Sc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrPSc and PrPC are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V136R154Q171 allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6–8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrPC, only ovine PrPSc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background.Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrP Sc) of the host-encoded prion protein (PrPC). Different PrPSc conformational variants or ‘strains’ can propagate in the same host, giving distinct biological phenotypes. Like other pathogens, prions can transmit from one species to another, representing a zoonotic risk. A barrier, commonly referred to as “species barrier”, limits prion cross-species transmission. This barrier is supposed to reflect the steric incompatibility between invading prion PrPSc and PrPC of the infected host. Rabbit is one of the species that exhibit a pronounced resistance to prions. To gain insights on the molecular determinants of the relative resistance of this species to prions, we generated transgenic rabbits expressing sheep PrPC and assessed their experimental susceptibility to sheep scrapie prions, as routinely done with transgenic mouse models of prion disease. All transgenic rabbits developed a typical prion disease within 200 days, whereas wild type rabbits remained healthy more than 700 days after inoculation. These data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background.Prions are infectious pathogens causing irremediably fatal neurodegenerative diseases in human and in farmed or wild animals. They are formed from abnormally folded assemblies (PrP Sc) of the host encoded cellular prion protein (PrPC). During TSE pathogenesis, PrPSc seeds, acquired through infection or arising from spontaneous conversion of wild type or mutant PrPC, are believed to template the conformational change of host PrPC to nascent PrPSc forms. This autocatalytic polymerization process leads to deposition of injurious deposits into the brain. PrPSc particles are thought to be the major if not the sole component of TSEs infectious agent or prion [Sc biochemical properties. There is compelling evidence that prion strain diversity reflects stable differences in PrPSc conformations, at the level of the tertiary and/or quaternary structure [Scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, transmissible mink encephalopathy (TME) or Creutzfeldt-Jakob disease (CJD) in humans belong to a group of fatal neurodegenerative diseases referred to as transmissible spongiform encephalopathies (TSEs) or prion diseases . TSEs aror prion . Distinctructure –5.C and the infecting prion strain type(s) [Sc in the brain and, sometimes, extraneural tissues of the new host. Their concomitant absence would usually suggest a resistance to infection or a disease incubation time exceeding that of the exposed host life span [Sc-templated conversion of PrPC by assays such as the protein misfolding cyclic amplification  , 4. The ife span –9. Rabbiife span , 10. In n  revealesources –14). Yet. YetC ansources . Transgenes can be introduced in animals knocked out for their own Prnp gene or on a murine wild-type background  act as major determinants, the V136R154Q171 allele conferring the highest susceptibility [Numerous transgenetic studies in mice have demonstrated that mouse resistance to foreign prions can be abrogated by introducing in the murine genome the corresponding reviews: , 16). Vireviews: , strongllication –19. In stibility –22. Expetibility .136R154Q171 ovine PRNP allele develop a TSE syndrome upon experimental inoculation with scrapie. Disease occurred at full attack rate, with no apparent interfering effect of the rabbit wild-type PrPC.In this study, we report that rabbits engineered to express transgenetically the Vth of November 1998.All the experiments involving animals were done in strict accordance with the European Community Council Directive 86/609/EEC. The French committee for GMO,-formerly the governmental "Commission de Génie Génétique" that belonged to the French Ministry for Education, Research and Technology-, has approved the creation and experimental infection with prions of the rabbit transgenic animal model. The agreement number is 2992-II. It was issued the 12136R154Q171 allele of the Prnp gene was cloned in a bacterial artificial chromosome (BAC) vector previously used to generate the tg338 mouse line [PRNP gene, was purified following a Not1 digestion and microinjected into New Zealand White rabbit embryos. Approximately 700 hundred embryos  were microinjected . 600 embryos were implanted into 30 pseudo-pregnant females. There were 28 live births, of which one was transgenic, as identified by PCR. Rabbit DNA was extracted by digesting ear fragments using proteinase K-SDS method. Digestion was followed by a DNA precipitation by ethanol. The two primers used to identify the sheep PRNP gene were TAGGCAGTTGGATCCTGGTT and CCCTATCCTACTATGAGAAA. The primers used to identify the control endogenous αS1-casein gene were CACTCCCTTGTTGAAAACTCTCCTCAG and ATTTTGTGGTTTCAGATCAACCAATAGG.A fragment of the sheep genome containing the Vuse line , 24. ThiPRNP RNA in ovine PrP transgenic rabbits (TgOv), RT-qPCR analyses were performed on 3 animals per line, as previously described [PRNP ovine and rabbit sequences, resulting in the amplification of 100 bp long fragments, respectively. Two sets of primers were used for rabbit PRNP, owing the existence of two variants (with or without exon 2). The sequences of the specific primers were as follows: forward primer: 5’- TCATGGTGAAAAGCCACATAGG-3’, reverse primer: 5’- CCTCCGCCAGGTTTTGGT-3’ for ovine PRNP; forward primer: 5’-TCCTCTCGGCAGCTGTCAT-3’, reverse primer: 5’-GCTTCGGCCGCTTCTTG-3’ for Rabbit PRNP without exon 2; forward primer: 5’-AGAGGCCCCAGTCCAGTGTA-3’; reverse primer: 5’-CACTCCACGTGGCCACAA-3’ for Rabbit PRNP with exon 2. These primers were chosen so that they are located in different exons. The primers for β-actin were as follows: forward: 5’- CCGCATGCAGAAGGAGATCA-3’; reverse: 5’-AGAGCGAGGCCAGGATGGA-3’. For each sample, RNA concentration normalization was achieved using RT-qPCR on β-actin, as previously described [(CtPRNP-Ctβ-actin).To estimate the concentration of ovine and rabbit escribed . Two micescribed . Quantifescribed . It is tfast strain. As controls, six tgOv animals were injected similarly with 50 μL of a 10% brain homogenate from a healthy tg338 mouse.Eighteen tgOv rabbits  and 6 wild type (WT) rabbits  aged 103 to 132 days  were selected for the experiment. Among them, twelve individually identified tgOv animals and six WT rabbits were injected with 50 μL of a 1% (wt/vol in 5% glucose) brain homogenate from tg338 mice infected with the LA21K fast strain has been obtained through serial transmission and biological cloning by limiting dilutions of the Langlade field scrapie isolate /g of tg338 brain [The inocula were prepared in a class II microbiological cabinet using disposable equipment, with strict safety rules, and immediately inoculated to the animals by the intracerebral route, at the level of the right parietal cortex (depth: 1 cm). Inoculations were performed under general anesthesia by injecting a mixture of xylazine  and ketamine  by the intramuscular route. All inoculations were carried out in compliance with ethics and animal welfare according to regulation requirements. The LA21K Toulouse ) to tg3338 brain .Rabbits were housed in individual cages, in a dedicated biosafety level-3 facility. They were monitored using a video system and monitored daily for clinical signs and food consumption, by different investigators. Any death arising during the experiment was recorded and animals were necropsied. At clinical stage, rabbits were sacrificed by carbon dioxide suffocation and autopsied for brain and spleen collection. One half of each brain and spleen was stored at –20°C for immunoblotting and biochemical analyses, while the other half of organs was fixed in neutral-buffered 4% formalin for 1 week before paraffin-embedding for immunohistochemistry and histology.C or PrPSc by Western blotting as previously described [Brains and spleens were analyzed for the presence of either PrPescribed . Brieflyescribed , the secescribed ), which C in brains and spleens of rabbits and sheep were compared. Immunoblots revealed with the mAb Sha31 were quantified using the Alpha-Ease software . Brain samples were diluted and PrP amounts were calculated according to a dilution curve of full-length recombinant ovine PrP , used as a reference.Expression levels of PrPRabbit brain sections (5 μm thick) were treated as described previously . BrieflyPET blot were performed using a method previously described . ImmunodParaffin-embedded sections were mounted on glass microscope slides and stained with hematoxylin and eosin.Sc reactive bands were cut, rinsed and then reduced with dithiothreitol and alkylated with iodoacetamide. Samples were incubated overnight at 37°C with 12.5ng/μl trypsin  in 25 mM NH4HCO3 [The equivalent of 6 mL of 20% brain homogenate from tgOv and WT rabbit, treated or not with proteinase K and denatured (see above) were resuspended in 100 mM NaCl, 10 mM EDTA, 10 mM TrisHCl pH 7.8, 0.5% DOC, 0.5% Igepal (Sigma). Monoclonal