ROS are produced during immune responses as signaling molecules as well as anti-microbial agents. ROS-sensitive TRPM2 channels are widely expressed in cells of the immune system and located on the cell surface as a Ca2+ influx pathway in macrophages, monocytes, neutrophils, lymphocytes, and microglia but preferentially within the lysosomal membranes as a Ca2+ release mechanism in dendritic cells; ROS activation of the TRPM2 channels, regardless of the subcellular location, results in an increase in the intracellular Ca2+ concentrations. Recent studies have revealed that TRPM2-mediated ROS-sensitive Ca2+ signaling mechanisms play a crucial role in a number of processes and functions in immune cells. This mini-review discusses the recent advances in revelation of the various roles the TRPM2 channels have in immune cell functions and the implications in inflammatory diseases.Transient receptor potential melastatin 2 (TRPM2) proteins form Ca Several Ca2+ signaling mechanisms are well known in immune cells. Activation of the T-cell receptor, B-cell receptor, and Fc receptors, which are coupled to phospholipase C γ (PLCγ), or the PLCβ-coupled chemokine receptors, generates inositol-1,4,5-trisphosphate (IP3) to activate the IP3 receptors and release Ca2+ from the endoplasmic reticulum (ER). Reduction in the ER Ca2+ elicits store-operated Ca2+ entry through Ca2+ release-activated Ca2+ channels to stimulate Ca2+ influx and increase the intracellular Ca2+ concentrations ([Ca2+]i) , neutrophils, T and B lymphocytes, and natural killer cells, play a critical role in orchestrating both innate and adaptive immune responses to microbial pathogens, environment irritants, and danger molecules released from damaged cells. Microglia represents the residual macrophages in the central nervous system (CNS) responsible for immune responses to nerve damages. Intracellular Casphate IP to activ+ influx .2+ (2+-permeable cationic channels gated by intracellular ADP-ribose (ADPR) upon binding to the unique NUDT9 homology (NUDT9-H) domain in the distal C-terminus  proteins, which are commonly divided based on sequence relatedness into TRPC , TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), and TRPML (mucolipin), but all have the same membrane arrangement and form cationic channels with a majority permeating Ca2+ , 9. The 2+ ; each su2+ . Two semterminus , 13  and ERK-dependent nuclear translocation of transcription factor nuclear factor-κB (NF-κB). In human U937 monocytic cells, H2O2-induced CXCL8 production was strongly dependent of extracellular Ca2+ and TRPM2 expression, and more specifically TRPM2-mediated Ca2+ influx was important in elevating the [Ca2+]i as a signal for sequential activation of proline-rich tyrosine kinase (Pyk2), which is sensitive to Ca2+, Ras and ERK (2O2-induced Ca2+ influx and CXCL2 production were remarkably attenuated in monocytes from the TRPM2-KO mice. Migration of neutrophils from the wild-type (WT) mice was enhanced in culture medium preconditioned by H2O2-treated monocytes from the WT but not TRPM2-KO mice. CXCR2 is known to have a critical role in inducing ulcerative colitis (2+ influx is important in ROS-induced CXCL2/CXCL8 production by monocytes (Table Human chemokine C-X-C motif ligand 8 (CXCL8) or mouse functional homolog CXCL2, produced by immune cells, plays an important role in recruiting neutrophils to the sites of infection and inflammation . For CXC and ERK . Consist colitis . In resp colitis . Neutropes Table  and neutProminent CXCL2 production and neutrophil infiltration were also observed in the inflamed paw of the WT mice induced by carrageenan injection or in the site of nerve injury . Both CXListeria monocytogense (Lm), a model of listeriosis  signaling pathway  in THP1 monocytic cells was significantly attenuated using short hairpin RNA (shRNA) to reduce the TRPM2 expression   –46. As dfunction   damage mechanisms have been proposed , oxtrophils . A recentrophils . TRPM2 dtrophils , 55, 56.trophils   provides a protective mechanism by limiting oxidative stress-induced tissue damage during inflammation and sepsis , 58. A r2+, Ca2+-sensitive protein kinase C α (PKCα), and membrane potential  induced substantial CXCL8 production but modest cell death in U937 cells (2+]i and decrease in cell viability (2O2 concentrations (0.3–1 mM) strongly reduced the viability of RAW264.7 and macrophages from the WT mice and such cell death was reduced by using PJ-34, a PARP inhibitor, and also reduced by TRPM2 deficiency (2+ homeostasis and cell death.The most well-established role of the TRPM2 channels in diverse cell types is to mediate ROS-induced cell death . This wa37 cells . TNF-α c37 cells . Treatmeiability  (Table 1ficiency  (Table 1ficiency , in whic2+-permeable cationic channels in immune cells, studies have made important progress in better understanding their locations and functions in the immune cell functions. Studies in combination with mouse models have demonstrated the importance of the TRPM2 channels in immune cells at the system level; the TRPM2 channel activity is clearly crucial for both innate and adaptive immunity but excessive TRPM2 channel activity contributes significantly toward the pathogenesis of inflammatory diseases. The ongoing and future studies, with the aid of specific TRPM2 inhibitors that hopefully become available in the near future, will provide a more mechanistic insight into the TRPM2 channels in humans under physiological and pathological conditions that ultimately will enable us to explore the therapeutic potential of this amazing ion channel.Since the discovery of TRPM2 channels as ROS-activated CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The results demonstrated that 4 mM [Ca2+]o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, [Ca2+]o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, [Ca2+]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca2+]o. Moreover, [Ca2+]o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca2+]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.Porcine bone marrow mesenchymal stem cells (pBMSCs) have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium ([Ca Bone marrow mesenchymal stem cells (BMSCs) not only regulate hematopoietic and other stem cells niches but also have multipotential capacities to differentiate toward osteocyte, chondrocyte, adipocyte, and myocyte , perform2+]o) modulates cell proliferation in various cells, such as myeloma cells [2+]o exerts its role in regulating cell proliferation either via calcium influx through calcium channels or by activating the calcium-sensitive receptor (CaSR) [2+]o or (CaSR) . CaSR isR) [2+]o . SeveralR) [2+]o , osteoblR) [2+]o , and preR) [2+]o  and playR) [2+]o .2+]o always leads to the increase of intracellular calcium ([Ca2+]i) and subsequent activation of the intracellular signaling pathway, which regulates cell proliferation. The MAPK signaling pathway, which consists of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun kinase (JNK), and p38 MAPK, plays an important role in controlling cell proliferation in mammalian cells o is involved in the regulation of cell proliferation, the role of [Ca2+]o in pBMSCs proliferation and the possible mechanisms underlying this process remain unclear. Thus, the present study was designed to investigate the effects of [Ca2+]o on pBMSCs proliferation by determining the cell numbers, cell cycle progression, and expression levels of proliferative marker genes. In addition, we sought to explore the underlying mechanisms involved in this process, including the contribution of CaSR and the relevant intracellular signaling pathway. Our results revealed that the enhanced pBMSCs proliferation in response to [Ca2+]o was associated with the activation of the CaSR and ERK1/2 signaling pathway.Although many studies described that o for 5 days to investigate the effects of [Ca2+]o on pBMSCs proliferation. In addition, the cells were treated with 4 mM [Ca2+]o and/or 0.1 μM NPS2143 or 1 μM U0126 for 5 days to explore the role of CaSR and ERK in [Ca2+]o-induced proliferation of pBMSCs, respectively.pBMSCs were isolated and purified from the bone marrow of postnatal Landrace pigs aged between 5 and 7 days as we previously described . The purAfter pBMSCs were cultured with different treatments for 5 days, the cells were then incubated with Cell Counting Kit-8 (CCK-8) reagent (0.1 mg/well) for 4 h. The number of viable cells was assessed by measuring the absorbance at 450 nm using a Synergy 2 Multi-Mode Reader .6 cells/per 25 cm2 flask and cultured in presence of 4 mM [Ca2+]o and/or CaSR antagonist NPS2143 supplementation for 5 days. Cell cycle status was determined by measuring cellular DNA content following staining with propidium iodide using flow cytometry as previously described [pBMSCs were seeded at 1 × 10escribed . Briefly2+]o and/or CaSR antagonist NPS2143 for 12 h and then loaded with Fluo 3-AM (2 μM) in the HEPES buffered salt solution (HBSS)  for 30 min at 37°C. The loaded cells were rinsed three times with HBSS. Intracellular calcium ([Ca2+]i) levels were then determined by flow cytometry using a BD FACSAria2 flow cytometer  at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Data analysis was performed using the FlowJo software.The pBMSCs, plated on a 12-well plate, were cultured with 4 mM o on pBMSCs proliferation, the cells were incubated in DMEM/F12 supplemented with various concentrations  of calcium acetate for 5 days, with the final concentrations of [Ca2+]o in the medium up to 1 (control), 2, 4, and 6 mM. The results of CCK-8 assay showed that [Ca2+]o significantly stimulated pBMSCs proliferation in a dose-dependent manner, with similar promotive effects observed at 4 and 6 mM [Ca2+]o  enhanced by 4 mM [Ca2+]o (2+]o markedly (P < 0.05) increased the protein expression of CaSR  alone had no effect on pBMSCs proliferation. However, NPS2143 significantly (P < 0.001) abolished the promotion of pBMSCs proliferation induced by 4 mM [Ca2+]o. In addition, the results of cell cycle progression analysis via flow cytometry revealed that 4 mM [Ca2+]o markedly (P < 0.05) increased the proportion of pBMSCs in the S phase and the proliferation index (PI) of pBMSCs and decreased the proportion of pBMSCs in the G0/G1 phase. However, NPS2143 eliminated the effects of [Ca2+]o on cell cycle distribution and PI  and PCNA but decreased the mRNA expression of p21, the inhibitor of cyclin-dependent kinase . As expected, [Ca2+]i, which was indicated as the percentage of gated cells (P < 0.001) increased in response to 4 mM [Ca2+]o, with an increase pattern similar to that of CaSR protein expression induced by 4 mM [Ca2+]o  increase of the p-ERK/ERK ratio, indicating the activation of the ERK1/2 signaling pathway , was applied to inhibit the kinase activity of MEK1/2 and thus to prevent the activation of ERK1/2 in our study. We determined that the significant increase of p-ERK/ERK ratio in response to 4 mM [Ca2+]o was reversed by U0126  alone had no effect on the proliferation of pBMSCs. However, U0126 could eliminate the promotion of pBMSCs proliferation induced by 4 mM [Ca2+]o  slowed the rate of porcine osteoblasts proliferation [2+]o on BMSCs proliferation might be attributed to the various species and/or culture conditions. In addition, the heterogeneous characteristic of BMSCs should also be considered for the different effects of [Ca2+]o on BMSCs proliferation. The pBMSCs used in our study were positive for mesenchymal surface markers CD29 and CD44 and negative for hematopoietic marker CD45 and for the adhesion molecule CD31 and were able to differentiate into adipocytes and myocytes o on pBMSCs proliferation in low oxygen tension should be further investigated in future study.In the present study, we determined that o on pBMSCs proliferation. In our study, we determined that [Ca2+]o significantly decreased the ratio of G0/G1 phase and increased the percentage of the S phase and the PI in pBMSCs, compared with those