangements are just beginning to emerge. Importantly, only one 3D atomic structure of a CML is present in the Protein Data Bank (PDB), i.e., the Ca2+-loaded form of the N-terminal domain of CML34 from Arabidopsis thaliana (PDB code 1TIZ) obtained by protein nuclear magnetic resonance (NMR) spectroscopy [2+-bound forms of CMLs are still missing. Nevertheless, it has been possible to obtain useful data about the dynamic properties of CMLs using combined biochemical approaches, which include isothermal titration calorimetry (ITC), NMR, circular dichroism (CD), and fluorescence spectroscopy. The emerging scenario is that CMLs are a highly diverse family of proteins that act generally, but not always, as Ca2+ sensors, and provide a wide variety of physiological responses to Ca2+.Empirical data on the affinity of Catroscopy , while X2+ sensors. The majority of the examples given within come from members of the Arabidopsis CML family since there is more biochemical, structural, and functional information for this family than for any other plant species.The present review discusses and summarizes the current knowledge on plant CMLs, from both biochemical and structural perspectives, and provides an overview of the principal techniques used to study these Ca2+ has the ability to provide interactions that are dominated by ionic forces [2+ with pentagonal bipyramid geometry. In particular, the Ca2+ ion is coordinated in the canonical EF-hand to carboxylate oxygens from residues 1 (+X), 3 (+Y), 5 (+Z) and 12 (-Z), carbonyl oxygen from residue 7 (-Y) and bridged water at position 9 (-X). A glycine residue at position 6 is highly conserved, allowing the loop to encompass the Ca2+ ion, which is a critical feature for high affinity binding [As a metal ion, Cac forces . Accordi binding   is glutamate in most Ca2+-binding EF-hand motifs, thereby providing bidentate chelation. Though non-coordinating residues, glycines at position 4 and 6 determine in large part the flexibility in the Ca2+ site. Hydrophobic amino acids  are predominantly present at position 8  are substituted by positively charged polar residues  that can perturb the network of interactions needed for efficient binding. At position 12, the substitution of glutamate with aspartic acid residue is very frequent among CMLs  that helps form the large hydrogen-bonding network upon Ca2+ binding [6 functions as an indicator of the Ca2+-bound state in an EF-hand. Using ITC and NMR data, the existence of three functional Ca2+ binding sites has been demonstrated for Arabidopsis CML42 [2+ ions, despite the presence of three EF-hand motifs [2+-binding sites, respectively, out of the four predicted EF-hands [2+-binding sites, as has been done for CML14 [Only a few CMLs have been incontrovertibly demonstrated to function as Ca binding . Thus, Gis CML42  and CML4is CML42 , and fouis CML42  and CML3is CML42   [2+ signaling system might be endowed with greater flexibility as a consequence of the different Ca2+ binding affinities of the various isoforms of CML. In this regard, a range of Ca2+ sensors for several Ca2+ signatures is likely to represent a crucial aspect for Ca2+ signal transduction. In addition, it would further appear that most CMLs have an apparent affinity for Ca2+ which differs substantially from CaM itself, leading to the possibility that they might be activated differently during Ca2+ spikes. The different binding affinities of Arabidopsis CML36 and CaM for Ca2+ ions would be critical in fine-tuning each isoform to specifically stimulate the activity of the common target, the Ca2+-dependent ATPase isoform 8 (ACA8), in different conditions [2+-affinity which is not in the range of canonical CaMs [2+-binding sites with moderate Ca2+ affinity and a third one with very low Ca2+ affinity (Kd ~ 10 mM). This low/modest affinity has been attributed to hydrophobic amino acid substitutions within the EF-hands, especially in EF-hands 1 and 4 [Kd, it is questionable whether rgs-CaM can work as a true Ca2+ sensor in vivo, even if its affinity for Ca2+ could increase in the presence of targets. Indeed, association of CaM with its targets is known to stabilize its Ca2+-bound conformation, increasing the affinity for Ca2+ [ITC analysis has underscored that M range) ,31,33,35cal CaMs . In part 1 and 4 . Howeverity Kd ~  mM. Thisfor Ca2+ ,38. 