antibody Sha31 coupled to magnetic beads  was added at the rate of 100 μL beads/240 mg tissue equivalent and reacted for 2 hours at 37°C. Beads were collected with a magnet, washed twice with PBS buffer and denatured in Laemmli buffer (10 min at 99°C). Samples were loaded on a 12% acrylamide gel, before electrophoresis and either immunoblotted  or silver stained . The silver stained gels were compared to the western blot and protein bands that were at the same molecular masses than the PrPhttp://www.matrixscience.com). The mass tolerance was 0.2 Da for both precursor and fragment ions.For CapLC system coupled to Q-TOF Ultima Global , the digested peptides were loaded on a precolumn  and desalted. Peptide separations were conducted on a C18 column . Peptides were eluted with a 5–60% linear gradient with water/acetonitrile 98/2 (v/v) containing 0.1% formic acid in buffer A and water/acetonitrile 20/80 (v/v) containing 0.1% formic acid inbuffer B. Mass data were acquired using one MS survey followed by MS/MS scans on the 3 most intense ions detected. Data were processed using ProteinLynx Global server 2.2. The peptide and fragment masses were matched in database (nrNCBI) using MASCOT software . Peptide separations were conducted on a Zorbax 300-SB C18 column, 75 μm i.d x 150 mm . Buffer A consisted of water with 0.1% formic acid while buffer B was 84% acetonitrile with 0.1% formic acid. Separation was performed by applying gradient of 15–55% B for 60 minutes at a flow rate of 400 nL/min. Mass data were acquired using one MS survey (m/z 500–2000) followed by MS/MS scans on the 3 most intense ions detected using Collision Induced Dissociation fragmentation mode. Identification was then performed with Bioworks 3.2  software. MS/MS spectra were searched against the non-redundant Uniprot database (2006_12) and analysed using TurboSEQUEST . Search parameters included differential amino acid mass shifts for oxidized methionine (+16 Da) and carbamidomethylation on cystein (+57 Da). The output data were evaluated in term of Xcorr magnitude up to 1.7, 2.2 and 3.5 for charge states 1+, 2+ and 3+, respectively.For Ettan MDLC system  coupled to LTQ Linear Ion Trap Mass Spectrometer , each sample was desalted using Zorbax 300-SB Cfast prions, and from uninfected tgOv rabbits. After 2 days, the medium was removed; the cells were rinsed in phosphate-buffered saline (PBS) and split into 25 cm2 flasks. Each week, one flask was used for subpassaging, whereas another was used to prepare a cell lysate for PrP content analysis (see above). The total protein content was estimated by using a protein assay kit (bicinchoninic acid assay (BCA); Pierce).Confluent Rov cells  werThe Swiss-Prot accession numbers for the proteins mentioned in the text are sheep (P23907) and rabbit PrP (Q95211).PRNP transcription unit. This insert has already been used to produce various mouse transgenic lines that express the PrPVRQ allele [PRNP transgene (period of observation > 700 days).One transgenic rabbit founder animal was obtained following microinjection of an ovine BAC DNA insert encompassing the entire Q allele . For aniPRNP RNA relative to that of rabbit PRNP RNA was estimated by RT-qPCR analyses on brain tissue extracts of tgOv rabbits and WT rabbits. In WT and tgOv rabbits, two rabbit PRNP RNA variants were found, as expected, resulting from the splicing (or not) of exon 2. The values were cumulated to obtain the total concentration of rabbit PRNP transcripts. In tgOv rabbits, ovine PRNP RNA levels were 1.5–2 fold higher than those of rabbit PRNP RNA . Rabbit PRNP RNA levels were similar between WT and TgOv rabbits, suggesting that expression of ovine PRNP has no interfering effects on the transcription of rabbit PRNP.The concentration of ovine C in tgOv rabbits. Immunoblots analyses indicate that the total levels of PrPC in brain of tgOv rabbits were approx. 1.5–2 fold higher than those found in the brain of their WT counterparts or sheep carrying the VRQ allele . The behavioral and clinical signs were invariant from animal to animal. The first behavioral signs were a drop in food consumption and restlessness. Early neurological signs  presented any clinical signs during the time course of the experiment  was readily detected in all infected tgOv rabbits Sc. Proteres distribution in the brain was examined by PET blot analyses and immunohistochemistry. The greatest levels of PrPres deposits were observed in the thalamus, hippocampus and frontal cortex of scrapie-sick TgOv rabbits  indicated that spongiosis was prominent in the thalamus . Mild sp rabbits .Collectively, these data indicate that scrapie infected tgOv rabbits exhibited the major clinical, biochemical and neuropathological hallmarks of TSEs.C had been converted in scrapie-sick tgOv rabbits, immunoprecipitated brain extracts of healthy and infected tgOv rabbits,—treated or not with PK-, were analyzed by two techniques of mass spectrometry. Both analyses allowed detecting PrP fragments in the different gel bands corresponding to the western blot immunoreactive bands. In PK-treated, LA21K fast infected tgOv rabbit brains, five fragments were identified: ESQAYYQR; GENFTETDIK; VVEQMCITQYQR; GENFTETDIKIMER; EHTVTTTTKGENFTETDIK. Only one fragment VVEQMCITQYQR was identified in mock-infected tgOv rabbit, while no fragments were obtained from WT rabbits. All these fragments were located within the C-terminal part of the PrP protein and two could be assigned without ambiguities to the sheep PrP sequence -, necessitated a relatively high number of PMCA rounds as compared to other prion sources [Sc in the brain of diseased tgOv rabbits, secondary passage to transgenic mice expressing rabbit PrPC are planned Click here for additional data file.S2 VideoThe animals were euthanized at that stage.(MOV)Click here for additional data file.S3 Video(MOV)Click here for additional data file.


Ejection and suction of fluid between the t-tubules and the extracellular compartment during isometric contraction were observed. We also examined the influence of t-tubule morphology and mitochondrial location on the electrophysiology and mechanics of the cardiomyocyte. Our results confirm that the t-tubule structure is important for synchrony of Ca2+ release, and suggest that mitochondria in the sub-sarcolemmal region might serve to cancel Ca2+ inflow through surface sarcolemma, thereby maintaining the intracellular Ca2+ environment in equilibrium.In numerical simulations of cardiac excitation-contraction coupling, the intracellular potential distribution and mobility of cytosol and ions have been mostly ignored. Although the intracellular potential gradient is small, during depolarization it can be a significant driving force for ion movement, and is comparable to diffusion in terms of net flux. Furthermore, fluid in the t-tubules is thought to advect ions to facilitate their exchange with the extracellular space. We extend our previous finite element model that was based on triphasic theory to examine the significance of these factors in cardiac physiology. Triphasic theory allows us to study the behavior of solids (proteins), fluids (cytosol) and ions governed by mechanics and electrochemistry in detailed subcellular structures, including myofibrils, mitochondria, the sarcoplasmic reticulum, membranes, and t-tubules. Our simulation results predicted an electrical potential gradient inside the t-tubules at the onset of depolarization, which corresponded to the Na The three constituents are assumed to occupy simultaneously the same region. Mechanical and electrical balance equations and conservation equations are defined for each phase. We adopt the formulation of Okada et al. ; at each cytoskelThe balance equation for the mixture is,The balance equation for the fluid phase isAbove, after the pressure gradient (first term), we have the osmotic pressure (second term), the frictional force in the solid phase (third term), and the effect of the electrical potential gradient (fourth term). We now define an incompressibility condition for the mixture, such that a change in mixture volume equals outflow of fluid,The balance equation for the ionic phase resolves electrical and frictional forces on ions with ion conservation,The change in ion concentration (first term) is equilibrated with the ion flux due to convection (second term), diffusion (third term), electrical forces (fourth term), and subcellular reactions (fifth term).Finally, the electroneutrality condition isΠ is first Piola-Kirchhoff stress of mixtures, J is the volume change (determinant of the deformation gradient tensor), χ is the reference configuration, κS is the volume modulus of the solid phase, Ctotal is the concentration of all the ions, CF is the concentration of the charges captured by subcellular components and buffers, z is the valence of the ions, Φw is the fluid volume fraction, Dα is the diffusion coefficient of the solute α, K is the coefficient of permeability, φ is the osmotic coefficient, R is the gas constant, and T is the absolute temperature.We use the subcellular reaction functions from our previous 3D reaction–diffusion–mechanical model of the cardiomyocyte , where pi is pressure at node i and G is the conductance. Fluid conservation at node i is given byThe t-tubule