of control group. These data indicated that [Ca2+]o accelerated cell cycle progression from the G0/G1 phase to the S phase and promoted pBMSCs proliferation. It has been implicated that cyclin D and cyclin E are required for the transition from G1 to S phase of the cell cycle, whereas cyclin A is involved in the initiation and completion of DNA replication during the S phase [2+]o. In addition, the level of PCNA, an essential component for DNA replication machinery [2+]o. By contrast, p21, the inhibitor of cyclin-dependent kinase, was inhibited in the presence of [Ca2+]o. Thus, these findings indicated that [Ca2+]o stimulated pBMSCs proliferation by influencing the cell cycle progression and the expression levels of proliferative marker genes.The cell cycle progression and the expression levels of proliferative marker genes  were detected to elucidate the stimulatory effects of o elicits its effects on the regulation of cell proliferation either via calcium influx through calcium channels or by activating CaSR-mediated signaling pathway [2+]o-stimulated pBMSCs proliferation . In contrast, we found the expression of CaSR in pBMSCs, which was also expressed in mesangial cells [2+]o on pBMSCs proliferation, suggesting the involvement of CaSR in [Ca2+]o-promoted pBMSCs proliferation. In agreement, it has been demonstrated that activation of CaSR by [Ca2+]o contributes to the cell proliferation of human mesangial cells [2+]o-induced stimulation of CaSR inhibited the cell proliferation of human colon epithelial cells o can induce [Ca2+]i increment [2+]i in response to [Ca2+]o, which was reversed by CaSR inhibition with NPS2143. These finding indicated the involvement of CaSR in the [Ca2+]o-induced increase in [Ca2+]i during pBMSCs proliferation. Notably, it was reported that [Ca2+]i can promote cyclin E expression and the initiation of DNA synthesis at G1/S transition, thereby contributing to the completion of the cell cycle [2+]o. Together, these observations give rise to the idea that the [Ca2+]o-induced elevation of [Ca2+]i and the enhancement of pBMSCs proliferation were mediated, at least in part, by activation of CaSR.A large body of evidence has determined that o and the activation of ERK1/2 was abolished by NPS2143. In addition, the inhibition of ERK1/2 signaling pathway with U0126 reversed the stimulation of pBMSCs proliferation, the alteration of cell cycle distributions, and the expression levels of cell cycle marker genes, which were induced by [Ca2+]o. These results provided the evidence that [Ca2+]o stimulated pBMSCs proliferation, at least in part, via activation of CaSR and the linked intracellular ERK1/2 signaling pathway. However, Tfelt-Hansen et al. found that the activation of p38 MAPK and PI3K but not that of ERK1/2 by CaSR promoted the cell proliferation of rat Leydig cancer cells o was associated with activation of plasma membrane receptor CaSR, elevation of [Ca2+]i, and the enhancement of the intracellular ERK1/2 signaling pathway. These data may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.In conclusion, our findings demonstrated that CaSR was expressed in pBMSCs and that the enhanced proliferation of pBMSCs in response to [Ca2+]o.Supplementary Figure: Nifedipine (1 μM), an antagonist of VGCC, has no effect on the promotion of pBMSCs proliferation induced by 4 mM [Ca


We show increased intracellular [Ca2+] in all of the examined brain regions following ketamine treatment but an altered cytosolic Ca2+ level correlated with hyperlocomotor activity was only established for the cortex and striatum. Our findings also suggest that an imbalance in the expression between the calcium “on” and “off” systems contributes to the deregulation of brain Ca2+ homeostasis in our ketamine-induced model of experimental psychosis. Identification of the genes whose expression is affected by ketamine treatment indicates their involvement as putative etiological factors in schizophrenia.Chronic N-methyl-D-aspartate receptor (NMDAR) antagonist treatment can provide valuable neurochemical and neuroanatomical models of experimental psychosis. One such antagonist, ketamine, with its short half-time and well-documented psychotomimetic action, has cognitive effects resembling various aspects of schizophrenia-like symptoms. In order to obtain insights into possible relationships between Ca According to this theory, disturbed Ca2+ homeostasis and subsequent dysfunctions in Ca2+-regulated signal transduction cause schizophrenic symptomology. Indeed, postmortem studies have consistently reported structural and functional abnormalities in the prefrontal cortex, hippocampus and cerebellum and dysregulation of the striatal dopaminergic system in schizophrenia  underlies the induction of psychotic symptoms , whereas “off” systems tend to restore the resting Ca2+ level. Most of the extracellular Ca2+ enters the cell through voltage-dependent Ca2+ channels and store-operated Ca2+ channels (SOCs). Voltage-dependent Ca2+ channels encoded by the CACNA1A-S genes are key transducers of electrical excitability to intracellular Ca2+ transients, which, in turn, initiate neurotransmission by triggering vesicular release , which are nonselective plasma membrane cation channels with a preference for Ca2+ over Na+, are considered to be SOCs , ATP-dependent Ca2+ pumps (PMCA1-4), sarco(endo)plasmic reticulum Ca2+-ATPases  and secretory-pathway Ca2+-ATPases (SPCA1-2) located in the membrane of the Golgi apparatus. Furthermore, significant amounts of cytosolic Ca2+ can also be accumulated by mitochondria. This Ca2+ uptake undertaken by the mitochondrial calcium uniporter (MCU) shapes spatiotemporal Ca2+ signals and might trigger cell death . The 5×HOT FIREPol EvaGreen qPCR Mix was from Solis BioDyne (Lithuania). The M-MLV reverse transcriptase, Trizol reagent and Fluo-4 Direct Calcium Assay Kit were from Life Technologies (USA). We obtained the CytoTox-ONE assay from Promega (USA). The ATPLite 1 step Luminescence Assay System was purchased in Perkin-Elmer (USA). Primers were synthesized in the Institute of Biochemistry and Biophysics (Poland).n = 12) were injected intraperitoneally with ketamine (30 mg/kg) dissolved in physiological saline. Injections of ketamine for 5 consecutive days were shown to induce the behavioral and molecular changes related to schizophrenia-like alterations  was injected with saline only (0.5 ml/kg). All animal experimentation was conducted in accordance with Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. All the procedures used were also approved by the Institutional Animal Care and Use Committee at Medical University of Lodz.The 10– to 12-week-old male Wistar rats  were group-housed in laboratory cages and kept under a controlled temperature (23 ± 2 °C) with a 12-h light/dark cycle and with food and water provided ad libitum. For 5 consecutive days, randomly chosen rats  and were allowed to explore the arena freely for 10 min. Then, locomotor activity was continuously measured over 20 min by using a webcam connected to the ANY-maze video tracking system . The stereotypic behavior was evaluated by counting the amount of turning, weaving and bobbing. Weaving and head bobbing were assessed by counting the neck wave up and down and left and right. Turning was measured by counting the number of turns around. The cumulative stereotypy was determined for each animal and was expressed as the sum of each of the scores over 20 min. Behavioral tests were performed between 10 a.m. and 3 p.m.2+ measurements, brains were processed essentially as described in Rothman . Following a 15-min incubation at 37 °C, samples were suspended in 1 ml trypsin-free buffer A and passed through a Pasteur pipette with a flame-narrowed tip. Then, cells from particular areas were centrifuged twice at 200g for 5 min. The pellets were suspended in buffer B  and used for experiments.Immediately after behavioral testing and no longer than 3 h following final ketamine administration, animals were decapitated by using a guillotine. Brains were freshly dissected and used immediately or were frozen for further use. For Ca Rothman . In brieFollowing dissociation, cellular viability was assessed by means of two independent assays. The CytoTox-ONE assay was used to measure lactate dehydrogenase (LDH) released into the extracellular medium. For this purpose, 100 μl cell suspension in buffer B , where Kd = 345 nM.Cell suspension (100 μl) obtained as in -ΔΔCt method  housekeeping gene to normalize the data.Total RNA from dissected brain regions such as the cortex, cerebellum, hippocampus and striatum was extracted with Trizol Reagent following the manufacturer’s instructions. Single-stranded cDNA was synthesized from 1 μg total RNA with oligo(dT) primers in a 20-μl mixture by using M-MLV Reverse Transcriptase. Real-time quantitative polymerase chain reaction (qPCR) was carried out with the Abi Prism 7000 sequence detection system (Applied Biosystems) by using the EvaGreen qPCR Mix and results were analyzed with accompanying software. Amplifications were generated over 15 min at 95 °C followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The same conditions were used for all analyzed genes. A melting curve was performed to assess the specificity of the primers listed in Table P < 0.05 was considered as statistically significant. Correlations were assessed by using Pearson’s correlations and were made by using GraphPad Prism software version 5.The data are shown as means ± SEM. The number of animals used in particular experiments is given below each figure. The results were analyzed by using STATISTICA 8.0 (StatSoft). The two-tailed unpaired Student’s t-test (or the Mann–Whitney U Test) and one-way analysis of variance  were used when appropriate. P = 0.02; Fig. P = 0.016; Fig. P = 0.019; Fig. P = 0.013; Fig. P = 0.042; Fig. P = 0.041; Fig. P = 0.024; Fig. P = 0.038; Fig. Administration of subanesthetic doses of ketamine (30 mg/kg) promoted behavioral alterations significantly different from those observed in the saline-treated group. Ketamine-injected rats exhibited continuous locomotion with a quick gait. Statistical analysis revealed considerably higher locomotor activity in this group, as reflected by the significant increase in the total distance travelled  was observed in cells isolated from the striatum, followed by a change in cerebellar cells by ∼40 nM (P = 0.004). The resting [Ca2+]c in the cortical and hippocampal cells was higher by ∼20 nM . In addition, a positive correlation was found between the locomotor activity in the rats and the increased calcium level in the cortex cells , calcium pumps , exchangers , participants in the store-operated Ca2+ entry mechanism  and the MCU.Since c and the changes in the expression of the members of the calcium-handling systems, we performed a correlation analysis. A significant negative correlation was revealed in the cortex for PMCA2 (r = −0.64, P = 0.047; Fig. r = −0.73, P = 0.023; Fig. r = −0.96, P = 0.036; Fig. r = 0.77, P = 0.04; Fig. r = −0.65, P = 0.043; Fig. r = −0.71, P = 0.048; Fig. r = 0.80, P = 0.049; Fig. r = −0.75, P = 0.032; Fig. r = −0.72, P = 0.017; Fig. r = −0.88, p = 0.031; Fig. 2+]c only for PMCA1 (r = −0.66, P = 0.034; Fig. r = −0.75, P = 0.016; Fig. r = −0.78, P = 0.026; Fig. To verify the potential relationship between increased c in all of the examined brain regions; however, an altered cytosolic Ca2+ level is correlated with hyperlocomotor activity only for the cortex and striatum.Assuming that schizophrenia is a disease of calcium signaling, the essential question to be asked is whether the up-regulation or down-regulation of Ca2+ flow result in the compensatory down-regulation of Ca2+ sequestering proteins (Lidow 2+ entry through glutamate-independent pathways and Ca2+ mobilization from internal stores (Lidow 2+]c increase in a large population of neurons. This increase can, in turn, inhibit NMDAR causing the dopamine D2 receptor-dependent potentiation of the