2+, Mg2+ is another physiologically important ion for plants. In plant cells, the free cytosolic concentration of Ca2+ and Mg2+ in the resting state has been reported to be about 100 nM and 0.5–2 mM, respectively [2+ are relevant. Indeed, since Ca2+ and Mg2+ have similar properties, Ca2+-binding proteins must be able to discriminate between the two cations against a 102–104 -fold excess of Mg2+. However, it has been shown that Mg2+ binding to EF-hands is important physiologically, and in reality more than one role has been hypothesized for the binding of Mg2+ [2+. Considering this possibility, binding of Mg2+ might play a functional role by shifting an activation curve to higher concentrations of Ca2+ while inactivating other enzymes at resting levels of Ca2+. In some Ca2+-binding proteins, it is known that Mg2+ binding has a function that is distinct from Ca2+ [It is worth noting that, in addition to Caectively ,40. This of Mg2+ ,40. Thesrom Ca2+ .Arabidopsis CMLs show heterogeneous behavior towards Mg2+ ion binding: in CML19 [2+, while in CML14 [2+ in the presence of Mg2+ indicates that this cation can compete directly for Ca2+ binding [2+ by 5-10 fold. For CML16 [2+ binding seems to impede the binding of Ca2+ to at least one EF-hand. Mg2+ also affects the affinity for Ca2+ in CML36 which possesses two Ca2+/Mg2+ mixed sites with high affinity and two Ca2+-specific sites with low affinity [2+/Mg2+ mixed sites for Mg2+ and Ca2+ are suggestive that both these EF-hands are normally occupied by a divalent cation during the resting state. This ensures that CML36 is in a folded ion-bound structure at all concentrations of Ca2+. After a stimulus-induced Ca2+ increase, Mg2+ is displaced and the dominant state of the protein becomes Ca2+-bound [2+ binding does not preclude the ability of CMLs to functionally respond to Ca2+.in CML19  none of in CML14 , CML15, in CML14  the weak binding ,43,44,45or CML16 , Mg2+ biaffinity . The obs2+-bound . This fi2+ has been measured exclusively in the presence of Mg2+ to mimic physiological conditions. While the approach appears to be theoretically valid, the study of Ca2+ binding in the absence and presence of Mg2+ may be crucial for understanding protein functionality. Indeed, performing NMR and ITC titrations of apo-CMLs with Ca2+ or Mg2+, as well as titration of Ca2+ in CMLs saturated with Mg2+, will provide crucial information on the possible competition between the two ions and on the influence of Mg2+ binding on Ca2+ affinities.It is worth pointing out that, in some cases ,31, the 2+ spikes due to a specific stimulus or through selective interaction with precise targets. Preserving multiple CML proteins may be essential in complex organisms to guarantee that the many Ca2+-dependent processes occur with the appropriate spatial-temporal resolution. This hypothesis may also explain the presence of 12 highly homologous (>70% identity) pairs of proteins [2+.The biochemical data on CMLs, albeit limited, give added credit to the hypothesis that the heterogeneity in the organization and composition of the EF-hands in CMLs is at the basis of their functional diversity, either by allowing activation at specific Caproteins  that couproteins  appears CML genes, including their intron/exon organization, has significant differences from that of CAMs. Indeed, the majority of CML genes are intron-less, while those of CAMs are intron rich [It is also interesting to note that the structure of the ron rich ,16,18,92ron rich  and rece2+ binding has been looked at in terms of signal transduction, focusing on Ca2+-induced conformational changes and what effects these may have on interactions with specific targets. This allows distinguishing Ca2+ sensors from what is generally referred to as “Ca2+ buffers” , which 2+ is bound. This is largely due to exposition of a hydrophobic region that is rich in Met residues  which were previously embedded within the protein. Through changing exposed surfaces, it is interesting that CaM regulates more than 300 proteins, including kinases, phosphatases, receptors, pumps, and channels [2+-induced changes in surface hydrophobicity can be utilized for purification of many recombinant Ca2+ proteins by using hydrophobic interaction chromatography (HIC). The Ca2+-dependent binding to phenyl-sepharose can be considered as a first step in studying a Ca2+ sensor protein. Along with this, the finding that CaM has increased mobility in electrophoresis if Ca2+ is present is, in fact, a defining property that can be used as