structure is modeled using simplified triphasic truss elements to reduce computational cost. The mechanical formulation uses the general truss element, and the balance and conditional equations for the ion phase are the same as those for the solid element. Fluid is driven by the pressure difference across the edges of the truss element. Therefore, fluid velocity is no longer an independent variable. The fluid velocity in the truss element connecting nodes At the open end of the t-tubule, fluid exchange between the truss element and solid element (extracellular space) followsi is written as follows,On other nodes, the t-tubule facing the intracellular solid dilates according to the pressure difference. Volume conservation at node We used the three-myofibril model to evaluate the significance of triphasic theory by comparing with our reaction–diffusion simulation findings. Using the same model parameters, the equivalent mesh, boundary conditions for isometric contraction and the initial conditions for the steady state in the 1 Hz reaction–diffusion simulation, a 1 s excitation–contraction cycle was simulated and compared. In the reaction–diffusion model, the extracellular space was not meshed as it was assigned constant bulk extracellular ion concentrations. In the triphasic model, ion concentrations on the nodes at the boundary of the extracellular space were fixed at the same constant values. We used their steady-state values from the reaction–diffusion model operating at 1 Hz as the initial conditions for the triphasic model to save the computational time.To determine the diffusion coefficient in the t-tubule, the simulation predictions of ion diffusion in t-tubules and the resultant changes in membrane currents were compared with the experimental data of Swift et al.  and Shep+]o (from 5.4 to 8.1 mM) under voltage-clamped (−80 mV) control. Shepherd and McDonough (Ca) caused by rapid changes in the extracellular calcium concentration ([Ca2+]o) (from 0.45 to 1.8 mM) in voltage-clamped (−45 mV) guinea pig ventricular cardiomyocyte at various intervals prior to the activation of ICa.Swift et al.  measuredcDonough  recorded+ concentration ([K+]o) from 5.4 to 8.1 mM under voltage clamped (−80 mV) conditions while applying various diffusion coefficients to the interior of the t-tubule. We then selected the diffusion coefficient value that gave the best fit to the experimentally observed time course of membrane current. Using this value, we simulated the experiment by Shepherd and McDonough . To simulate the voltage clamp protocol, we fixed the electrical potential of the nodes at the boundary of the extracellular space to zero. The electrical potential of the nodes on the M-line in the cell core were fixed at the intended voltage . To simulate the experiments of Swift et al. , which assures their individual convergence  Xeon CPU (2.6 GHz). The calculation took 150 h for 1 s simulation of the quarter model.2+ concentrations and membrane currents of the triphasic simulation (solid line) and the reaction–diffusion–contraction simulation (gray broken line). We can see that general electrophysiological phenomena are reproduced by the triphasic model. The differences in the concentrations of the ions and metabolites between the two simulations were less than 1%. The contributions of the electrical and convectional terms on the change of intracellular ion concentrations were 2 and 0.02%, respectively, in the maximum compared with the diffusional term.A comparison of the triphasic model with the prior reaction–diffusion model was previously reported in Japanese  from 5.4 to 8.1 mM under voltage clamped conditions. The legend values are rates of applied diffusion constant inside the t-tubules relative to those of cytosol. The diffusional restriction delayed changes in the intra-t-tubule [K+], which also caused gradual changes in the membrane currents. In Figure +] diffusion constant of 72 μm2/s, corresponding to 0.08 times the cytosolic value and 0.04 times the aqueous phase  using a one-dimensional simulation.We simulated Swift et al.'s  experimeIn the experiment, the membrane current of the de-tubulated myocyte changed gradually. This is in marked contrast to our simulation predictions of almost instantaneous variation Figures . The reaCa current during step changes in voltage from −45 to 0 mV for several step intervals  after changing extracellular [Ca2+]o from 0.45 to 1.8 mM. As the timing was prolonged, the maximum inward current progressively increased, thus showing good agreement with the experimental results.We used the parameter values from our simulations of the Swift experiments in the subsequent simulations of the Shepherd experiments. Figures 2+]i) after the onset of excitation  current, which triggers the Ca2+ release from nearby JSR; diffusion of Ca2+ provokes Ca2+ release from the remote JSRs. The relationship between delays in the timing of Ca2+ release in these JSRs and the distance from the nearest t-tubule or sarcolemma is shown in Figure 2+ within 20 ms. This delay was strongly correlated with the distance in most cases. The JSRs exhibiting a larger delay are located in the bottom-right region  exhibited larger amplitudes than the others. The difference in peak timing of force development among the myofibrils was less conspicuous compared with that of Ca2+ transient . Nonetheless, the third sub-membrane myofibril produced an approximately 15% larger force amplitude than the others  between the deep and surface regions during the first 0.8 ms of action potential. However, the magnitudes of these currents were small at this time. The more marked differences in INaCa and ICa observed after 0.7 ms were not homogeneous along the t-tubule due to the difference in intracellular [Ca2+] caused by the distance from the Ca2+ release unit.Ion currents at locus 1 at depths of the t-tubule (solid line) and at locus 5 near the surface (broken line) are shown in Figures 2+ distribution  as several studies estimated similar values for different ions (Yao et al., +] and [Ca2+] in the extracellular solution (Shepherd and McDonough, In our previous study, we validated our reaction–diffusion–contraction finite element model by reproducing the variation in timing of the averaged membrane potential, the concentrations of the ions and metabolites, the local Ca2+ wave-induced depolarization (Fujiwara et al., The triphasic extension of the model successfully reproduced the field stimulation-induced depolarization reported by Sharma et al. . Our fin2+ from the JSRs increases in proportion to the distance from the sarcolemmal or t-tubule membrane. This confirms previous experimental and modeling finding that the well-developed and dense t-tubule structure is important for synchrony of Ca2+ release (Louch et al., In the present study, we improved our modeling capability by extending the segment to be half a sarcomere in length and spanning quarter of the cross section to contain 12 myofibrils. The larger model enabled us to include randomness in the t-tubule morphology and in the mitochondrial location. Our results demonstrated that a delay in the release of Ca2+ the fastest. Cytosolic [Ca2+] surrounding the mitochondria is maintained at a higher level at rest and at a lower level during excitation compared with [Ca2+] at loci without mitochondria, as mitochondrial Ca2+ exchange occurs through uniporters and Na+–Ca2+ exchangers. This higher [Ca2+] under resting conditions facilitate the Ca2+ release from JSR adjacent to mitochondria.We also found that among JSRs distant from the membrane, those located adjacent to mitochondria released Ca2+ dynamics in the sub-sarcolemmal region. The myofibrils next to sarcolemma but without SSM were exposed to higher peak Ca2+ than the other myofibrils. At the myofibrils next to the sarcolemma, Ca2+ flows in from the sarcolemma diffusing radially, as well as from the Z-line diffusing longitudinally. Without this diffusional restriction from the presence of SSM between the myofibril and sarcolemma, the Ca2+ level in the sub-sarcolemmal region increases compared with the interior region. The myofibrils next to the sarcolemma, but without SSM, develop approximately 15% greater force than other myofibrils. Inhomogeneous force development between sub-sarcolemmal and the interior region is not favorable for mechanical efficiency, and may even cause shearing within a cell. It is conceivable that SSM might serve to cancel the Ca2+ inflow through surface sarcolemma and average the intracellular [Ca2+] environment.Mitochondria play another role in CaMcNary et al. , 2012 deIn the present study, we have semi-quantitatively evaluated the intra-t-tubular diffusion coefficient and the relationship between t-tubular morphology, mitochondrial location, and the distribution of force development. Although the advancement of experimental technologies now enables us to visualize ions and molecules in living cells, its spatial resolution is still limited. Electron microscopy with proper molecular markers can provide high-resolution images, but they are snapshots of inactive cells. Detailed three-dimensional simulation enables studies beyond the limit of current experimental technologies.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.