ecytosolic calcium signal. The dopamine system is known to interact closely with Ca2+ signaling and both phasic dopamine release and the activity of D2 receptors are dependent on the Ca2+ influx or Ca2+ release from internal stores (Lidow 2+ level and schizophrenia-like symptoms suggested in our study is additionally highlighted by recent findings showing the ketamine-induced potentiation of dopamine release and the reduction of GABA secretion from rat striatum (Grasshoff et al. The phenomenon of calcium homeostasis dysregulation can be partially deduced from the ketamine-induced model of schizophrenia. Namely, a reduction of NMDAR drive and a concomitant decrease in Cans Lidow . Blockades Lidow . Therefoes Lidow . The con2+ homeostasis by blocking NMDAR located on GABAergic neurons in the striatum and prefrontal cortex; this might influence Ca2+-dependent dopamine release resulting in increased neural activity. Among GABAergic neurons, prefrontal cortical parvalbumin interneurons seem to be highly sensitive to NMDA antagonists. Chronic exposure to ketamine leads to a reduction of GAD67 and parvalbumin expression in parallel with the activation of NADPH oxidase and superoxide accumulation. Blocking of NADPH oxidase prevented these actions, a finding that indicates this enzyme as a source of parvalbumin interneuron vulnerability. Therefore, psychotomimetic effects generated by NMDAR antagonists can be mediated, at least in part, by oxidative stress produced by NADPH oxidase (Behrens et al. Based on the well-established model of ketamine-induced experimental psychoses, we demonstrated a correlation between altered calcium signaling in the cortex and striatum and the generation of behavioral abnormalities. The prefrontal cortex stands out as one of the main regions affected in schizophrenia and the postnatal inactivation of this structure in rats promotes behavioral alterations similar to ketamine-evoked psychotic symptoms (Usun et al. So far, most of the reports have focused on the relationship between defective postsynaptic glutamatergic neurotransmission and schizophrenia-related symptoms. However, a growing body of evidence has also revealed presynaptic glutamatergic alterations, particularly connected with proteins involved in synaptic vesicles fusion and exocytosis (Oni-Orisan et al. 2+]c. In order to avoid an exaggerated analysis, we decided to limit our discussion of the potential effect of ketamine on brain calcium homeostasis only to these genes. In the cerebellum, hippocampus and striatum, ketamine administration leads to a down-regulation of PMCA3. This PMCA isoform and PMCA2, whose expression is reduced in the cortex, are the fastest PMCA isoforms and their presence is thought to sensitize neuronal cells even to subtle changes in [Ca2+]c. Although the data regarding specific PMCA3 function are scarce, several studies of neuronal cultures have shown that the reduced expression of either PMCA2 or PMCA3 decreases Ca2+-clearing potency making cells unable to maintain Ca2+ homeostasis (Fernandes et al. 2+ transient to the baseline and the compromised ability to oppose the influx of calcium through the plasma membrane capacitive channels have recently been attributed to dysfunctional PMCA3 (Zanni et al. Also of interest, among the analyzed genes encoding calcium-handling factors, we found only a few whose expression is correlated with increased [Ca2+]c level in these brain structures, suggesting a compensatory change by sequestration of calcium to intracellular compartments. However, the changes in the Ca2+ content in the internal stores depend upon the balance between the activity of SERCA pumps and Ca2+ leakage or release back into the cytosol. A massive calcium efflux from the ER is not the reason for the increased [Ca2+]c in ketamine-treated brains observed in our study, as ketamine has been shown to decrease the release of internally stored Ca2+ (Xiang et al. 2+ into the lumen of the ER aimed at reducing the abnormally high cytosolic Ca2+ level. A similar reason can be suggested for the up-regulation of PMCA1 in the striatum and cerebellum, PMCA4 in the cortex and NCX3 in the hippocampus. The beneficial role of PMCA and SERCA overexpression on the reduction and increase in the ER [Ca2+] level, respectively, has previously been demonstrated in vivo (Brini et al. Increased expression of SERCA3 in the cortex, hippocampus and striatum was correlated with a higher [Ca2+ current (Yamakage et al. 2+ channels have been found to be engaged in mesolimbic-mediated behaviors and in the hippocampal formation of spatial memory in rodents (Bhat et al. Calcium signaling through CACNA1 has been implicated in several brain disorders and the CACNA1c variant has been associated with schizophrenia (Lajtha et al. 2+ homeostasis in ketamine-treated rats might be caused by an imbalanced expression between the calcium “on” and “off” mechanisms. The key changes correlating with higher [Ca2+]c involve the dysfunctional expression of the calcium extruding systems, with a prevalence of PMCA2 and PMCA3. Although various rescue mechanisms, i.e., the increased expression of constitutive PMCA isoforms or SERCA pumps, are activated in discrete brain regions, a finding possibly related to their functional specificity, they are still inefficient with regard to the restoration of the balance of calcium ions. Consecutively, an abnormal calcium signal can be linked with behavioral abnormalities; such a correlation has been revealed here for the cortex and striatum. The identification of genes whose expression is affected by ketamine treatment, in conjunction with the changes reported in schizophrenia, indicate their involvement as putative etiological factors of this disease.In conclusion, we have shown that the inability to maintain brain Ca


Exserohilum rostratum in patients receiving contaminated steroid injections resulted in 64 deaths, receiving a lot of press and briefly bringing into the public eye the difficulty of treating systemic fungal infections [Aspergillus fumigatus, Candida spp. , Cryptococcus neoformans, Pneumocystis carinii, and dimorphic fungi that cause endemic mycoses . Fungal pathogens pose an especially high risk to individuals with compromised immunity, and this population of susceptible hosts is growing [The recent outbreak of fungal meningitis caused by fections . What isfections . The fun growing ,4. There growing –8.albicans Candida species [C. neoformans, and echinocandin resistance is increasing in some Candida species [Despite the extensive list of fungal pathogens and the increasing frequency of their occurrence, we have at our disposal only a very limited number of antifungal drugs. The past two decades have seen the emergence of two classes of antifungals: those that target ergosterol synthesis (the azoles) and those that target cell wall β-1,3 glucan synthase (echinocandins). Of the azoles, the triazoles have gained importance as alternatives to the more toxic amphotericin B. Triazoles are fungistatic and their continued use has resulted in an increase in triazole resistance among formerly sensitive species and a rising number of disease cases caused by intrinsically azole-resistant non- species . Echinoc species . ClearlyA. fumigatus [C. albicans [C. neoformans [B. dermatitidis, and H. capsulatum [An ideal drug target is a fungal-specific protein that (1) functions as a virulence factor or is essential for fungal viability and (2) is absent from the host organism, such that its inhibition causes no toxicity in the host. The low number of drugs in our antifungal armamentarium is in part due to the relative evolutionary relatedness of fungi and mammals and the resulting paucity of fungal-specific proteins that meet these criteria. One class of molecules that do fit both of these criteria is comprised of proteins that function in the so-called two-component signal transduction pathways. These signaling pathways are based on the transfer of phosphoryl groups among their components (phosphorelays) and are one of the primary means by which bacteria and fungi sense and respond to environmental cues. Two-component signal transduction pathways present attractive targets for antifungal drug discovery because they exist in prokaryotes, plants, and lower eukaryotes but not in mammalian cells. Furthermore, while the genes encoding two-component system proteins are frequently not essential for viability, multiple studies have demonstrated the importance of two-component signal proteins for virulence in fungal pathogens, including umigatus , C. albialbicans –13, C. noformans , B. dermpsulatum   and a response regulator (RR) protein . In respDepending on the system and the specific factors involved, the initial HK phosphorylation may occur either in response to stress or in response to removal of stress, with the ultimate outcome of either activating or down-regulating the transcription of stress response genes. A comprehensive list of all known fungal two-component signal transduction proteins and their roles in virulence-related processes can be found in Despite the acknowledged attractiveness of two-component systems as drug targets and their well-understood molecular mechanisms, thus far no two-component system inhibitor has reached the clinic, even though efforts to identify inhibitors of bacterial two-component systems stretch back approximately two decades. Most of the in vitro efforts to identify such inhibitors have focused on searching for compounds that could prevent HK phosphorylation activity . HoweverSaccharomyces cerevisiae strain heterologously expressing C. albicans Nik1 [While in vitro screens can identify compounds that act via a desired molecular mechanism, these compounds may not be active in cellular or organismal context. For example, several identified HK inhibitors with good activity in vitro failed to inhibit bacterial growth in vivo because they were sequestered in membranes and other lipid-rich compartments . Anotherans Nik1 . Howeverans Nik1 .C. albicans, compromising the two-component system can be achieved by deleting one of the two copies of the gene encoding a HK or RR, creating a heterozygous mutant. In a haploid fungus, such as Candida glabrata, a temperature-sensitive allele of a two-component system gene can be utilized for the same purpose. This approach has been used to identify compounds that affect a variety of cellular pathways, including ergosterol biosynthesis, the actin cytoskeleton, and protein folding [A conceptually different type of in vivo screening approach applied in both bacteria and fungi uses a strain where the function of a particular pathway is compromised by mutation; as a result, the screen strain shows exacerbated sensitivity relative to the wild-type strain to compounds that specifically inhibit this pathway ,26. BecaC. albicans, S. cerevisiae, and C. neoformans [Another in vivo approach can take advantage of the fact that two-component signal transduction systems regulate gene expression, either via activating downstream MAP kinase cascades or by RR proteins acting as transcription factors . Thus, ioformans –29. ThusThe limited data available from studies of two-component proteins in fungal pathogens have revealed the critical functions of these proteins in adaptation to stress, regulation of virulence factors, and sensitivity to antifungal drugs, underscoring the importance of these signaling pathways in fungal pathogenesis. These features, together with the absence of two-component pathways in animals, make these proteins very attractive targets for antifungal drug discovery. Because two-component systems are found in all major fungal pathogens, drugs targeting these factors may have broad spectra. Recently developed tools for phosphohistidine analysis are likely to facilitate in vitro screening efforts, while complementary searches for pathway-based inhibitors may identify compounds that specifically inactivate two-component signal transduction in vivo.


Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families  based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1  and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed  in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium. Vascular endothelium, a thin-cell monolayer lining blood vessels walls, is ideally positioned to detect and transduce biochemical and physical information. Endothelial cells (ECs) are essential regulators of vascular tone, cellular adhesion and vascular smooth muscle cell (SMC) proliferation. The wide variety of stimuli sensed by ECs necessitates an intricate web of intracellular signalling components, including phosphoinositide-signalling involving phospholipases C (PLC) as main players.2+)-dependent phosphodiesterases that hydrolyze phosphatidyl-inositol bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) plus inositol trisphosphate (IP3) 16] and δhe other –22. Therrd or 4th order MA, PA and MCA) were isolated from 3–4 months old C57BL/6 mice. Prior to experimentation, arteries were carefully cleaned of adipose and connective tissue in cooled HEPES solution.All animal manipulations were performed in accordance with the Canadian Council on Animal Care guidelines. The Montreal Heart Institute Animal Research Ethics Committee approved all animal studies (Protocol number: 2011-33-01). Resistance arteries: . As control, total RNA was isolated from brain and testis using RNeasy Lipid tissue Mini Kit (Qiagen) and from blood using QIAmp RNA blood Mini kit (Qiagen). The purity and concentration of RNA were assessed using a Nanodrop spectrophotometer. Next, reverse transcription was performed using iScript kits (Bio-Rad) according to manufacturer’s instructions. Primers  and 5 ng2, 10mM HEPES, 10 mM glucose, 2 mM CaCl2, 1.0 mg/ml DTT, 0.1% BSA and with enzymes 0.62mg/ml (MAECs) or 0.31mg/ml (PAECs and MCAECs) papain , 1.5mg/ml (MAECs) or 0.75mg/ml (PAECs and MCAECs) collagenase . Arterial segments were incubated in digestion solution for 25 min at 37°C followed by a triple wash in enzyme-free solution and then gently triturated. SMC-deprived EC tubes were visually identified with a Zeiss Axiovert A1 microscope and individually collected with patch micropipettes to ensure the purity of the isolated ECs. ECs mRNA was extracted and purified  before amplification using MessageBOOSTER Whole Transcriptome cDNA synthesis kit for qPCR (Epicentre Biotechnologies). Reverse transcription was performed using iScript kit (Bio-Rad) according to the manufacturer’s instructions. The purity of the isolated ECs was further assessed by qPCR analysis of the relative abundance of endothelial specific (CD31) and SMCs specific (SM22) mRNA (CD31/SM22 ratio) in MAECs, PAECs and MCAECs , ECs were freshly isolated from MA, PA and MCA as previously described by Socha et al . Cell did MCAECs . qPCR waen face) on a Sylgard block. Arteries were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 then blocked with 4% normal donkey serum. Following overnight incubation at 4°C with primary antibodies  in 0.1% Triton X-100, arteries were incubated for 1h with an Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody (Invitrogen). DAPI was applied to stain nuclei prior mounting for confocal imaging. Autofluorescence of the internal elastic lamina was also acquired. Fluorescence emissions were detected using a Zeiss LSM 510 confocal microscope . All images were deconvolved with Huygens professional software using experimentally determined point spread functions (PSFs) and reconstructed with Zen 2009 Light Edition.MA were cut longitudinally, and pinned . Tissue was then lyophilized, disrupted and heated at 95°C for 10 minutes in SDS-gel sample buffer  ,25. FinaData are presented as means ± SEM. One-way ANOVA with Turkey’s multiple comparisons test was used to compare the means; P<0.05 was considered significant.Expression of PLC isoforms was initially determined in freshly isolated arteries from 3 different vascular beds: MA, PA and MCA Figs  and 4A. vs. SMC markers , γ(1–2), δ, ε, ζ and η(1–2) [2+ [PLCs are crucial components of the phosphoinositide signalling pathway through their hydrolysis of PIPd η(1–2) . PLC iso1–2) [2+ . For exa1–2) [2+ ,29. Thes1–2) [2+ . For exa1–2) [2+ ,31. Theset al.: PLCβ1 and ε are expressed in native ECs from all vascular beds tested but not in HUVECs. However, β2 is absent from freshly isolated murine ECs but is expressed in HUVECs. Also, η1 and η2 are expressed in HUVECs but were not detected in the 3 types of native ECs. These discrepancies might be explained by the alteration of endothelial phenotype maintained in culture [Recently, Lo Vasco and coll. Report that of 10out of 13 PLC isoforms are expressed in HUVECs, a cultured ECs model . Interes culture . In vitr2+, suggesting a potential role downstream of changes in endothelial Ca2+ dynamics [et al. [Employing qPCR allowed us to identify the PLC isoforms expressed specifically in freshly isolated ECs from MA, PA and MCA. Three of the four known PLCβ isoforms were detected in all ECs tested: PLCβ1, β3 and β4. Moreover, PLCβ3 expression was also found at the protein level with immunoblots and confocal imaging. This is an interesting finding since PLC β3 is involved in the VEGF-dependent inhibition of vascular permeability . Howeverdynamics ,42. Howedynamics  but its  [et al. . Finally [et al. , while P [et al.  and PLCη [et al. . In summvs. resistance) or vascular bed, including vascular permeability, hemostasis or vasomotor tone in response to the specific requirements of the perfused organ. For example, pulmonary vasculature is exposed to a low-pressure, high flow of oxygen-deprived blood. Therefore, the proteins expressed in ECs will have varied in order to adapt to the needs of their particular vascular bed of origin [ECs are heterogeneous both in structure and function across the vascular tree under normal or pathological conditions ,21. For f origin . We dete2+ dependant vasoregulatory signals. In fact, the release of Ca2+ through IP3 receptors in the ER membrane is stimulated by the IP3 generated upon activation of membrane receptors leading to activation of PLC. PLC are known to regulate Ca2+ levels and thus might be involved in the modulation of intracellular Ca2+ dynamics. In 2013, De Bock and coll. reviewed the signalling pathways including PLCs pathway leading to intracellular Ca2+ elevation in blood-brain-barrier ECs [2+-dependent, they vary with respect to their sensitivity to Ca2+. PLCδ and η are the most sensitive to Ca2+ and are involved in potentiating Ca2+ signalling [2+ oscillations [Endothelial PLC might be involved in the modulation of Carier ECs . Althouggnalling ,48. In allations .3 production results in the release of Ca2+ from intracellular stores [3R is the only ER Ca2+ channels expressed in these cells [3 is generated through activation of G protein-coupled receptors or tyrosine kinase receptors which activate PLCβ and γ respectively [2+. Therefore, specificity of the Ca2+ signal is often achieved through spatial and temporal compartmentalization of Ca2+ signals. Several different patterns of Ca2+ dynamics have recently been described in ECs: Ca2+ pulsars, TRPV4- and TRPA1-sparklets or wavelets, each generated by a specific pathway [2+ pulsars are MEP localized signals consisting of a spontaneous Ca2+ release from IP3 receptors [2+ pulsars. However, specific PLC activation could be responsible for localized Ca2+ signalling such as Ca2+ pulsars. Cytoplasmic gradients of IP3 would result in a limited activation of IP3R and restricted propagation of the signal. Although our immunochemistry data showed a homogeneous distribution of PLCβ3, γ2 and δ1 in ECs, PLC partners and modulators could be heterogeneously distributed within the cell. Microdomains would then result from these associations as well as the distribution of proteins involved in Ca2+ sequestration. For example, Gonzales and coll. have recently shown colocalization of PLCγ1 with IP3R, TRPM4 and TRPC6 in VSMC from cerebral arteries and elegantly demonstrated its requirement for pressure-induced membrane depolarization and myogenic vasoconstriction [2+ sparklets are clustered in EC microdomains at MEP where they are modulated by PLC isoforms [2+ pulsars [In ECs, increased IPr stores  and the se cells . IP3 is ectively . Multipl pathway ,12,14,53eceptors . Limitedtriction . Moreoveisoforms ,55. As f pulsars . MoreoveIn summary, in this study we established for the PLCs that are expressed in native endothelium and freshly isolated ECs. We showed that 8 out of the 13 mammalian PLC isoforms are expressed in MAECs and 10 in PAECs and MCAECs. These results represent an important step forward in our understanding of the intracellular signalling pathways and their role in the regulation of endothelial microdomains. Further investigation of the subcellular distribution and biological function of these PLC isoforms is required in order to have a better understanding of their relative involvement in regulating endothelial function.S1 FigQuantitative real time PCR analysis of mRNA expression levels of CD31, an endothelial-specific marker, and SM22, a smooth muscle cell-specific marker. (A) Pie chart illustrating the relative expression of CD31 to SM22 (CD31/SM22 ratio) in mesenteric arteries (MA) and in endothelial cells isolated from mesenteric arteries (MAECs). (B) Pie chart illustrating CD31/SM22 ratio in pulmonary arteries (PA) and in endothelial cells from pulmonary arteries (PAECs). (C) Pie chart illustrating CD31/SM22 in middle cerebral arteries (MCA) and in endothelial cells from middle cerebral arteries (MCAECs). n = 3.(TIF)Click here for additional data file.