another simple approach when investigating putative Ca2+ sensors [2+-dependent electrophoretic mobility shifts via SDS-PAGE  to Ca2+/Mg2+ mixed sites appear to guide the change from a molten globule apo-structure to a stable holo-protein. However, when examining the position of peaks in the forms complexed with Mg2+ and Ca2+, it is clear that the conformational changes in CML36 induced by binding of Ca2+ are distinct from those induced by Mg2+, in agreement with its hypothesized function as a Ca2+ sensor [Significant information on the structural rearrangements that CMLs undergo upon addition of metals can be obtained by 2D po-CML19 , CML42 [po-CML19 , and CMLpo-CML19 , while s globule . Cation + sensor .2+ binding ability can also be obtained by CD spectroscopy in the far-UV region. Multiple lines of evidence have indicated that CMLs contain substantial α-helical structure as for CaM. Nevertheless, in contrast to CaM for which a distinct increase in ellipticity has been observed in the presence of Ca2+, the behavior of CMLs is somewhat more variable. CD data for Arabidopsis CML15 [2+ has almost no impact on secondary structure. A modest effect on the CD spectrum upon addition of Ca2+ was also observed in tobacco CML (rgs-CaM) and soybean CML27 [Arabidopsis CML16 [2+ increases the overall helical content. Rice CMLs (OsCMLs) also have heterogeneous behavior in terms of structural changes in CD spectroscopy with some members of the family displaying small changes  and others showing large increases in molar ellipticity  following binding of Ca2+ [2+ sensors is much more complex than originally believed. For CaM, variations in the response to Ca2+ binding mainly involve helix reorientation, and not merely a change in α-helical content [Arabidopsis CMLs (4.2%) does not differ substantially from that in CaM, suggesting that CMLs could share a conserved and analogous mechanism of action with CaM. Nonetheless, it should be noted that the Met content in Arabidopsis CMLs ranges from 0.9% to 8.6% and that the amount of exposed hydrophobic surfaces, in the apo- and holo-forms of CMLs, do indeed vary when considering the different family members, as demonstrated by studies with the fluorescent probe anilino-8-naphthalene sulfonate (ANS). CML36 (2.4% Met) is similar to CML15 (4.5%) and CML16 (5.0%) in that they show a significant degree of hydrophobic exposure even when Ca2+ is not present and only a relatively small increase in hydrophobicity is observed when Ca2+ is bound [2+ [2+ atom without changes in exposed hydrophobicity, and therefore it does not behave like a classical Ca2+ sensor [2+ binding site is unlikely to be compatible with a buffer function. The behavior of CML14 could point out a role of Ca2+ for target binding of CML14 that differs from the classical switch-like role with exposure of the interfacial hydrophobic regions. Only the identification of the interaction partners of CML14 will elucidate its molecular mechanisms of action. Notably, rice CMLs also exhibit a broad spectrum of hydrophobic characteristics as measured by ANS fluorescence [2+ sensors and further implies that the binding of a target to a CML might be based on a recognition mechanism that is more specific than just generalized exposure of hydrophobic residues.Crucial structural information on CML proteins and their Cais CML15  is reminis CML15  and CML4is CML15 , since tan CML27 ,103. Howis CML16 , CML37 [is CML16 , CML39 [is CML16 , and CMLis CML16  the bind of Ca2+ . It is s content , uncover content ,105. In  content ,107,108.is bound ,35. On tound [2+ ,31,32,82+ sensor . On the rescence . This st2+ signals, and molecular properties of the two EF-hand proteins . Therefore, CMLs might be able to carry out interactions that are common among the different members of the protein family, in addition to interactions that are specific to individual members. Moreover, the interaction of CML proteins with other CML family members has been documented, which could be significant in terms of Ca2+ signaling events [The recognition of targets for CaM/CML and better appreciation of the impact of CaM/CML-binding on biological processes are primary goals in untangling the broader role of CaMs/CMLs. Up to now, several CML targets have been identified by protein microarray analysis, in addition to genetic and in vivo studies. The protein targets identified to date include transcription factors, protein kinases, metabolic