Na+/Ca2+ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na+/Ca2+ exchangers are divided into three groups based upon stoichiometry: Na+/Ca2+ exchangers (NCX), Na+/Ca2+/K+ exchangers (NCKX), and Ca2+/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na+/Ca2+ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na+/Ca2+ exchangers across the phylum Nematoda. In this analysis we identify Na+/Ca2+ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na+/Ca2+ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.Na Na+/Ca2+ exchangers are divided into three groups in animals based upon their stoichiometry: Na+/Ca2+ exchangers (NCX) which exchange sodium for calcium, Na+/Ca2+/K+ exchangers (NCKX) which exchange sodium for potassium and calcium, and Ca2+/Cation exchangers  which exchange sodium or lithium for calcium +/Ca2+ exchangers have been shown to regulate calcium exchange at the cell membrane, endoplasmic reticulum, mitochondrion, and at the nucleus +/Ca2+ exchangers contain a tandemly repeated protein motif, the alpha repeat, which invariably occurs in two blocks of five transmembrane domains separated by a cytoplasmic loop; with some variations, the residues of the alpha repeat are conserved among all three classes of Na+/Ca2+ exchanger and in all organisms; and where perturbed experimentally, these residues have been shown to be crucial for exchanger function +/Ca2+ exchangers contribute to the normal physiology of a wide variety of cells and tissues. Na+/Ca2 based exchange is considered the principal method of Ca2+ removal during heartbeat NaCaenorhabditis elegans, a total of ten Na+/Ca2+ exchanger genes have been identified in the C. elegans genome (designated ncx-1 to ncx-10) ncx-1, ncx-2 and ncx-3); ncx-4 and ncx-5 encode for proteins that belong to the NCKX branch; and ncx-6 – ncx-10 encode for CCX representatives in C. elegans. Here we set out to characterize structural and taxonomic specializations within the family of Na+/Ca2+ exchangers across the phylum Nematoda. We sourced members of the Na+/Ca2+ exchanger family in the following twelve nematode species  were searched for NCX, NCKX and CCX protein sequences with bidirectional BlastX and BlastP searches using WormBase ver.WS243 C. elegans and C. briggsae were calculated using DnaSP ver.5 http://ohalloranlab.net/NemCX.htmlProteins were aligned using the multiple sequence alignment software MUSCLE v3.8.31 Caenorhabditis japonica strain DF5081 was maintained at 20°C by mating males and females on NGM plates seeded with E. coli strain OP50 Heterorhabditis bacteriophora strain TTO1 animals (kindly provided by John Hawdon) were cultured in vivo at 25°C in Galleria mellonella (wax moth) larvae using standard protocols H. bacteriophora were harvested by obtaining G. mellonella cadavers 6 to 8 days post-infection and cutting them open in a Petri dish containing M9 buffer, and the emerging nematodes were washed twice with distilled water. Female adult nematodes were obtained by dissecting G. mellonella cadavers 5 to 7 days post-infection in a Petri dish containing M9 buffer and females were picked using an aspirator.2, 0.45% IGEPAL, 0.45% Tween-20, 0.01% gelatin) was added to the tube and then frozen at −80°C for 10 mins followed by incubation at 60°C for 1 hr followed by 95°C for 15 mins. In the case of H. bacteriophora, animals were crushed into a fine powder using a pestle and mortar. Tubes were then centrifuged at 13,000 rpm for 1 min and ∼50 µl gDNA supernatant isolated for PCR. For H. bacteriophora, an extra step of phenol-chloroform purification was performed by adding a 1∶1 volume to gDNA supernatant. Total RNA was isolated from mixed stage animals using 1 ml Trizol Reagent . A 20 G syringe (Becton-Dickinson 3 ml syringe) was used to break down material and 200 µl of chloroform was added to isolate RNA from the sample. Tubes were vigorously shaken followed by centrifuging at 13,000 rpm for 10 min. The clear supernatant mixed with 1 volume of 70% EtOH was then cleaned using an RNA mini kit . The concentration and purity of the RNA samples were determined using spectrophotometry and ethidium bromide visualization of intact 18S and 28S RNA bands after agarose gel electrophoresis. Total RNA was treated with DNase  by incubating at 37°C for 30 min followed by 65°C for 10 min, and reverse transcribed (RT) with MMLV reverse transcriptase  in 5× RT polymerase chain reaction (PCR) buffer , 4 U RNase inhibitor, 10 mM each of the dNTPs. The final PCR products were electrophoresed on 1.5% agarose gels. Primer pairs used for Caenorhabditis japonica were CJ-F TACGTGAGCCATGGACATCACA and CJ-R TCGATACGTTGGATTGAGAATC, and primer pairs used for Heterorhabditis bacteriophora were: Hba-F TGCTCTACTCCTTGCTTCGTGCCCCGTA, Hba-R GGGAGTTACATTCATGGCATTTGGCAATGG using the following cycling conditions for C. japonica: 95°C for 3 mins, 95°C for 30 sec, 56°C for 30 sec, and 72°C extension for 3 mins 30 sec for gDNA and 1 min for cDNA; and the following cycling conditions for H. bacteriophora: 95°C for 3 mins, 95°C for 30 sec, 56°C for 30 sec, and 72°C extension for 1 min 40 sec for gDNA and 1 min for cDNA.Genomic DNA was isolated by harvesting animals and collecting in a 1.5 ml tube. 200 µl of Lysis buffer  and mammals  were used to search various databases and resources: WormBase ver. WS243 Strongyloides ratti, Haemonchus contortus, Heterorhabditis bacteriophora, Caenorhabditis elegans, Caenorhabditis brenneri, Caenorhabditis japonica, Caenorhabditis briggsae, Caenorhabditis remanei, Pristionchus pacificus, Brugia malayi, Loa loa, and Ascaris suum. All hits were filtered through InterPro Na+/Ca2+ exchangers were mostly conserved across all species examined . We also conducted a sliding window analysis of nucleotide diversity between C. elegans and C. briggsae NCX genes using DnaSP ver. 5 ncx-1 and ncx-3 sequences , and slightly elevated levels of diversity for ncx-2 (average π score  = 0.369) (ncx-2 does not hold for other Caenorhabditis pairs: for example using C. elegans and C. japonica the average ncx-2 π score  = 0.173 (sampling variance  = 0.007), and using C. elegans and C. remanei the average ncx-2 π score  = 0.11 (sampling variance  = 0.003). Next, we tested specific sites for positive selection using REL Caenorhabditis taxa, and from this analysis we did not detect any sites undergoing positive selection. We also tested for episodic diversifying selection using MEME and detected two significant (p<0.01) sites: codon 455, which is positioned close to the second calcium binding domain (CBD2) between TM5 and TM6, which detects local intracellular calcium levels, and codon 925 which is located after TM9 in the intracellular loop that connects with TM10 (see Methanococcus jannaschii (NCX_Mj) has been resolved C. elegans NCX-1) and a representative from the NCX-3 cluster (A. suum GS_14034) for in silico structural analysis. For NCX-1 from C. elegans 32% of the residues  were modelled at 100% confidence and yielded 33% alpha helical and 20% beta strand structures. The A. suum GS_14034 NCX was modelled at 100% confidence for 36% of the sequence (289 residues) including 36% alpha helical and 22% beta strand structures. In each case the single highest scoring modelling template was the resolved NCX_Mj structure (PDB: 3V5U) from Liao et al. A. suum GS_14034  . HoweverTM10 see . The cryGS_14034 . In the  742–748 .Caenorhabditis species grouped together although we did not detect an NCX-5 member for C. japonica , and a global dN/dS value of 0.115 using MEME (p<0.01). A sliding window of nucleotide diversity was generated for each NCKX gene pair between C. elegans and C. briggsae and revealed similar patterns of DNA polymorphisms across each exchanger  (p<0.01).Members of the NCKX family exhibited much diversity at the protein level and broadly assembled into representatives of NCX-4 and NCX-5 clusters. In each cluster, the japonica . In all  = 0.22) . Next, wC. japonica two tandem predicted genes, Cja11479 and Cja38547, were annotated as separate protein-coding genes, and at the translated protein level we found that each gene was predicted to encode only one half of a single CCX protein .In analyzing the CCX group, we noted that for  protein . We obse protein . Interesis locus . We obseCaenorhabditis genus , and a global dN/dS value  = 0.1550 using MEME (p<0.01). We examined nucleotide diversity for each CCX gene and observed unique patterns of diversity  example at codon 754 of C. briggsae NCX-7 in close proximity to the second transport repeat domain.The CCX nematode phylogeny revealed the most unexpected reconstruction, most notably is the gene expansion specific to the is genus . Within de level , at the de level , which civersity  but simiiversity  for eacht domain . We idenCaenorhabditis genus we observed significant lineage specific expansions within the NCLX exchanger group, suggesting the possibility of relatively recent gene duplication events. Within the five Caenorhabditis species we examined, the NCLX-type genes ncx-6 and ncx-7 are positioned in tandem sequence within their respective physical maps; the ncx-8 and ncx-9 NCLX-type genes are also in tandem sequence