XAB2 genetic variants with the risk of non-small cell lung cancer (NSCLC) using a tagging approach.XPA-binding protein 2 (XAB2) interacts with Cockayne syndrome complementation group A (CSA), group B (CSB) and RNA polymerase II to initiate nucleotide excision repair. This study aims to evaluate the association of XAB2 gene were selected by Haploview software using Hapmap database. Genotyping was performed using iPlex Gold Genotyping Asssy and Sequenom MassArray. Unconditional logistic regression was conducted to estimate odd ratios (ORs) and 95 % confidence intervals (95 % CI).A hospital-based case-control study was conducted in 470 patients with NSCLC and 470 controls in Chinese population. Totally, 5 tag single nucleotide polymorphisms (SNPs) in XAB2 genotype with rs794078 AA or at least one rs4134816 C allele were associated with the decreased risk of NSCLC with OR (95 % CI) of 0.12 (0.03–0.54) and 0.46 (0.26–0.84). When stratified by gender, we found that the subjects carrying rs4134816 CC or CT genotype had a decreased risk for developing NSCLC among males with OR (95 % CI) of 0.39 (0.18–0.82), but not among females. In age stratification analysis, we found that younger subjects (age ≤ 60) with at least one C allele had a decreased risk of NSCLC with OR (95 % CI) of 0.35 (0.17–0.74), but older subjects didn’t. We didn’t find that XAB2 4134816 C > T variant effect on the risk of NSCLC when stratified by smoking status. The environmental factors, such as age, sex and smoking had no effect on the risk of NSCLC related to XAB2 genotypes at other polymorphic sites.Unconditional logistic regression analysis showed that the XAB2 tagSNPs (rs794078 and rs4134816) were significantly associated with the risk of NSCLC in Chinese population, which supports the XAB2 plays a significant role in the development of NSCLC.The  Worldwide, lung cancer harbored the highest incidence and mortality rates among all malignant cancers , 2. Non-Nucleotide excision repair (NER) is the major DNA repair pathway to remove bulky DNA lesions induced by UV light and environmental carcinogens . NER hasIn vitro, when cells treated with DNA-damaging agents, enhanced interaction of XAB2 with RNA polymerase II or XPA was observed, which suggesting DNA damage-responsive activity of the XAB2 . Light and heavy smokers were categorized by using the 50th percentile pack-year value of the controls as the cut points . Statistical analysis was performed using the SPSS version16.0 . A P value of < 0.05 was considered as statistically significant. Gene-smoking interaction was analyzed by GxEscan (http://biostats.usc.edu/software).The P = 0.832 for gender, and P = 0.470 for age). There were also no significant differences in the distribution of smoking status between cases and controls. However, the heavy smokers (≥25 pack-year) accounted for 63.4 % in cases and only 49.2 % in controls, which suggested that cigarette smoking was a prominent contributor to the risk of lung cancer. Among ever-smokers, 46,8 % (96) and 41.8 % (79) are former smokers in lung cancer cases and controls, respectively. Of 470 NSCLC patients, 37.9 % (178) were adenocarcinoma, 50.6 % (238) was squamous cell carcinoma, and 11.5 % (54) were other types, including large cell carcinoma (n = 49) and mixed cell carcinoma (n = 5).The demographic characteristics of all participants are presented in Table XAB2 gene were presented in Table P > 0.05 for all).The position and minor allele frequency (MAF) of the 5 selected tag SNPs in XAB2 genes, two SNPs were identified to be associated with the risk of NSCLC. For XAB2 rs794078 G > A polymorphism, we found that AA genotype carriers had a significantly decreased risk for developing NSCLC  in comparison to those with GG genotype. For XAB2 rs4134816 T > C polymorphism, just one CC genotype was found among all individuals, so we combined CT with CC genotype together for further analysis. Our data showed that the subjects with rs4134816 CT or CC genotype had a decreased risk of NSCLC compared with those carrying TT genotype with OR (95 % CI) of 0.46 (0.26–0.84). We didn’t find that any other selected SNPs were associated with the risk of NSCLC.The observed genotype frequencies in participants and the association of genotypes with the NSCLC were presented in Table XAB2 polymorphisms with the risk of NSCLC (Table P = 0.034). Our data also showed that younger subjects (age ≤ 60) with at least one C allele had a decreased risk of NSCLC with OR (95 % CI) of 0.35 (0.17–0.74), but older subjects didn’t. However, there was no gene-environment interaction observed (P = 0.094). We didn’t find that XAB2 4134816 C > T variant effect on the risk of NSCLC when stratified by smoking status. The environmental factors, such as age, sex and smoking had no effect on the risk of NSCLC related to XAB2 genotypes at other polymorphic sites  in Nucleotide excision repair is the main mechanism for removing the bulky DNA adduct from damage DNA for preventing carcinogens-induced mutagenesis , 23. Sev−/− mice were increased susceptible to chemically induced skin cancer [TC-NER, as one of important sub-pathways in NER, only repairs the lesions in the transcribed strand in active genes. There are several major proteins involved in TC-NER in human cells, including CSA, CSB, XPA and XAB2. Studies have showed that the deficient of these nucleotide excision repair proteins contributed to the risk of various cancers. Animal experiments showed that the CSB played an important role in the cellular response to stress and CSBn cancer . A case-n cancer .XAB2 is a key factor in TC-NER, which is composed of 855 amino acids and contains 15 tetratricopeptide repeat motifs. By interacting with CSA, CSB, RNA polymerase II and XPA, XAB2 conducted the multiple functions in the process of transcription and TC-NER [d TC-NER , 20. Micd TC-NER . Over exd TC-NER .XAB2 variants on the risk of lung cancer. This is the first study to investigate the association of XAB2 polymorphisms with the risk for developing cancer. There were several studies to evaluate the role of XAB2 genetic variants in complex autoimmune disease. For example, Briggs et al. conducted a case-control study to evaluate the correlation between XAB2 rs4134860 T > C variant and the risk of multiple sclerosis (MS) and found an increased risk of MS among rs4134860 CC genotype carriers [XAB2 rs4134860 T > C polymorphism with the risk of NSCLC. In another study, researchers analyzed the impact of several polymorphisms in DNA repair genes on the prognosis of colorectal cancer patients and didn’t find the association of XAB2 rs794078 G > A variant with the cancer prognosis [XAB2 rs794078 AA genotype had 88 % decreased risk of NSCLC.Studies have demonstrated that the polymorphisms, which located in NER genes or regulatory sequences, may affect DNA repair capacity and further increase likelihood of cancer development. In the present study of NSCLC in Chinese, we used a relatively comprehensive selection of SNPs and found the significant effects of carriers . In thisrognosis . In presXAB2 polymorphisms in the development of NSCLC stratified by smoking status. We observed that a 49 % protective effect for XAB2 rs4134816 variant was evident only for non-smokers, but not for smokers. The exact mechanism of how cigarette-smoking effects on DNA repair capacity posted by XAB2 polymorphism is unknown. One possible explanation may be that the protective effect of XAB2 variant allele might be evident in non-smokers with low levels of oxidative damage. Similar pattern of genetic effects have been observed for DNA repair gene XRCC1 (X-ray repair cross-complementation group 1) at low smoking exposure, but not at high smoking exposure [As we know, the magnitude of the effect of smoking far outweighed all other factors leading to lung cancer , 32. Manexposure .XAB2 rs4134816 C genotype among men, but not among women. Genetic variants in NER genes are associated with variability of lung cancer risk. Letkova and his colleagues investigated the polymorphisms of selected DNA repair genes, including XPC, XPD, hOGG1 and XRCC1, and found the different risks of developing lung cancer when stratified by gender, which further supporting our current findings [XAB2 rs4134816 T > C genetic variant among subjects aged 60 years or younger. Using Cox proportional hazard model, Gauderman et al. estimated the age-specific genetic incidence rate and found that the estimated proportion of lung cancer patients with high-risk allele exceeds 90 % for cases with onset at age 60 years or less and decreases to approximately 10 % for cases with onset at age 80 years or older. These findings suggested the contribution of age in the development of cancer [When stratified by gender, our study showed a 61 % protective effect of findings . Our pref cancer . The numOur study has its limitation. Due to the moderate sample size and the lack of related phenotypic and functional assays, large studies and functional evaluations are still need to be conducted in the future.XAB2 gene in this NSCLC case-control set. We found the evidence of significant association with the risk of NSCLC for two tag SNPs (rs794078 and rs4134816) in XAB2 gene in Chinese population. These results further supported that XAB2 play a significant role in the development of NSCLC.In conclusion, we have genotyped 5 tag SNPs in


We evaluated the role of Iks remodeling in VF susceptibility using a tachypacing HF model of transgenic rabbits with Long QT Type 1 (LQT1) syndrome.Remodeling of cardiac repolarizing currents, such as the downregulation of slowly activating KIn vivo telemetry demonstrated steepening of the QT/RR slope in LQT1 with TICM . In vivo electrophysiology showed that LQT1-TICM had higher incidence of VF than LMC-TICM . Optical mapping revealed larger APD dispersion  and steep APD restitution in LQT1-TICM compared to LQT1-sham . LQT1-TICM developed spatially discordant alternans (DA), which caused conduction block and higher-frequency VF . Ca2+ DA was highly dynamic and preceded voltage DA in LQT1-TICM. Ryanodine abolished DA in 5 out of 8 LQT1-TICM rabbits, demonstrating the importance of Ca2+ in complex DA formation. Computer simulations suggested that HF remodeling caused Ca2+-driven alternans, which was further potentiated in LQT1-TICM due to the lack of IKs.LQT1 and littermate control (LMC) rabbits underwent three weeks of tachypacing to induce cardiac myopathy (TICM). 2+. Our results strongly support the contention that the downregulation of IKs in HF increases Ca2+ dependent alternans and thereby the risk of VF.Compared with LMC-TICM, LQT1-TICM rabbits exhibit steepened APD restitution and complex DA modulated by Ca Ventricular arrhythmia is a significant cause of mortality in heart failure (HF) patients –3. Elect+ channels are critical in cardiac repolarization, and their downregulation is thought to play a major role in HF-related APD prolongation. The most consistently downregulated K+ channels in HF are the transient outward potassium current (Ito) [Ks) [to rapidly inactivates during the plateau phase of action potentials, it is thought to have minimal impact on APD in large mammals [Ks can play an essential role as repolarization reserve [to and IK1, several depolarizing currents are upregulated, including late Na+ current [+/Ca2+ exchanger current (INCX) [Ks downregulation in HF in conjunction with other ion channel remodeling may further accentuate APD prolongation and promote arrhythmogenesis.Voltage-dependent Knt (Ito)  and slowto) [Ks) , 11, 12. mammals , 14. In  reserve –18 in ac reserve –21. Besi current –24 and Nt (INCX) –28. HencKs downregulation in HF may not necessarily be arrhythmogenic. Due to slow deactivation [Ks becomes larger during fast heart rates [Ks contributes APD shortening at short diastolic intervals to form the characteristic APD restitution curve [Ks may be effective against repolarization shortening in the setting of fast heart rate and flattened APD restitution, thereby protecting against reentry formation [Ks can promote post-repolarization refractoriness and enhance wavebreaks in VF [Ks in HF remodeling may be compensatory and beneficial by preventing repolarization shortening and post-repolarization refractoriness. Overall, the reduction of IKs in HF can have either a pro-arrhythmic or an anti-arrhythmic effect depending on its amplitude and relative contribution to repolarization and restitution. Intracellular Ca2+ could also play a role in modulating IKs current