enzymes, and transport proteins ,110,111.2+ binding. In addition, electrostatic interactions between CaM and a target CaM binding domain can lead to stabilization of a CaM-target complex [2+, which reveals its versatility in terms of signaling [CaM-binding domains (CaMBDs) normally share similar secondary structures consisting of short (12–30 amino acids) sequences of amino acids with a tendency to form α-helices . These s complex ,113. The complex ,113,114.Multiple sequence alignment between CaMs and CMLs highlights two major differences that may be associated with an important impact on structure and target interactions. First, CMLs are widely variant in length compared to CaM, and have an N- or C-terminal extension in which signal sequences are not always readily found . These eArabidopsis, CML5 and CML11 from tomato [Lotus japonicus [Arabidopsis) have pairs of cysteines that can form disulfide bonds that affect the structural properties of the protein, e.g., allowing dimerization, and target binding. Since the EF-hand is normally present in pairs, dimerization could explain the existence of CMLs with odd numbers of functional EF-hands.In the interaction with target, the presence of secondary modifications is crucial since these can play particularly important roles in protein function and regulation. Different CMLs  harbor aponicus ,15,16,172+ sensors will require the study of their regulation. A major challenge will be to evaluate the structural properties and functional aspects of target binding. Certainly, the 3D structures of the apo- and holo-forms and of the complex with their target will be needed to categorically address these issues, to compare the recognition mode, and get deeper insight into the structural diversity of CML-binding to their target regions. Besides X-ray crystallography and NMR spectroscopy, cryo-electron microscopy (cryo-EM), which emerged as a remarkably successful technique for protein structure determination in the latest years, can also provide useful information on CML-target complexes (provided that CML-target complexes of sufficient size are studied). Notwithstanding, an interesting approach to study the interactions between CaM/CML-target is by identification of the CML-binding region in the target and synthesizing the corresponding peptide. Different biophysical techniques, including fluorescence, NMR and CD spectroscopy as well as ITC, SEC and native-PAGE, in fact, can be used to perform thorough structural and energetic characterization of the CML-peptide interaction and its Ca2+ dependence. Such approaches have been applied for many Ca2+ sensors, and not only in plants [Arabidopsis CML members the only CML-target complexes for which a detailed biochemical description has been achieved are CML19-RAD4 [Thus, greater knowledge of CML-target complexes is needed, and understanding the specific roles of Can plants ,120,121.L19-RAD4  and CML3L19-RAD4 .2+-saturated CML19 [A first simple analysis is monitoring the complex formation between CML and the target peptide via native PAGE. Indeed, upon incubation of the protein with increasing molar ratio of the peptide the appearance of a new band with a lower mobility than that of free CML is a clear indication that a protein-peptide complex has been formed ,32,33. F2+-saturated CML19 to RAD4p caused a significant increase in the fluorescence intensity of the peptide and a blue shift of maximum emission wavelength from 353 to 333 nm, indicating that the only Trp in the peptide entered a more hydrophobic environment and confirming that RAD4p interacts with Ca2+/CML19 [Next, Trp fluorescence spectroscopy can give crucial information on the stoichiometry and binding strength, as well as the mode of binding. Trp is often considered as an intrinsic fluorescent probe to follow conformational changes. Several binding regions in CaM/CML proteins contain a lone Trp residue , whereas2+/CML19 .2+ [2+ has an unordered structure. Addition of the peptide to the protein usually leads to an increase in the dichroic signal. A smaller rise in ellipticity signal could be associated to conformational changes of the Ca2+ sensor itself, but the major contribution usually comes from the peptide, changing from random coil to α-helical following interaction with the protein [2+-CML19, indicating that RAD4p might be induced to adopt α-helical structure [2+-saturated CML19, the NMR spectrum of the protein also