in the C. elegans genome, and ncx-10 is closely linked on the same arm of chromosome V in C. elegans; in C. briggsae ncx-8, ncx-9, and ncx-10 genes are all located within 15 kb of each other on chromosome V; in C. brenneri and C. remanei, ncx-9 and ncx-10 are within 10 kb and 3 kb of each other respectively. This linkage organization for subsets of NCLX-type exchangers may lend support to the hypothesis that some of these genes have arose relatively recently within Caenorhabditis species. While these apparent serial and parallel gene duplication events may be relatively recent in terms of nematode evolution, data from our group suggests that at least in the case of ncx-6 and ncx-9, these genes are contributing to important neuronal functions at the behavioral and developmental levels , suggesting that these duplicated genes have acquired neo-functionalized roles in the animal. It will be interesting moving forward to characterize mutants in exchangers of the other NCLX-type genes in an effort to understand how sequence specificity may lend itself to functional specializations within this Na+/Ca2+ exchanger subtype. Furthermore, searching more nematode genomes as they become more annotated will add more resolution to the timing of gene accretion within the NCLX subtype by also testing the alternative possibility that these NCLX-type duplicates may have been lost in other nematode lineages. It was also surprising that we did not detect NCLX-type orthologs within the genomes of the Clade III nematodes  that we examined. Gene loss is one possibility to explain this observation, however, it is unexpected considering the central role NCLX proteins have been shown to play in mammalian systems +/Ca2+ exchanger duplicates is in the case of NCX4 Within the +/Ca2+ exchangers are central regulators of calcium homeostasis in a wide variety of cell types. Not surprisingly, defects in Na+/Ca2+ exchange have been implicated in numerous diseases and pathologies including epilepsy, multiple sclerosis, Parkinson's disease, Alzheimer's disease, as well as brain ischemia +/Ca2+ exchanger genes from a broad spectrum of nematodes, and using this sequence data to reconstruct molecular phylogenetic relationships and tease apart the selective pressures shaping this family of proteins. From our analyses we uncover a pervasive theme of constraint across the Na+/Ca2+ exchanger family and reveal a significant level of heterogeneity within subtypes of this family. Specifically, in the case of the NCLX subtype of Na+/Ca2+ exchangers we observed lineage specific expansions as well as possible gene loss. Together, these findings reveal a complex picture of Na+/Ca2+ transporters in nematodes that suggests an incongruent evolutionary history of an important family of proteins that provide central control of calcium dynamics.Na


The cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC.in vivo.Cancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers’ analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results high/+/CD24low/–, stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A  levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment.PEITC attenuated proliferation of CD44The anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients. Despite considerable improvement in cancer diagnosis and therapy, relapse and metastasis are still common . HoweverPhenethyl isothiocyanate (PEITC) is a dietary compound derived from common vegetables such as watercress, broccoli, cabbage, and cauliflower . We and Apoptosis, or programmed cell-death, is essential to maintaining tissue homeostasis, and its impairment is implicated in many human diseases, including cancers . The tumin vivo anti-tumorigenicity effects of PEITC were evaluated in a xenograft mouse model.In the present study, we investigated the efficacy of PEITC in attenuating the growth of sphere-forming cervical CSCs isolated from HeLa cells (hCSCs) as well as its ability to up regulate death receptors for TRAIL-mediated induction of apoptosis. Furthermore, the Phenethyl isothiocyanate , 99%, was diluted in dimethyl sulfoxide  to make 0.5–20-mM stock concentrations and was further diluted in media to obtain 2.5–20-μM final concentrations, which are achievable following oral administration in human  and have2-humidified atmosphere at 37°C. HeLa cells were trypsinized with TrypLE  and then sub-cultured with a 1:5 splitting ratio when the cells reached about 90% confluency. From the parental HeLa cells (termed simply as HeLa in the rest of the document), hCSCs were cultured following a modified protocol described by Gu et al. . The manufacturer’s instructions were followed, and treatments were compared with vehicle control (DMSO-treated cells) at 490 nm in a BioTek Synergy H4 multimode plate reader .A standard colorimetric method (MTS assay) was used to determine the number of viable cells in samples. For cell-proliferation assays, HeLa and hCSCs were cultured for 4 days, and an additional 9 ml of sphere culture medium was added for another 5 days, as described in the sphere culture section. Viable cells were harvested and counted with a hemocytometer before seeding into 96-well microplates at a density of 2 × 106) were seeded in each well of a 6-well plate and incubated overnight at 37°C in a 5% CO2 incubator. Old culture medium was replenished by culture medium containing either 10-μM or 15-μM concentrations of PEITC for 5 hours. The cells were then treated with 10 ng/ml human recombinant TRAIL or with 10 ng/ml TNFα  for additional 1-hour incubation. Cell harvesting and immunoblotting were carried out as we previously reported [hCSCs (1 × 10reported . BrieflyPrkdcscid/J) mice , 17 weeks old, were randomly grouped into five groups (four mice per group) in specific pathogen-free (SPF) housing at a constant temperature of 24–26°C with a 12-h:12-h light/dark cycle. All mice were allowed to acclimatize for 1 week and were provided with sterile food and water ad libitum. HeLa and hCSCs were cultured, trypsinized, washed, pre-treated with 10 μM PEITC where indicated, and resuspended in PBS at the concentration of 1 × 107 cells/ml before injecting into the mice. Each mouse was subcutaneously injected at the neck scruff with one injection of PBS , HeLa (1 × 106), HeLa pretreated with 10 μM PEITC (1 × 106), hCSCs (1 × 106), or hCSCs pretreated with 10 μM PEITC (1 × 106). The cell number in each injection was consistent with the study previously carried out by Gu et al. [2 asphyxiation 21 days post-treatment, and blood was collected through cardiac puncture immediately after sacrifice. Excised tumor and lung samples were kept in 10% formalin for subsequent histopathological examination. The average tumor number or mass per injection was calculated by dividing each group’s total number of tumors or total mass by the number of mice in that group.Animal studies were carried out in accordance with the guidelines of, and, using an approved protocol by, the Institutional Animal Care and Use Committee (IACUC), South Dakota State University . Twenty female non-obese diabetic, severe combined immunodeficient   for examSince hCSCs are of human origin, ELISA was carried out to assess the presence of human vascular endothelial growth factor A (hVEGF-A), which promotes tumor angiogenesis in a host. The collected mouse blood samples were kept in a slanted position at room temperature for 1 hour, followed by 4°C for 24 hours, and then centrifuged at 5000 rpm for 5 minutes. The Platinum ELISA kit  was used to quantify the hVEGF-A present in each serum sample (pg/ml) from a single mouse, according to the manufacturer’s instructions.t test was applied to compare two groups receiving similar treatments. Data were expressed as means ± SEM. Experiments were repeated at least three times. The significance of differences between means is represented by asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.Statistical analyses were carried out using Sigma Plot software . Statistical significance between the groups was assessed by multiple mean comparisons using one-way analysis of variance (ANOVA) followed by a post-hoc Dunnett’s test. Student’s In this report we used the HeLa cervical cancer cell line to isolate and characterize hCSCs following a previously described sphere culture method , which fPEITC attenuated the formation of primary hCSC spheres in a concentration-dependent manner Figure A. AdditiTo investigate a potential pro-apoptotic effect of PEITC in triggering hCSC growth inhibition, we carried out western blot experiments on hCSCs treated with different doses of PEITC in the presence or absence of TRAIL and TNFα. We observed an increased expression of cPARP with higher doses of PEITC (15 μM) following exposure for 5 hours, which was further augmented by the presence of 10 ng/ml TRAIL, which indicated elevated levels of endogenous caspase-mediated apoptosis in hCSCs Figure A. After To further understand the characteristics of PEITC in the extrinsic apoptosis pathway in hCSCs, we carried out flow cytometry analyses of DR4 and DR5 death receptors. Since both PEITC- and DMSO-treated hCSCs were treated