density, as higher levels of intracellular Ca2+ increase IKs current [Alternatively, Itivation , the amprt rates , 30, 31.on curve . Steep Aon curve –36, and ormation , 38. In ks in VF . Therefo current .Ks in HF-related arrhythmias by inducing HF by a tachypacing protocol [Ks vs. their LMCs in which tachypacing induced ~55% downregulation of IKs [Ks significantly increases arrhythmogenesis by increasing APD dispersion and promoting spatially discordant alternans (DA). This finding emphasizes the importance of IKs remodeling in promoting arrhythmogenesis in failing hearts.Here we investigated the role of Iprotocol  in a traprotocol  and compn of IKs . We founAll animal experiments were performed in accordance with the local guidelines of the institutions and only after approval by the Institutional Animal Care and Use Committee (IACUC) at Rhode Island Hospital, in accordance with the Institute for Laboratory Animal Research (ILAR) Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Adult male New Zealand white rabbits were selected from LMC and LQT1 lines and age-matched.Tachypacing-induced cardiomyopathy  was usedFollowing a week recovery period, the rabbits underwent a three-week rapid pacing protocol . After anesthesia with ketamine and xylazine, the chests of rabbits were shaved and ECG leads were attached for simultaneous recording of ECG and echocardiography. Two-D echocardiogram images (Hewlett Packard 5500) were obtained using a 7.5 mHz probe, and both long- and short-axis views were used, similar to human echocardiograms. The M-mode was obtained from the short-axis view. Analysis of LV and RV dimensions, left atrium, wall thickness, valve function, and LV ejection fraction (by Simpson’s planimetry method) were performed by a blinded echocardiographer. LQT1 and LMC rabbits were monitored using telemetric ECG devices to calculate QT/RR ratios  . TelemetThe EPS protocol was modified from a previously established protocol from our lab . Studies+ current recording were the same as before [Ca,L was obtained in Tyrode solution before K+ current recording; holding potential was—50 mV, and test potentials were—40 to +40 mV with 10-mV steps lasting 250 ms. ICa,L was defined as the difference of peak and steady-state current at the end of the pulse. E-4031 (5 μM) and chromanol 293B (30 μM) were used for isolating IKr and IKs, respectively. Tetrodotoxin (20 μM) and CdCl2 (0.2 mM) were added as needed to block Na+ and Ca2+ currents.Isolation of cardiomyocytes by standard enzymatic techniques and patch-clamp recordings were performed as described previously . Apical s before . ICa,L w.kg-1 IM), xylazene (15 mg.kg-1 IM), ketamine (60 mg.kg-1 IM), pentothal (35 mg.kg-1 IV), and heparin (200 U.kg-1). After an appropriate level of anesthesia was achieved, rabbits were euthanized via beating-heart harvest. The hearts were retrogradely perfused through the aorta with (in mmol.L-1) 130 NaCl, 24 NaHCO3, 1.0 MgCl2, 4.0 KCl, 1.2 NaH2PO4, 5 Dextrose, 25 Mannitol, 1.25 CaCl2, at pH 7.4, gassed with 95% O2 and 5% CO2. Hearts were placed in a chamber to maintain temperature, and 5 μmol.L-1 blebbistatin was added to the perfusate to reduce movement artifact.Rabbits were injected with buprenorphene (0.03 mg/kg IM), acepromazine max. Data was filtered using a spatial Gaussian filter (3×3 pixel), and first/second derivatives  were calculated using a polynomial filter . Pixels with low signal-to-noise ratio determined by (dF/dt)max (lower than 3×σ of baseline) and outliers of pixels determined by Grubbs’ test were removed from the analysis  [max-APDmin [The activation and repolarization time-points at each site were determined from fluorescence (F) signals by calculating (dF/dt) pixels) . APD disx-APDmin .2+ duration was performed by comparing odd and even beats as described in [Alternans analysis of APD and Caribed in , 50. Theribed in . Brieflyal lines .2+ channels and Na+-Ca2+ exchange properly distributed in space. The ryanodine receptors and L-type Ca2+ channels were simulated using stochastic algorithms. To model LMC-TICM, we reduced IKs by 50%  to compare the means of two groups and the Mann-Whitney test to compare values not normally distributed. Fisher’s exact test was used for categorical variables. All data are presented as means ± standard deviation, and a p value < 0.05 was considered significant.For normally distributed values, we used Student’s in vivo ECG monitoring to examine restitution kinetics from free-moving animals, 4) in vivo EPS for VF inducibility, and 5) optical mapping of isolated hearts to investigate VF mechanisms (Experiments were carried out in the following sequence: 1) TICM protocol to create HF, 2) echocardiographic studies to verify TICM phenotype in different genotypes, 3) chanisms .Four cohorts of rabbits were studied: LMC sham pacing (LMC-SH-P), LMC-TICM, LQT1 sham pacing (LQT1-SH-P), and LQT1-TICM. After three weeks of tachypacing, we verified HF phenotypes in TICM rabbits based on LV ejection fraction. TICM rabbits exhibited a significant reduction in mean LV ejection fraction (LVEF (%), n = 5; LMC-SH-P: 59±7; LMC-TICM: 35±6, LQT1-SH-P; 62±7, LQT1-TICM: 32±9, see Ks was significantly downregulated in LMC-TICM  regardless of progress in deterioration of LV function and the reduction of IKs in LMC-TICM. Due to the Iks-blocking properties of inhaled isoflurane (the anesthetic used for sedation in EPS), VERP measurements here should be interpreted with caution. Both LMC-TICM (3 of 11) and LQT1-TICM (6 of 11) animals were found to be inducible for VF during the programmed stimulation protocol, while sham-operated animals were not inducible  changed gradually in space, while Ca2+ transient changed more abruptly. The alternation of Ca2+ transient recovery between odd and even beats was greater than that of APD (see panel C). This effect can also be seen in panel A, middle trace (b), where APD alteration between odd and even beats are not prominent, while Ca2+ exhibits marked alternation between odd and even beats.2+ handling [DA can be created by two competing mechanisms: conduction alternans or tissue heterogeneities such as heterogeneous Cahandling . Theorethandling  have proin vivo experiments, LMC-TICM and LQT1-TICM both demonstrated increased propensity to VF under ramp pacing protocol during optical mapping , n = 5; LMC-TICM: 12.59±1.47, LQT1-TICM: 15.65±1.75, *P<0.05, panel C). The basic relationship between baseline APD and VF frequency was lost  as in vivo EPS .Fig 6 shows the results of a computer model of ventricular myocytes, which includes a detailed spatiotemporal Ca2+ cycling system , followed by formation of the APD nodal line (3rd column), suggesting that Ca2+ instability promotes complex Vm DA. As expected, abolishing Ca2+ transient with ryanodine reduced the incidence of DA (6 of 9) and VF induction in LQT1-TICM. Some LQT1-TICM hearts still demonstrated DA under ryanodine (3 of 9 hearts), though their dynamics were markedly different before ryanodine perfusion. The nodal lines of Vm DA under ryanodine were closely related to the activation pattern  still exhibited DA after ryanodine perfusion. The nodal lines after ryanodine administration indicate abnormal conduction, as they are aligned with wave fronts and move towards the pacing sites, unlike the case in LQT1-TICM . AbnormaNa , 62, 65.Ks, it is surprising to see even higher-frequency VF in LQT1-TICM compared to LMC-TICM. The higher VF frequency in LQT1-TICM may be related to its steep APD restitution. As a result, refractoriness and wavelength can be substantially shortened at short CLs such as in VF, allowing high-frequency VF in LQT1-TICM. The cause of APD shortening at rapid heart rate is not clear. Harada et al.[CaL. Further studies are needed to understand the mechanism underlying APD shortening and high-frequency VF in LQT1-TICM.Our study demonstrated that VF in LQT1-TICM exhibited significantly higher VF frequency despite prolonged APD. Since LQT1-TICM hearts lack Ida et al. providedKs, is highly arrhythmogenic. Abnormal Ca2+ handling in failing hearts promotes Ca2+ alternans, and lack of IKs enhances the effect of Ca2+ alternans on APD alternans, complicating the dynamics of DA and VF. This study emphasizes that the repolarization reserve provided by IKs is an important modulator of Ca2+-driven DA and VF.Our results strongly support the contention that lack of repolarization reserve in HF, particularly reduction in IS1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S1 Moviem and Ca2+. Bottom panels: Vm and Ca2+ nodal lines. The nodal lines of LQT1-TICM demonstrated complex patterns including genesis, vanishing, wiggling, and moving. The spatiotemporal patterns of nodal lines of action potential and Ca2+ transient were also complex. This movie corresponds to Top panels: V(MP4)Click here for additional data file.S2 Moviem, Right panel: Vm nodal line in LQT1-TICM heart after ryanodine. This movie corresponds to Left panel: V(MP4)Click here for additional data file.


Although intracellular disruptions of cytosolic and mitochondrial calcium, and in particular low cytosolic calcium ([Ca2+]c) buffering and a strong interaction between metabolic mechanisms and [Ca2+]i have been identified predominantly in motoneuron impairment, the causes of these disruptions are unknown. The existing evidence suggests that the mutant superoxide dismutase1 (mtSOD1)-mediated toxicity in ALS acts through mitochondria, and that alteration in cytosolic and mitochondria-ER microdomain calcium accumulation are critical to the neurodegenerative process. Furthermore, chronic excitotoxcity mediated by Ca2+-permeable AMPA and NMDA receptors seems to initiate vicious cycle of intracellular calcium dysregulation which leads to toxic Ca2+ overload and thereby selective neurodegeneration. Recent advancement in the experimental analysis of calcium signals with high spatiotemporal precision has allowed investigations of calcium regulation in-vivo and in-vitro in different cell types, in particular selectively vulnerable/resistant cell types in different animal models of this motoneuron disease. This review provides an overview of latest advances in this field, and focuses on details of what has been learned about disrupted Ca2+ homeostasis and mitochondrial degeneration. It further emphasizes the critical role of mitochondria in preventing apoptosis by acting as a Ca2+ buffers, especially in motoneurons, in pathophysiological conditions such as ALS.Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disorder characterized by the selective degeneration of defined subgroups of motoneuron in the brainstem, spinal cord and motor cortex with signature hallmarks of mitochondrial Ca Amyotrophic lateral sclerosis (ALS) is an incurable, adult-onset, deadly neurodegenerative disorder distinguished by the progressive degeneration of a defined motoneuron (MN) population in the brain stem, spinal cord and motor cortex. ALS leads to paralysis, atrophy and death within 5 years . Current2+ into mitochondria and low content of Ca2+ binding proteins which modulate physiological as well as pathophysiological processes and may render MNs selectively vulnerable to degeneration i buffering and a robust interaction between metabolic processess and [Ca2+]i have been related with selective and severe MN damage resulting from excitotoxic stress and disruptions of cellular and mitochondrial Ca2+ homeostasis i, which subsequentially leads to increased nitric oxide formation. Peroxynitrite, produced by the reaction of superoxide anions and nitric oxide, can consequently lead to oxidative disruption [2+]i homeostasis and free radical production. For example, in human ALS (hALS), overexpression of glutamatergic synapses leading to excessive Ca2+ influx has been linked with MN degeneration i where low CB-D28k transfected neuronal cells display a significant (~2 times) reduction in the peak amplitude of the sustained [Ca2+]i increase compare to high CB-D28k transfected neuronal cells (data not shown). τ in CB-D28k transfected cells is also slower (~60s) compared to non-transfected cells where baseline recovery time is ~30-35 s while showing little differences in the area under the time - concentration curve (AUC). This observation is in good agreement with the ∁low buffering hypothesis” which states that low buffer capacity allows for rapid Ca2+ dynamics during physiological activity, but represents a significant risk factor during ALS-related MN disease [in-vitro cell culture models have shown