underwent considerable changes with some peaks undergoing chemical shifts, and new peaks appearing, thereby confirming that the interaction between CML19 and the peptide leads to a unique, stable structure [Far-UV CD spectroscopy can complement Trp fluorescence as a basic tool to study the interaction of CML with peptide, since many CaM/CML-binding peptides are placed into an amphipathic helix after binding Ca2+ ,123,124. protein ,110,125.tructure . Followitructure .Finally, it should be mentioned that the thermodynamic parameters of peptide binding to CMLs can be determined using ITC, which also gives crucial information about the dominant forces in the association of the peptide with the specific CML (electrostatic versus hydrophobic interaction). However, up to now, such an approach has never been used to study the energetics of CML-target interactions.2+-CML complexes, will undoubtedly provide more insights into the molecular basis of the activity of CMLs.Clearly, there is a lack of biochemical and biophysical characterization on the binding of CMLs to their targets (and/or peptides). Further studies of the interaction of CMLs with several natural peptide targets, as well as CaM-specific targets, in addition to solving the structure of Ca2+ and Mg2+) binding and structural properties. One of the major challenges will be obtaining 3D structures of the holo- and apo-CMLs in isolation and in complex with targets. Moreover, there is a need to expand the knowledge about the roles of post-translational modifications on CMLs which are strongly related to the biological activity of proteins. Multiple channels of evidence have indicated that CMLs have the biochemical properties of Ca2+ sensors. Globally, biochemical and structural analysis of CMLs will provide insight into how such a vast array of CMLs proteins can coexist, without apparent redundancy, and how they make a distinct contribution to cellular signaling while being different from CaM and other Ca2+ sensors.While not exhaustive, we have attempted to summarize the recent advances in our understanding of the features of CMLs from biochemical and structural points of view . To lear


Although imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), 25–30% patients do not respond or relapse after initial response. Imatinib uptake into targeted cells is crucial for its molecular response and clinical effectiveness. The organic cation transporter 1 (OCT1) has been proposed to be responsible for this process, but its relevance has been discussed controversially in recent times. Here we found that the multidrug and toxin extrusion protein 1 (MATE1) transports imatinib with a manifold higher affinity. MATE1 mainly mediates the cellular uptake of imatinib into targeted cells and thereby controls the intracellular effectiveness of imatinib. Importantly, MATE1 but not OCT1 expression is reduced in total bone marrow cells of imatinib-non-responding CML patients compared with imatinib-responding patients, indicating that MATE1 but not OCT1 determines the therapeutic success of imatinib. We thus propose that imatinib non-responders could be identified early before starting therapy by measuring MATE1 expression levels. The response rate varies within different stages of the disease.3 Besides other mechanisms of resistance such as mutations in the BCR-ABL1 fusion gene,4 the transport of imatinib into its targeted cells has been proposed to be crucial for the clinical effectiveness.7 As an organic cation, imatinib has to be actively translocated across the cell membrane. Several clinical trials as well as in vitro studies investigated the role of the organic cation transporter 1 (OCT1) in this process. Although some studies suggested a critical role of OCT1 in regulating imatinib efficacy,7 other studies could not confirm these findings.9 The role of OCT1 for imatinib uptake has mostly been inferred from inhibition studies with prazosin and amantadine. This approach has been criticized recently, as these substances do not specifically inhibit OCT1 but are potential inhibitors of other imatinib uptake pathways distinct from OCT1.10 Although OCT1 is generally capable to transport imatinib, it might have minor relevance for a transport under clinical conditions. Indeed, in vitro studies have shown a moderate affinity of OCT1 to imatinib12 as well as low expression levels in mononuclear and CD34+ cells.13 OCT1 belongs to the SLC22A