with TRAIL , we expected to see greater induction of DR4 and DR5 in PEITC + TRAIL-treated cells compared to TRAIL treatment alone. We observed that PEITC induced overexpression of DR4 in comparison with the DMSO control . An equal number of cells (1×106) containing either HeLa cells or hCSCs (each with or without 10 μM PEITC pre-treatment) developed different tumor loads in each group of NOD/SCID mice. The average tumor number per injection was observed to be much higher in the hCSC group (1.75) than in the HeLa group (0.5), while PEITC pre-treatment helped lower tumor formation in both hCSC (1.75 vs. 0.5) and HeLa (0.5 vs. 0.33) groups of mice than in controls , followed by hCSCs treated with PEITC  and untreated HeLa cells (1.08 pg/ml), while we did not detect any hVEGF-A in HeLa cells treated with PEITC Figure C. To seein vitro hCSC proliferation and in vivo tumorigenicity as well as stimulating intracellular receptors that mediate TRAIL-induced apoptosis.Cervical cancer is the second-most-frequent female malignancy worldwide . ConcurrAccording to the CSC concept of carcinogenesis, CSCs represent novel and translationally relevant targets for cancer therapy, and the identification, development, and therapeutic use of compounds that selectively target CSCs are major challenges for future cancer treatment . It is pPEITC treatment in hCSCs reduced proliferation and sphere formation and expressed higher levels of cPARP, indicating elevated levels of apoptosis, which is possibly through caspase activation by isothiocyanate in treated cancer cells as reported previously . At simiin vivo, we carried out xenotransplantation in immune-compromised mice. Mice receiving untreated hCSCs produced the highest numbers of tumors and also showed greater invasiveness, as confirmed by the presence of lung metastases. However, given the short 3-week duration of the experiment, metastasis was found in only one of the four animals in the hCSC group but in no other animal in the remaining groups. We observed a marked reduction in tumorigenicity in mice that had received a PEITC-treated hCSC inoculum, and the outcome was comparable to the HeLa-injection group. It should be noted here that the sphere culture approach to isolation of hCSCs that we used in the study followed by cell-surface marker-based characterization helps to identify CSC-enriched subpopulations but did not enable unambiguous isolation of all of the CSCs.We observed that PEITC likely sensitized TRAIL but not the TNFα pathway while inducing apoptosis. Although TNF-α can trigger apoptosis in some solid tumors, its clinical usage has been limited by the risk of lethal systemic inflammation . By compin vitro, and PEITC-treated hCSC xenotransplants were less tumorigenic in a relevant mouse model. PEITC, in combination with TRAIL, upregulated the death receptor-induced extrinsic pathway of apoptosis and resulted in the increase in cPARP proteins. It should be noted that in the current study we did not evaluate the individual effectiveness of TRAIL against hCSCs, but TRAIL is currently in clinical trials in the US (NCT00508625). Importantly, PEITC is anti-proliferative in both HeLa cancer cells and hCSCs, suggesting that it may contribute to eradication of cancer more efficiently than compounds targeting either CSCs or regular cancer cells alone. Collectively, our data strongly justify future clinical trials of PEITC, individually or in combination with recombinant TRAIL therapy, for improved treatment outcomes in cancer patients.We have provided the first evidence that PEITC is effective in abolishing human cervical CSCs


Olea europaea L.), is believed to have therapeutic potentials for various diseases, including tumors. We obtained an oleuropein-enriched fraction, consisting of 60% w/w oleuropein, from olive leaves, and assessed its effects on intracellular Ca2+ and cell viability in mesothelioma cells. Effects of the oleuropein-enriched fraction on Ca2+ dynamics and cell viability were studied in the REN mesothelioma cell line, using fura-2 microspectrofluorimetry and MTT assay, respectively. Fura-2-loaded cells, transiently exposed to the oleuropein-enriched fraction, showed dose-dependent transient elevations of cytosolic Ca2+ concentration ([Ca2+]i). Application of standard oleuropein and hydroxytyrosol, and of the inhibitor of low-voltage T-type Ca2+ channels NNC-55-0396, suggested that the effect is mainly due to oleuropein acting through its hydroxytyrosol moiety on T-type Ca2+ channels. The oleuropein-enriched fraction and standard oleuropein displayed a significant antiproliferative effect, as measured on REN cells by MTT cell viability assay, with IC50 of 22 μg/mL oleuropein. Data suggest that our oleuropein-enriched fraction from olive leaf extract could have pharmacological application in malignant mesothelioma anticancer therapy, possibly by targeting T-type Ca2+ channels and thereby dysregulating intracellular Ca2+ dynamics.Malignant mesothelioma is a poor prognosis cancer in urgent need of alternative therapies. Oleuropein, the major phenolic of olive tree ( Malignant mesothelioma is a poor prognosis cancer with worldwide increasing insurgence, arising from the pleura and other mesothelial tissue and showing close association with asbestos exposure. An effective therapeutic approach for the disease is currently lacking, while alternative treatments are urgently needed . Among aOlea europaea L.) is a main temperate fruit crop, mostly destined to oil production i rises followed by a prompt recovery of the baseline level upon washout  contained in the extract  i [2+]i spikes that also in this case were inhibited by Ca2+-free medium. Moreover, NNC-55-0396 also hindered the hydroxytyrosol-induced [Ca2+]i spikes, with 80% inhibition at 10 μM hydroxytyrosol and 65% inhibition at 100 μM hydroxytyrosol  as positive control, since we had previously shown that this compound induces Ca2+ rise in REN cells via T-type Ca2+ channels [2+]i spikes with an estimated EC50 of 69 μM  was slightly, but not significantly, stronger than that of standard oleuropein (IC50 = 25 μg/mL). Both substances were slightly more cytotoxic than EGCG (IC50 = 33 μg/mL)  .2+ are new. The activity of oleuropein was slightly lower than that of the oleuropein-enriched fraction, possibly due to the occurrence in the fraction of other secoiridoid esters known to be present in olive leaves, such as demethyloleuropein, ligstroside, and oleuroside i oscillations, suggesting that intracellular Ca2+ dysregulation determining transient [Ca2+]i rise may also interfere with tumor development, possibly leading to cell death [2+ channel activity, could be promising tools for the inhibition of cancerous cell growth.T-type Caepilepsy , while tepilepsy , 29. Henepilepsy , 31. Howechanism . Also, cll death . Accordi2+ channel, thereby inducing blood pressure lowering [2+ channels.In apparent contrast with the present data, it has been previously shown that an olive leaf extract exerts antagonistic effects on high-voltage-activated L-type Calowering , 35. A c50 for incubations of 48–72 h is 20 μg/mL or lower [50 of 22 (17–29) μg/mL in terms of oleuropein. We can then estimate IC50 of 36.7 (28.3–48.3) μg/mL for the oleuropein-enriched fraction as a whole. Hence, our oleuropein fraction is close to the cytotoxicity threshold established by NCI and could therefore be appropriate for the development of synergistic treatments allowing reducing the doses of highly toxic conventional drugs, as previously suggested for EGCG [More insight into the possibility of using the oleuropein fraction in antitumor treatment for mesothelioma is provided by our cytotoxicity tests, indicating that the antiproliferative activity of the oleuropein fraction essentially depends on its major constituent oleuropein. The United States National Cancer Institute plant screening program has established that a plant extract can be deemed to have a cytotoxic effect if ICor lower . Our ole50 of 22 7–29 μg/mfor EGCG .μg/mL (0.01 μM) for oleuropein, while its main metabolite hydroxytyrosol can reach values of about 0.15 μg/mL (1 μM) [2+ could be easily achieved upon clinical administrations of oleuropein, followed by its conversion into the more effective metabolite hydroxytyrosol.There is no established standard dose of oleuropein for humans, but in acute toxicity studies, oleuropein and hydroxytyrosol have shown no lethality or adverse effects in mice up to doses of 1000 and 2000 mg/kg, respectively. In addition, studies conducted on humans with olive extract or its polyphenolics have shown no undesired effects , 39. PhaL (1 μM) . These v2+ dynamics occurring via T-type Ca2+ channels. This kind of effect suggests a possible use of the oleuropein fraction from olive leaves in the treatment of mesothelioma.We have shown a method to obtain oleuropein-enriched olive leaf extract fractions that could be of industrial interest, because extraction is generally competitive with chemical synthesis for complex molecules like oleuropein. In addition, we have highlighted a novel mechanism of action for oleuropein and its metabolite hydroxytyrosol, consisting in a dysregulation of Ca