that elevated [Ca2+]i buffer concentration reduces ALS specific MN damage providing further support in favor of the notion that increased buffer concentrations create beneficial protection i to MNs death is not clear though these processes have been mostly credited to mitochondrial Ca2+ accumulation or glutamate excitotoxicity. Whether the change in ΔΨm is simply an inevitable consequence of an unusually enormous Ca2+ load or there is other downstream pathway contributing to glutamate response is not known yet, however depolarisation of ΔΨm is one consequences of it. Data also suggested the role for other factor in addition to Ca2+ such as nitric oxide synthase (NOS) inhibitors. Results also suggest that mitochondrial damage arbitrated at least in part by Ca2+ induced MPTP opening and may contribute to surge of neighbouring cell activation. In conclusion, we believe that two of the most important features for MNs in ALS are: (i) low buffer capacity generates exceptionally large Ca2+ domains, but not in case of serious end stage ALS like symptoms and (ii) in ALS vulnerable MNs buffering capacity critically depends upon the domain size of mitochondria and ER. Therefore, we proposed a model where a portion of MN mitochondria interacts with areas of high [Ca2+] around influx sites due to low buffering induced excitotoxicity shown in Figure In the absence of adequate cytoplasmic buffering, or in cases where the existing cytoplasmic buffering system is overwhelmed, there is a shift in mitochondrial Ca2+ homeostasis results from compromised voltage-dependent calcium influx c concentrations in neurons compromises mitochondrial integrity and function by inducing enhanced production of ROS from mitochondria i in many systems i elevation, indicating a relative inability to sequester Ca2+ in the MNs of SOD1G93A mice as compared to WT lettermates. These results indicate that the conventional Ca2+ storing function of mitochondria is dominating over ER Ca2+ accumulation in these MNs. These results are in good agreement with the “hotspot” hypothesis that suggests that mitochondria preferentially accumulate Ca2+ at microdomains of elevated Ca2+concentration ([Ca2+]i), predominantly near ER Ca2+ release sites and other Ca2+ channels. Accordingly, mitochondria can affect both Ca2+ release from the ER and capacitative Ca2+ entry across the plasma membrane, thereby shaping the size and duration of the intracellular Ca2+ signal in MNs of WT and SOD1G93A mice. These events determined by the Ca2+ sensitivity of the Ca2+ channels and capability of mitochondria to remove Ca2+ from the subcellular microdomain at the opening of the ion channel. This effect has been confirmed in-vitro, but the condition appears markedly different in various cell models i signals i transients; even during small cytosolic Ca2+ increases, mitochondria are known to take up more than 50% of intracellular Ca2+. Outsized and enduring Ca2+ microdomains around Ca2+ influx sites increase the danger of toxic Ca2+ buildups and a successive activation of Ca2+ dependent neurodegenerative pathways under excitotoxic conditions. Excitotoxicity associated with these influx domains is primarily suppressed by mitochondrial contributions, rather than ER Ca2+ uptake i signaling varies at sub cellular level in and around microdomain? (c) In the presence of mtSOD1 gene, what are the consequences of alterations in mitochondrial function on Ca2+ homeostasis and ERMCC? Numerous developments and improvements in visualization of diseased MNs and spatiotemporal resolution of mitochondria-ER calcium signaling cascades have the potential to bring novel insights. MN possess numerous Ca2+ channels that cause rapid Ca2+ influx because of comparatively weak [Ca2+]i buffering, results in mitochondrial Ca2+ overload and strong ROS generation in mtSOD1 Mice. Further studies of ER-mitochondria calcium cycle (ERMCC) with targeted calcium probes show a rapid and dramatic increase in free intra mitochondrial and ER calcium. The defects in mitochondrial assembly and vacuoles derived from defective mitochondria found in post mortem ALS patients further support the suggestion. Design of novel antioxidant strategies to selectively target the oxidative stress and redox imbalance is the other evenue need to be explored. Furthermore, check on selective loss of MNs causing the discharge of pro-apoptotic proteins which activate enzymes leads to activation of apoptotic pathways and apoptotic cells observed in MNs of ALS is beneficial. Dysregulation between mitochondria-ER and ERMCC are well known features and therapeutic drugs aiming to stabilize these cycles reduce ROS and oxidative stress and may be effective in wide range of MN diseases. Another nice direction would be to develop so called “smart drugs” and “combination therapies or multi drug therapies” which have mutifactorial imact on disease mechanism due to mulfactorial nature of ALS disease. Furthermore, it is hypothesized that disrupted Ca2+ homeostasis and oxidative stress induced ROS have a vital role in propagating injury by increasing the excitability of MNs and by targeting neighboring glia. Perhaps as a consequence, excitotoxicty builds up with increased activity-dependent Ca2+ influx and associated mitochondrial Ca2+ cycling. Given the mitochondrial disturbances, Ca2+ buffering becomes inefficient and increase in cytosolic Ca2+ levels. Protective options are to elevate the resistance of MNs to high intracellular Ca2+ concentrations by inducing defense mechanism and/or to inhibit the downstream apoptotic and death cycle pathways activated by increased intracellular Ca2+ concentrations. However, severely impaired MNs are not amendable to taking functional advantage of neuronal protection in ALS.In spite of rigourous research for years there are several important questions still unanswered; these include: (a) does the expression of mtSOD1 at physiological levels causes morphological and structural abnormalities of mitochondrial assembly and its calcium buffering capacity? (b) At pathophysiological levels, does the mitochondria-ER and EMRCC CaTherefore perhaps we should focus on new tools such as recently discovered genes that cause ALS and induced pluripotent stem cells taken from ALS patients and derived into MNs to identify potential cytosolic pathways and barriers that could lead to MN degeneration in ALS. Forthcoming studies will hopefully add to the understanding of why these processes preferentially damage MNs and the role non-cell autonomous cell death might play. In conclusion, data indicates that ALS is a multifactorial disease and therefore a combined therapeutic interference (combination therapy) with many facets of target site both at the MNs and glial cells will be most likely essential for survival of ALS patients. While keeping in mind the previous failures in clinical trials for ALS, further studies in this direction to better understand the pathogenesis of cell death in ALS and targeted therapy therefore will be of great interest.


CSF3R, the gene encoding G-CSFR. These fall into distinct classes, each of which is associated with a particular spectrum of myeloid disorders, including malignancy. This review details the various CSF3R mutations, their mechanisms of action, and contribution to disease, as well as discussing the clinical implications of such mutations.Granulocyte colony-stimulating factor is a cytokine able to stimulate both myelopoiesis and hematopoietic stem cell mobilization, which has seen it used extensively in the clinic to aid hematopoietic recovery. It acts specifically via the homodimeric granulocyte colony-stimulating factor receptor (G-CSFR), which is principally expressed on the surface of myeloid and hematopoietic progenitor cells. A number of pathogenic mutations have now been identified in  Granulocyte colony-stimulating factor  augments the production and function of neutrophilic granulocytes, which play an essential role combatting infection, especially those of a bacterial or fungal nature –5. G-CSFThe biological actions of G-CSF are mediated via docking to a homomeric receptor found on the surface of target cells, granulocyte colony-stimulating factor receptor (G-CSFR)  . The G-CCSF3R, have now been described. These mutations can be placed into a number of distinct classes that relate to the type of mutation and their biological and clinical consequences  mutation  activation  mutation was initially described as a late somatic mutation in the development of AML in an SCN patient already bearing an alternate G-CSFR mutation . Howevernovo AML , 48, 49,novo AML . G-CSFR novo AML , which cnovo AML , 48, butnovo AML , 48, witnovo AML , but notnovo AML , 48.de novo AML (The p.Thr617Asn (p.Thr640Asn) mutation was first identified in a single case of AML . Furthernovo AML , 51. Hownovo AML . Interesnovo AML , furthernovo AML . Lineagenovo AML . The p.TFinally, an in-frame three nucleotide deletion has been identified in MDS that replaces two amino acids with an alternate residue, p.Asn630Lys,Arg631del . This mutation resulted in prolonged signaling following ligand stimulation  and pGln731* (p.Gln754*) (CSF3R (82%), followed by RAS (~50%) and monosomy 7  , but medGln754*) –60. HoweGln754*) , 62. Indnosomy 7 , and whemutation . Howevermutation , progresex vivo   . The priclass IV , which eCSF3R SNP that is present in ~6% of the population leads to a p.Glu785Lys (p.Glu808Lys) amino acid substitution in the intracellular region of the G-CSF, which predisposes individuals to high-risk MDS (A risk MDS . Interesrisk MDS , providirisk MDS , 83.CSF3R mutations. However, the available data are complicated, making conclusions difficult. One study reported no significant relationship between age of MDS/AML onset and G-CSF dose or duration of therapy (CSF3R mutation, such as those leading to CNL. A number of pharmacologic agents are now available that target signaling pathways downstream of the G-CSFR (Figure Granulocyte colony-stimulating factor has proven to be an effective therapy in a range of life-threatening conditions or to aid in the recovery of medical treatments, such as in the treatment of neutropenia following chemotherapy. However, the evidence suggests that G-CSFR mutations contribute to several disorders, including in settings where G-CSF may be used therapeutically. It has been suggested that use of G-CSF in SCN may allow the selective expansion of clones containing truncating  therapy . However therapy . However therapy . Thus it therapy . HoweverR Figure , providiR Figure , 48.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Store-operated Ca2+ entry (SOCE) channels are important Ca2+ influx mechanisms in many cells. SOCE involves release of Ca2+ from intracellular pools followed by Ca2+ entry. The best-characterized SOCE channels are the Ca2+ release-activated Ca2+ (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca2+ uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels  were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca2+ release mechanism. Passive depletion of ER Ca2+ stores with thapsigargin resulted in a significant raise in [Ca2+]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca2+ entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca2+ uptake in enamel formation.Dental enamel formation requires large quantities of Ca Ameloblasts are polarized cells responsible for the regulation of Ca2+ transport during enamel formation. These cells form an epithelial barrier restricting the free flow of Ca2+ into the enamel layer where hydroxyapatite-like crystals are growing12+ and to avoid toxicity, these cells must tightly regulate Ca2+ influx and buffering, organellar Ca2+ release and sequestration, and Ca2+ extrusion. Ameloblasts express Ca2+ binding proteins in the cytoplasm and ER234562+-ATPases (SERCAs) pumps being involved in ER Ca2+ sequestration thus contributing to cytosolic Ca2+ buffering2+-ATPases (PMCA) as well as K+-dependent and K+-independent Na+/Ca2+ exchangers 789101112132+ in the formation of hydroxyapatite-like crystals, our understanding of the mechanisms employed by ameloblasts to mediate Ca2+ uptake and transport remains limited although biochemical data has suggested a “transcytosis” route for Ca2+ being channelled across the cell within the ER222Ca2+ release-activated Ca2+ (CRAC) channel protein STIM1 in murine enamel organ cells from a genome wide study2+ influx pathway in non-excitable and excitable cells that is activated following Ca2+ release from