family of polyspecific organic cation transporters that also includes OCT2 and OCT3, which have an overlapping spectrum of substrates.14 Experiments on model cells have shown that other members of this group also transport imatinib.15 Others and our group recently demonstrated that the multidrug and toxin extrusion protein 1 (MATE1), also belonging to the group of organic cation transporters, accepts imatinib as a substrate.12Although the tyrosine kinase inhibitor imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), some patients do not respond to imatinib or relapse after initial response.Transporters other than OCT1, especially MATE1, have not been investigated regarding their role for imatinib accumulation in targeted cells for CML treatment yet. Here we investigate transporters that might be relevant for imatinib uptake, and regarding their role in mediating cellular imatinib uptake, regulating molecular response and their correlation to the clinical therapeutic response to imatinib of CML patients.m glutamine at 37 °C in 5% CO2. HEK293 cells were stably transfected with single OCT16 and cultured as described.11 Overexpression of the single OCT was verified via real-time reverse transcription polymerase chain reaction (qRT-PCR)11Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of six healthy volunteers (German Red Cross) with a Ficoll density gradient assay  and used for experiments within 6 h. The ethics committee of the University of Münster approved these experiments. K562 cells were kindly provided by Prof. Müller-Tidow , Germany) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mRNA isolation and reverse transcription were performed with Qiagen RNeasy Midikit  and Invitrogen Super Script III , respectively. For amplification of the respective transporters and the analyzed housekeeping genes, the following specific primer pairs were used: OCT1 S, 5′-CATCATAATCATGTGTGTTGGCC-3′ and OCT1 AS, 5′-CAAACAAAATGAGGGGCAAGGCTT-3′ OCT2 S, 5′-CATTGAACTAAGAAGAGAGACCG-3′ and OCT2 AS, 5′-CCACAGTGTACAATAGACTCCA-3′ OCT3 S, 5′-GACAAGAGAAGCCCCCAACCTGAT-3′ and OCT3 AS, 5′-CACTAAAGGAGAGCCAAAAATGTC-3′ MATE1 S, 5′-GCAACCACACTTGGAGTGATGG-3′ and MATE1 AS, 5′-GAGCAGAATTCCCACTCCGAG-3′ GAPDH S, 5′-CAAGCTCATTTCCTGGTATGAC-3′ GAPDH AS, 5′-GTGTGGTGGGGGACTGAGTGTGG-3′. Quantitative real-time PCR was then carried out with ABI PRISM 7900 Sequence Detection System  using SYBR Green PCR Master Mix .17 Imatinib plasma levels and the unbound fraction were obtained from the literature.19 The half-maximal inhibitory drug concentration (IC50) was taken from previously published data, where IC50 values were obtained by inhibition of the well-known model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+).11 Similar IC50 values were obtained when inhibiting the uptake of 1-methyl-4-phenylpyridinium iodide (MPP+) by imatinib underlining the stability of this method.12To elucidate whether a transport is relevant under clinically used concentrations, peak plasma levels of imatinib were compared with apparent affinities of single OCT to imatinib as recommended by the International Transporter Consortium.m imatinib  for 10 min at 4 °C, where mainly passive diffusion of imatinib through the cell membrane is possible, and at 37 °C, where active transporter-mediated uptake occurs. The transport process of the investigated transporters was specifically inhibited by chemical substances at concentrations that are known to inhibit specifically the distinct transporters ,20 whereas 80 μm MPP+ (Sigma-Aldrich) was used for OCT1 and OCT2 coinhibition.23 Cells were then lysed and intracellular imatinib accumulation was quantified with high-performance liquid chromatography with UV detector as described previously.11HEK293, PMBC or K562 cells were incubated with 5 μsporters . MATE1-mM MATE1 small interfering RNA (siRNA)  or control siRNA . The transfection efficiency was verified by PCR and western blot analysis for MATE1 as described earlier.11 To assess the influence of MATE1 expression on imatinib-dependent biologic effects, transfected K562 cells were incubated with imatinib at different concentrations (5 or 10 μM) for 10 min and the activity of BCR-ABL1 protein was then detected by western blot analysis using anti-phospho-ABL  and GAPDH antibody (Sigma-Aldrich). Further, we evaluated the clonal growth of K562 cells stably transduced with lentiviral vectors (plKO.1-GFP) delivering a short hairpin RNA against