The magnitude of [Ca2+] increase caused by NMDA-receptor (NMDAR) and voltage-gated Ca2+ -channel (VGCC) activation is thought to determine both the amplitude and direction of synaptic plasticity by differential activation of Ca2+ -sensitive enzymes such as calmodulin. Ca2+ influx is negatively regulated by Ca2+ -activated K+ channels (SK-channels) which are in turn inhibited by neuromodulators such as acetylcholine. However, the precise mechanisms by which SK-channels control the induction of synaptic plasticity remain unclear. Using a 3-dimensional model of Ca2+ and calmodulin dynamics within an idealised, but biophysically-plausible, dendritic spine, we show that SK-channels regulate calmodulin activation specifically during neuron-firing patterns associated with induction of spike timing-dependent plasticity. SK-channel activation and the subsequent reduction in Ca2+ influx through NMDARs and L-type VGCCs results in an order of magnitude decrease in calmodulin (CaM) activation, providing a mechanism for the effective gating of synaptic plasticity induction. This provides a common mechanism for the regulation of synaptic plasticity by neuromodulators.The key trigger for Hebbian synaptic plasticity is influx of Ca 2+ influx which activates calmodulin and CaMKII. The influx of Ca2+ through voltage-dependent NMDA receptors and Ca2+ channels is regulated by Ca2+ -activated K+ channels (SK-channels) providing negative feedback regulation of postsynaptic [Ca2+]. Using 3-dimensional modeling of Ca2+ and calmodulin dynamics within dendritic spines we show that the non-linear relationship between Ca2+ influx and calmodulin activation endows SK-channels with the ability to “gate” calmodulin activation and therefore the induction of Hebbian synaptic plasticity. Since SK-channels are inhibited by several neuromodulator receptors including acetylcholine and noradrenaline, the gating of synaptic plasticity by SK-channels could represent a common mechanism by which neuromodulators control the induction of synaptic plasticity.Hebbian or associative plasticity is triggered by postsynaptic Ca Spike timing-dependent plasticity (STDP) is the classical manifestation of Hebbian plasticity where temporally correlated pre- and post-synaptic activity induces NMDA receptor (NMDAR)- and Castrength –3. The con (LTP) –6. Howevon (LTP) , 8, largasticity , 9 raisi spiking  highligh2+ /calmodulin-activated kinase II (CaMKII) and therefore on the transduction of Ca2+ signals by calmodulin (CaM)  transients. In turn, spatiotemporal [Ca2+] distributions are determined by endogenous Ca2+ buffering within spines which restrict Ca2+ to localized micro- or nano-domains  to the SK-channel cluster. EPSPs were simulated by representing synaptic-cleft glutamate concentration immediately after presynaptic stimulus as a step-pulse of 1mM amplitude with duration 1ms. bAPs were simulated by instantaneous rise of membrane potential to maximal depolarization (67mV), followed by double exponential-decay to resting potential. Diffusing Ca2+ was buffered by a low binding-capacity endogenous fixed-buffer (EFB) . In simulations involving calmodulin, the simple mobile-buffer model (Eq. 16) was replaced with a cooperative binding model of Ca2+ to the two calmodulin lobes . Parameter values, constrained by Ca2+ -imaging experiments, were tuned (in simulations using mobile-buffer binding kinetics for the relevant Ca2+ -indicator dye) by matching changes in model-predicted levels of Ca2+ -bound mobile buffer to changes in measured Ca2+ dye fluorescence.Solutions to the spine Car MATLAB , 21. Fulpossible . For spiibutions . The boug-scheme . The NMDgCaT = 0. pS and Lio of 20 ) and a mls gSK =  pS. AMPA2+] in postsynaptic dendritic spines is an important factor governing synaptic plasticity, we built a 3-dimensional, deterministic model of [Ca2+] dynamics within a single dendritic spine  from internal calcium stores. However, for CA1 spines, evidence suggests a limited role for calcium stores  gradient across the spine head with highest [Ca2+] near the Ca2+ channel clusters  . Application of this protocol rapidly established a [Caclusters . [Ca2+] 5000s−1) . The spa current . EPSP maVA VGCCs , causingCCs [2+] . The bAP-unblock . The glostimates .2+] -signal magnitude, rise and decay time, and spatial extent are perturbed by Ca2+ buffer changes  signals are restricted to channel nanodomains   levels. Furthermore, this priming effect on SK-channels was largely insensitive to the Ca2+ buffering capacity  , which icapacity .2+] transients elicited by combinations of stimuli, but not for [Ca2+] transients elicited by unitary stimuli. These results also suggest that SK-channels only influence global [Ca2+] when they are situated within ∼200nm of a Ca2+ source  generated in the spine in response to both unitary and compound stimuli. In particular, we focused on a STDP induction protocol—a single EPSP followed by two bAPs at 10ms intervals (EPSP-2bAPs)—which is associated with robust LTP at the Schaffer collateral synapse in CA1 of the hippocampus  up to 17-fold during STDP induction protocols associated with LTP, indicating that SK-channels effectively ‘gate’ the induction of STDP. Our results suggest that the mechanism behind this is ‘priming’ of SK-channels during EPSPs, by NMDAR activation, which effectively silence L-type VGCCs during subsequent bAPs. L-type VGCCs then provide a crucial Ca2+ signal for CaM (and therefore CaMKII) activation when SK-channels are inhibited. We have also shown that, in tandem, the distinctive Ca2+-binding properties of the CaM N- and C-lobes give the CaM molecule some sophisticated Ca2+ sensing properties.Our analyses show that SK-channels may play a central regulatory role in the induction of Hebbian synaptic-plasticity. 3-dimensional modeling of spine Ca2+ signal location in spines is an important determinant for synaptic plasticity, or—at the very least—that the amplitude and temporal characteristics of the spine Ca2+ signal is not sufficient to explain all plasticity observations. Many studies of spine Ca2+ signalling have concluded that the spatial localisation of Ca2+ sources is important for the ultimate biological role of the Ca2+ signal  showing that 3-dimensional modelling is capable of revealing roles of Ca2+ signalling that would be missed using a non-spatial model.3-dimensional modelling improves the biological plausibility and validity of our analyses. There is strong evidence to suggest that Canal  [2+] responses to unitary and compound stimuli. SK-channel activation following a single bAP is too slow for the inhibitory current to influence the fast-activating HVA L-type VGCC Ca2+ current. However, if a bAP occurs shortly after an initial ‘priming’ stimulus, then the SK-current effectively silences the L-type Ca2+ channels by repolarizing the membrane potential below the threshold voltage for L-type VGCC activation. Interestingly, fully-activated [CaCaM] signals were insensitive to SK-channel blockade for induction protocols not associated with plasticity (Given the highly effective endogenous buffering of Ca+ source . SK-chan(τ∼ 6ms)  which leasticity . On the 2+ thereby saturating endogenous buffers, which will have an effect on other Ca2+ interactions. Strong vs. weak clustering of channels could be viewed as effectively altering the VGCC ‘cluster’ conductance. We probe the effect of varying VGCC cluster conductance on the activation of CaCaM (see 2+ source in the cluster condition), the [CaCaM] signal is greatly reduced by changing the spatial distribution of the VGCC conductance. In a sense, when the VGCC current density is uniform over the entire surface of the spine, there is no VGCC nanodomain. In the case of uniform distribution of VGCCs the main conclusion of the paper, that SK-channels are powerful modulators of CaM activation still holds.Strong clustering of VGCCs will increase the local influx of CaaCaM see  and findaCaM see . This isaCaM see . As expe2+] signal during STDP LTP induction protocols leading to greatly enhanced CaM (and therefore CaMKII) activation. Thus, SK-channels may provide a common mechanism by which multiple neuromodulators can gate Hebbian plasticity enabling the switching of neuronal networks, such as the hippocampus, between LTP-competent and LTP-incompetent configurations.SK-channels are negatively regulated by neuromodulators such as acetylcholine and noradrenaline , 49, 50 S1 Table(PDF)Click here for additional data file.S1 Figx and Cx, represent calmodulin N- and C-lobe Ca2+ binding-sites. Binding sites are initially in state T, with associated binding rates kxon(T) and kxoff(T). Upon binding Ca2+, the lobe’s remaining Ca2+ binding site instantaneously switches to R state, with associated binding rates kxon(R) and kxoff(R).N(TIF)Click here for additional data file.S2 Fig2+] is defined as Ca2+ concentration at 50nm radial distance from channel cluster.Additional stimuli for (TIF)Click here for additional data file.S3 Fig2+] transients for EPSP+bAP stimulation protocol. Nanodomain [Ca2+] is defined as [Ca2+] at 20nm radial distance from channel cluster. Simulations use the simple binding scheme for Ca2+ to mobile Ca2+ buffer described in equation system 17. The first two columns show transients for simulations where EFB parameters were fixed and the concentration of the mobile buffer was varied. Mobile Ca2+ buffer parameter values were selected to represent a typical ‘fast’ mobile buffer and a ‘slow’ mobile buffer. Slow mobile buffer binding parameters were set to match estimates for calbindin [2+ buffer was set to 100μM calbindin and either the EFB binding forward rate or EFB affinity was fixed while the other varied.Comparison of global and nanodomain [Caalbindin  and fastalbindin . The end(TIF)Click here for additional data file.S4 Fig(A) Peak [Ca2+] gradients in Ca2+ channel nanodomains across buffer condition and spine stimuli. (B) Peak [Ca2+] gradients in Ca2+ channel nanodomains in the presence of EFB and 100μM calbindin. Each curve represents a different stimulation pattern. Dashed horizontal lines in all plots indicate SK half-activation parameter, Ks = 0.33μM.