the ER162+ causes the ER resident proteins STIM1 and STIM2 to interact with ORAI proteins, which form the pore of the CRAC channel in the plasma membrane, enabling localized and sustained Ca2+ entry1718STIM1 and ORAI1 genes, which are characterized by severely hypo-mineralized enamel13202+ uptake during enamel formation.Recent evidence gathered by our group first identified one of the components of the Ca2+ and phosphate, which are moved to the extracellular domain to supersaturate the enamel fluid and enable a vast increase in thickness of the enamel crystals13152223Stim1 and Stim2 genes as up-regulated transcripts in the maturation stage and we further confirmed these results by Western blot analysis of STIM1 and STIM2 proteins. The present study explores whether secretory stage enamel organ (SSEO) and maturation stage enamel organ (MSEO) cells are equipped with components essential to increases in Ca2+ handling capacity, and tests whether Ca2+ entry in SSEO and MSEO cells is mediated by CRAC channels.Enamel develops largely in two stages, the secretory and maturation stages. The continuously growing rodent incisor is an ideal model to study enamel development as a population of cells from both stages can be identified through life. In the secretory stage, ameloblasts are involved in the synthesis and secretion of enamel-specific proteins, forming an organic template for the growth of thin enamel crystals2+ concentration ([Ca2+]i) can be mediated by the release of Ca2+ from ER stores via IP3Rs or/and RyRs3R isoforms were identified in rat secretory stage enamel organ and maturation stage enamel organ cells by RT-PCR, however, RyRs expression levels were negligible compared to those of the IP3R transcripts (data not shown). This was also evident by immunofluorescence staining of the RyR proteins which showed very weak signals  cells in secretory stage, and papillary layer (PL) cells during maturation stage, also showed positive staining for the IP3R proteins  by RT-PCR in maturation stage enamel organ cells, which was also the predominant isoform .Refilling ER Ca isoform . We ther isoform . In secr isoform . These dStim and Orai isoforms in secretory and maturation stage enamel organ cells by RT-PCR  and pore subunit (ORAI1) of the CRAC channel. We also examined the contribution of STIM2 and investigated its localization by immunoperoxidase staining. STIM2 protein was expressed in both secretory and maturation ameloblasts with stronger signals in maturation  in maturation stage enamel cells (~230 nM) compared to secretory stage enamel cells (~135 nM) (2+ stores activates SOCE mediated by CRAC channels. We thus stimulated enamel cells with thapsigargin (1.25 μM) to block SERCA, a procedure that depletes Ca2+ from ER stores through poorly understood ER leaks292+ resulting in increased levels in [Ca2+]i due to Ca2+ release from ER stores (2+ release increased [Ca2+]i by ~40 nM. In maturation stage cells this increase was in the order of ~70 nM.Intracellular Ca~135 nM) . Depleti cells ~2 nM compaR stores . We foun2+ entry was subsequently analyzed upon readdition of extracellular Ca2+ to the cells. We identified a significant increase  in [Ca2+]i in both cell types demonstrating that enamel cells from secretory and maturation stages have SOCE. This increase in [Ca2+]i was however significantly higher in maturation stage enamel cells than in secretory stage cells  (2+]i following readdition of Ca2+ was ~115 nM in secretory stage cells and ~350 nM in maturation stage cells (Ca t-test) . The incge cells . These dge cells .2+ influx upon readdition of extracellular Ca2+ lar Ca2+ . These r2+ transport is critical for enamel development and yet the cellular mechanisms associated with Ca2+ entry remain a central question in enamel biology. The present study demonstrates that rat secretory stage enamel organ and maturation stage enamel organ cells express SOCE with CRAC channel properties. As Ca2+ release from the ER pools is required for SOCE activation, we investigated whether enamel cells are equipped with common receptors involved in ER-Ca2+ release and to compare possible differences among them. Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) are the main ER Ca2+ release mechanisms253R, revealing differences in cellular localization  signals detected in ameloblasts  significantly reduced Cadetected . This sm2+ buffering proteins232+ transport enabling us to refine hypotheses concerning Ca2+ transport in enamel formation. In enamel biology, the calcium transcytosis hypothesis of Hubbard2222+ movement in enamel epithelium. Instead, an active transport system is more likely562+ entry, transit and extrusion  act as shuttles of Ca2+ across cells. However, the demonstration that calbindin28 kDa-null mice lack a dental phenotype2+ entry points, supports the role of ER as a key component for Ca2+ transport as previously proposed22+ from the base to the apex42Data shown here combined with previous studies identifying luminal ER Ca2+ entry model in enamel cells as shown in 2+ extrusion during enamel mineralization.The data presented here allow us to propose a CRAC channel-regulated Ca2+ uptake via CRAC channels in primary enamel cells. These data together with the localization of STIM1 and ORAI1 throughout the ameloblasts highlights the possibility that the ER may act as a key organelle enabling Ca2+ transport across the cells as held by the calcium transcytosis model. These data greatly advance our understanding of the mechanism regulating Ca2+ transport during enamel formation, and warrant further investigations into the role of SOCE in enamel cell signaling and calcium transport.Our work provides the first functional evidence for Castratum intermedium cells may also be present. During maturation, papillary layer cells replace the stratum intermedium. Total RNA was extracted from rat secretory and maturation stage enamel organ cells and control tissues using RNeasyR Micro Kit  according to the manufacturer’s specifications. Reverse-transcribed PCR was performed using iScripttm cDNA Synthesis Kit . RT-PCR amplifications were set up in a total volume of 20 μl using 0.7 μg of cDNA, 250 nM forward and reverse primer using SsoAdvancedTM Universal SYBR Green Supermix . Cycling conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 39 cycles of 95 °C for 10 sec, 58 °C for 30 sec. Specificity of PCR products was confirmed by analysis of a melting curve. RT-PCR amplification were performed on a CFX ConnectTM Real-Time System  and all experiments were done in triplicate. The housekeeping gene beta-actin was amplified to standardize the amount of sample RNA. Relative quantification of gene expression was achieved using the ΔCT method. Primer sequences used can be found in the All animal protocols were approved by the Animal Care and Use Committee, and are in full compliance with Federal, State, and local laws and institutional regulations. Animals were maintained following NIH guidelines. Sprague Dawley rats or C57BL/6 mice were used as detailed below for each experiment. The use of rats was preferred over mice for the isolation and processing of enamel organ cells for Fura-2 AM analysis, RT-PCR and Western blot as previous studies had reported on anatomical landmarks in rats that allow us to independently isolate secretory from maturation stage enamel organs and because the larger size of rats makes this procedure easier443R1 , anti-IP3R2 , anti-IP3R3 , anti-SERCA2 , anti-RyR1 , anti-RyR2  and anti-RyR3 . Sections were incubated overnight at 4 °C. Negative controls were provided by incubating sections with Dako Diluent lacking primary antibody. After washing with PBS, sections were incubated for 30 min at room temperature using appropriate secondary antibodies from Invitrogen at 1:800 (Anti Rabbit IgG AlexaFluor488 and/or Anti-Mouse IgG Alexa Fluor555). After washing, slides were mounted with a coverslip using Prolong Gold Mounting Media  containing DAPI. Images were obtained using a Leica TCS SP5 II confocal microscope or a Nikon Eclipse 2000TE. The immunolocalization of Stim2 was performed by incubating with an anti-Stim2 antibody  using Vectastain ABC Kit (Vector Laboratories) and counterstaining with haematoxylin before mounting the slides with Vecta Mount Mounting Media (Vector Laboratories). Samples were imaged using a Nikon Eclipse E600 microscope with NIS Elements BR3.2 (Nikon) software. Western blot analysis of SERCA2 was performed as describedFor immunohistochemical analysis, rats and mice at 10 days postnatal were euthanized. Isolated mandibles stripped of soft tissues were immersed in formalin overnight at 4 °C and decalcified in 4.13% EDTA (pH 7.3) for 7–10 days and washed. Appropriate positive control tissues were also collected and processed as for dental tissues but without the decalcification step. Samples were embedded in paraffin for sectioning (5 μm thick). After deparaffinization, antigen retrieval was performed by cooking the slides for 20 min in 10 mM citric buffer (pH 6.0) followed by blocking for 1 hr with Dako Diluent . The following antibodies and dilutions were used: anti-STIM1 , anti-calnexin (a marker for ER) , anti-ORAI1  for 10 min at 37 °C in a 5%-CO2 incubator and manual disruption. Reaction was stopped by adding DMEM  containing 30% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% glutamine. For intracellular Ca2+ concentration ([Ca2+]i) measurement, isolated SSEO and MSEO cells were cultured in X-VivoTM 15  cell media containing 10% FBS, 1% penicillin/streptomycin, 1% glutamine and maintained at 37 °C in a 5%-CO2 incubator. Cells were used within 24 hrs after isolation.Rats (100–160 gram) were euthanized, mandibles were dissected out and the surrounding soft tissues removed. Isolated mandibles were transferred to Hanks solution (GIBCO) containing 1% Antibiotic-Antimycotic and kept on ice at all times. Isolated SSEO and MSEO cells were treated with 0.15% collagenase  in PBS for 1 hr at 37 °C in a 5%-CO2 incubator. To determine intracellular Ca2+ concentrations ([Ca2+]i), cells were loaded with Fura-2-AM  for 30 min at room temperature. Cells were then washed with X-VivoTM 15 cell media containing 10% FBS, 1% penicillin/streptomycin and 1% glutamine. Finally, cells were dispensed into 96-well black clear bottom plates. Fura-2 emission was detected at 510 nm after excitation at 340 nm and 380 nm using a FlexStation3 plate reader 2+]i was estimated from calibration curves by using the Calcium Calibration Buffer Kit#1  using the following equation: [Ca2+]i = Kd x ((R-Rmin)/(Rmax-R)) x (F380max/ F380min) as described by Grynkyewiczd is the dissociation constant of Fura-2 (Kd = 156 nM); R is the ratio of Fura-2 emission following excitation at 340 nm divided by the Fura-2 emission following excitation at 380 nm; Rmin was measured in nominally Ca2+ free buffer; Rmax was measured at saturating Ca2+ concentration (39 μM); F380max is the fluorescence intensity after excitation at 380 nm in Ca2+ free buffer; F380min is the fluorescence intensity at saturating Ca2+ concentration (39 μM). All experiments were performed at 25 °C. In order to measure SOCE, intracellular Ca2+ stores were depleted by inhibiting SERCA activity with thapsigargin  in cells kept in Ca2+ free Ringer solution followed by readdition of an equal volume of Ca2+-containing Ringer solution at the times indicated to a final concentration of 2 mM Ca2+. For some experiments, SSEO and MSEO stage cells were treated with the CRAC channel inhibitor Synta compound 66 4, 32.2 Hepes, 2 Na2HPO4, 4 CaCl2, and 5 glucose at pH 7.4 (extracellular Ca2+ buffer) or 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32.2 Hepes, 0.5 EGTA, 5 glucose, pH 7.4 (Ca2+-free buffer). Ca2+ data were acquired using SoftMax Pro software  and analyzed using GraphPad Prism v.5. Cells were analyzed for baseline [Ca2+]i, increase in [Ca2+]i after ER depletion and SOCE. The area under the curve (A.U.C.) following Ca2+ influx was analyzed using GraphPad Prism v.5.Secretory stage enamel organ (SSEO) cells and maturation stage enamel organ (MSEO) cells were kept in media for 4 hrs at 37 °C in a 5%-COn represents the number of independent experiments. Differences were tested for significance using Student’s unpaired two-tailed t-test or ANOVA. P<0.05 was considered statistically significant.Data are provided as mean ± SEM, How to cite this article: Nurbaeva, M. K. et al. Dental enamel cells express functional SOCE channels. Sci. Rep.5, 15803; doi: 10.1038/srep15803 (2015).