MATE1  or control mRNA (shscramble). Thereon, short hairpin-transduced K562 cells (3 × 103 cells) were plated in 1 ml of methylcellulose medium  without cytokines in quadruplicates, supplemented with imatinib (0–1000 nM) and the colonies were counted on day 7.MATE1 knockdown in K562 cells was performed with Hyperfect transfection reagent (Qiagen) according to the manufacturer's instructions using either 40 nBCR-ABL1 diagnostic procedures were performed in the Munich Leukemia Laboratory  and data were kindly provided by S Jeromin and T Haferlach. Upon diagnosis, MATE1-mRNA expression was quantified by qRT-PCR in total bone marrow (BM) cells as described above. All patients were treated with imatinib and MATE1 expression was compared between imatinib-responding and -refractory patients defined by major molecular response. BCR-ABL1/ABL1 ⩽10%  after 3 months of imatinib therapy was defined as optimal response, whereas poor response was defined by a BCR-ABL1/ABL1 >1% after 6 months of therapy (warning and failure according to the ELN 2013 guidelines24). Detailed patient characteristics are given in In total, 30 patients with BCR-ABL1 major transcript (p210)-positive CML were included in this study after written informed consent was obtained . post hoc test or unpaired Student's t-test were used for statistical analysis as indicated. Values are mean±s.e.m. with the following statistical significance levels: *P<0.05, **P<0.01, ***P<0.005 and NS=not significant.Data were analyzed with GraphPad Prism, V4.0 . One-way analysis of variance with Tukey's P=0.0013), OCT2-  and MATE1-overexpressing HEK cells , but not in OCT3-HEK cells  compared with wild-type HEK cells (0.53±0.03 nmol/mg protein)  . This in17 Along with these guidelines, transporters with a quotient of unbound plasma drug concentration (Cunbound) divided by IC50 >0.1 are defined as clinically relevant. Imatinib plasma levels for therapy regimens of 400 and 600 mg daily, the unbound fraction and the half-maximal inhibitory imatinib concentration to the respective OCTs (IC50) were obtained from the literature  and 5.8 (for 600 mg imatinib daily), suggesting a possible interaction at clinically used concentrations. Quotients for OCT2 were manifold lower compared with that for MATE1, but in contrast to those of OCT1 still in the range of a potential contribution under clinically relevant concentrations (0.2 for 400 mg and 0.6 for 600 mg imatinib daily).To elucidate whether this transport is relevant under clinically used concentrations, we compared imatinib plasma levels to apparent affinities of the transporters according to the recommendations of the International Transporter Consortium.terature .11, 18, BCR-ABL-positive CML patient in blast crisis.25 In both cell types mRNA for OCT1 , OCT2 , and MATE1  in PBMC and K562 cells, respectively, were expressed at similar levels  than at 4 °C . This suggests a transporter-mediated process as the main mechanism of imatinib uptake (27 The selective inhibition of MATE1 with pyrimethamine significantly reduced the imatinib uptake in PBMCs by 34±8% (P=0.0262), whereas coinhibition of OCT1 and OCT2 with MPP+ had no significant effect (P=0.6051)   . Most of to OCT1 . The exp=0.0005) . Consequ=0.0005) . These rBCR-ABL1 mutations,4 patients with these mutations were excluded from the analysis. OCT2 mRNA was not expressed at detectable levels in the BM of all patients. The expression of mRNA for OCT1 was low  and did not differ significantly between responders and non-responders.To further prove that MATE1 has an important role in regulating imatinib sensitivity in CML patients, we analyzed the transporter profile of total BM samples obtained from CML patients by qRT-PCR and correlated the expression levels with the major molecular response to imatinib treatment . As someCt=0.07±0.001) compared with imatinib responders   between MATE1 transporter mRNA expression and clinical response to imatinib treatment, suggesting that MATE1 levels might enable to predict whether CML patients are likely to respond to imatinib therapy  between OCT1 transporter expression and major molecular response  . We foun therapy . In contresponse .m) led to a colony reduction of 58% (1280 colonies without and 532 colonies with 250 nM imatinib) and 1 μm nearly totally inhibited colony growth . In contrast, the effects of imatinib were reduced when MATE1 was knocked down. Thus, there was no significant reduction of colony growth by imatinib in shMATE1-transduced K562 cells until a concentration of 1 μm was used (1266 colonies without and 560 colonies with 1 μm imatinib, reduction of 56% To underline the hypothesis that MATE1 expression is crucial for the response to imatinib, we examined the role of MATE1 for the reductive effect of imatinib on colony formation. Therapeutically used imatinib concentrations reduced colony growth in shscramble-transduced K562 cells concentration dependently. Imatinib (250 n28 Most studies showing that OCT1 mediates the cellular uptake and regulates the response to imatinib rely on inhibition experiments with prazosin or amantadine.28 This approach has been criticized recently and might explain the dichotomic results as both, the inhibition with prazosin and amantadine, are not specific for OCT1.10 We here show that although OCT1 can potentially transport imatinib, the affinity to imatinib is too low to allow a significant interaction at therapeutically reached plasma levels. Indeed, inhibition of OCT1 and OCT2, which we also identified as a potential imatinib transporter, by MPP+ had no significant effect of the uptake in PMBC. In contrast, MATE1 transports imatinib with a manifold higher affinity as shown in model experiments permitting an uptake at clinically observed plasma levels. Indeed, prazosin and amantadine have similar or even higher inhibitory affinities to MATE1 than to OCT1.27 The observed inhibitory effects of prazosin and amantadine on imatinib uptake and its effectiveness might thus be rather because of MATE1 than OCT1 inhibition.Besides other mechanisms of resistance, the transport of imatinib into target cells was proposed to regulate the effectiveness of imatinib treatment. The role of OCT1 in this process has been discussed controversially.14 To further prove the role of MATE1 for the cellular uptake and effectiveness of imatinib, we thus confirm the observed results of our model experiments with both specific inhibition of MATE1 by pyrimethamine and a MATE1 gene knockdown. Pyrimethamine was used at low concentrations, which significantly but not completely reduced imatinib uptake. However, we did not increase the pyrimethamine concentration to reach a total inhibition of MATE1 to guarantee specificity. This may explain the different degree of reduction in inhibition and knockdown experiment.Specific inhibition of single OCT is difficult as these transporters share various substrates.29The importance of MATE1-mediated imatinib uptake for therapeutic effects is supported by our transduction experiments, where MATE1 knockdown markedly reduced the effects of imatinib on c-ABL phosphorylation and colony formation. Both effects are essential in the pathogenesis of CML, and c-ABL phosphorylation has been shown to be a good predictor for the molecular response of CML patients to imatinib.31 However, imatinib is the most cost-effective tyrosine kinase inhibitor therapy for CML and, in contrast to dasatinib and nilotinib, is associated with less severe side effects. Screening tools such as a MATE1 expression analysis or detection of MATE1 mutations, which have been shown to rigorously change the functionality of MATE1,32 might thus help to individually choose the best, safest and economically reasonable therapy.Deep and early molecular response is suggested to be predictive of improved long-term outcomes in CML therapy. We therefore correlated MATE1-mRNA expression levels in CML patients with the molecular response to elucidate whether MATE1 can influence the response of CML patients to imatinib. There was a significant reduction of MATE1-mRNA expression in patients who did not respond to imatinib and a strong correlation of MATE1-mRNA levels in total BM cells with the molecular response of CML patients. Interestingly, this correlation was not observed for OCT1. Measuring MATE1-mRNA levels might therefore enable to predict whether CML patients are likely to respond to imatinib therapy. Clinical trials showed evidence that dasatinib and nilotinib generate faster and deeper short-term responses in first-line CML therapy compared with imatinib.In conclusion, we demonstrate that MATE1 is the major transporter for the cellular uptake of imatinib and crucial for the therapeutic success in CML patients. We suggest that the detailed analysis of MATE1 expression levels and mutations could be a predictor for the response to imatinib therapy.