(TIF)Click here for additional data file.S5 FigSK-channels were active and spatially coupled (50nm) to NMDAR cluster.(TIF)Click here for additional data file.S6 FigSK-channels were active and spatially coupled (50nm) to NMDAR cluster.(TIF)Click here for additional data file.S7 Fig(A) Concentration gradients of peak activated [CaCaM] in NMDA and VGCC nanodomains during EPSP-bAP induction protocols. Each curve represents different simulated values for CaM diffusivity. (B) Activated [CaCaM] is increased by SK-blockade across CaM diffusivities. Very immobile CaM results in heightened effect of SK-blockade on activated [CaCaM] . (C) Ratio of peak [CaCaM] for SK-channel conditions. Subscript 0 indicates that SK-channels were blocked and subscript 1 indicates SK-channels were active.(TIF)Click here for additional data file.S8 FigEPSPs were required for SK-channel activation since SK-channel cluster was spatially coupled to NMDAR cluster (50nm) for these simulations. Bar charts in end column summarize global and nanodomain peak [CaCaM].(TIF)Click here for additional data file.S9 Fig(A) Concentration gradients of peak activated [CaCaM] in NMDA and VGCC nanodomains during EPSP-bAP induction protocols. Each curve represents different simulated values for L-type channel cluster conductance. (B) Activated [CaCaM] is increased by SK-blockade across a range of L-type conductances although differences are enhanced as L-type conductance is increased . (C) Ratio of peak [CaCaM] for SK-channel conditions blocked or coupled to NMDARs. Subscript 0 indicates that SK-channels were blocked and subscript 1 indicates SK-channels were active.(TIF)Click here for additional data file.S10 Fig2+—source in the clustered case.Clustered VGCC condition refers to the VGCC arrangement described in the main text and illustrated in (TIF)Click here for additional data file.


One potential mechanism for this hyper-contractility is abnormal intracellular Ca2+ handling in airway smooth muscle cells from subjects with asthma compared to non-asthmatic controls by measuring: i) bradykinin-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation and intracellular Ca2+ concentration, ii) sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression, iii) mechanisms of cytoplasmic Ca2+ clearance assessed following instantaneous flash photolytic release of Ca2+ into the cytoplasm.We sought to compare intracellular Ca2+ concentrations, bradykinin-stimulated IP3 accumulation or intracellular Ca2+ responses. Quantification of SERCA2 mRNA or protein expression levels revealed no differences in ASM cells obtained from subjects with asthma compared to non-asthmatic controls. We did not identify differences in intracellular calcium kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation with or without SERCA inhibition. However, we did observe some correlations in subjects with asthma between lung function and the different cellular measurements of intracellular Ca2+ handling, with poorer lung function related to increased rate of recovery following flash photolytic elevation of cytoplasmic Ca2+ concentration.We found no differences in airway smooth muscle cell basal intracellular Ca2+ handling between airway smooth muscle cells from non-asthmatic and asthmatic subjects. Therefore, increased contraction of airway smooth muscle cells derived from asthmatic subjects cannot be fully explained by altered Ca2+ homeostasis.Taken together, the experimental results reported in this study do not demonstrate major fundamental differences in Ca Asthma, a chronic inflammatory disease, remains a major health care burden affecting over 300 million people worldwide. There is a subset of patients that do not respond adequately to traditional treatments, highlighting an urgent need for new therapies ,2. Asthm2+]i) plays a cardinal role in ASM contraction. An increase in intracellular ASM [Ca2+]i is likely to originate from the sarcoplasmic reticulum of the cell, with evidence of a lesser reliance on Ca2+-influx across the sarcolemma through voltage-dependent channels than other muscle types [2+]i handling, signalling or storage, as possible underlying mechanisms for this hyper-contractility [2+]i involves the movement of Ca2+ out of the cell through the sarcolemmal Ca2+-ATPase or Na+/Ca2+-exchangers, or into the sarcoplasmic reticulum through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). SERCA2 is the dominant SERCA isoform expressed in ASM, predominantly as the SERCA2B splice variant, and reduced SERCA2 in ASM from subjects with asthma has been reported and proposed to prolong the time for [Ca2+]i to return to baseline following stimulation [2+]i handling in ASM from asthmatics have been explored following agonist-induced activation. Such receptor-dependent changes in [Ca2+]i are often complex, involving complex signal transduction mechanisms that can modulate release and re-uptake of [Ca2+]i at multiple loci. Flash photolysis allows a ‘caged’ form of calcium to be released in a controlled, time-resolved manner to investigate [Ca2+]i recovery independently of a need for agonist addition [2+ across the sarcoplasmic reticular and/or sarcolemmal membranes to determine if potential asthma-related abnormalities in [Ca2+]i handling are observed independently of the need to perturb Ca2+ homeostasis via receptor-mediated mechanisms.Cytosolic calcium concentration  bradykinin-induced changes in intracellular calcium [Ca2+]i; (ii) SERCA expression, (iii) agonist-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation, and (iv) [Ca2+]i kinetics assessed following release of a Ca2+ load into the cytoplasm by flash photolysis and uncaging.We hypothesized that CaAsthmatic subjects (n = 20) and non-asthmatic controls (n = 18) were recruited from Leicester, UK. Asthma severity was defined by Global Initiative for Asthma (GINA) treatment steps  [2+]i, ASM cells plated at sub-confluence on coverslips were loaded with 2 μM Fura-2 AM (Molecular Probes/Thermo Fisher Scientific) in the presence of 2.5 mM probenecid (Sigma-Aldrich) and 0.04% w/v pluronic F127 (Molecular Probes/Thermo Fisher Scientific), and visualized using an inverted epifluorescence microscope (Nikon Diaphot 200). Changes in Fura-2 fluorescence (F) intensity were measured as a ratio, R, where R = F340 nm/F380 nm, such that R increases as [Ca2+]i increases [−1) in physiological saline solution  at 37°C.To measure changes in [Cancreases . Bradyki-∆∆Ct method [Real-time reverse transcription-polymerase chain reaction was performed . Relative quantification was done using the comparative 2t method  and expr3 accumulated rapidly after bradykinin addition and was maximal at 10 sec. Incubations were terminated by rapid aspiration and addition of trichloroacetic acid (0.5 M). Acid extracts were neutralized and IP3 accumulation measured using a competitive binding assay as described previously [3 accumulation is expressed as pmol mg−1 of cell protein.ASM cell monolayers in 24 well plates were stimulated with different concentrations of bradykinin (0.001-10 μM). Preliminary experiments demonstrated that IPeviously . IP3 acc2+ transients were monitored using an Olympus confocal laser scanning inverted microscope (FV1000). The single scan head was used to monitor changes in [Ca2+]i by detecting fluo-4 fluorescence with the 488 nm line of a multi-line argon laser (at ~2% maximum output) using ‘round-trip’ mode, with emission occurring at 528 nm and a wide confocal aperture setting of 374 μm. Near instantaneous switching allows photo-activation/uncaging, with a 405 nm laser flash without bleaching or response saturation, then switching back to 488 nm monitoring to capture the subsequent recovery of the cell towards basal [Ca2+]i iper se.ASM cells plated at sub-confluence on coverslips were co-loaded with 2 μM Fluo-4 AM and 2.5 μM nitro-phenyl-EGTA AM  in the  [Ca2+]i ,21. Inte6 ASM cells resuspended in 144 μl of serum-free medium with stimulus as required. The gels were added to 24-well plates (PBS/0.5% BSA) and left to polymerize at 37°C for 90 min. The gels were then detached and suspended in 500 μl of serum-free medium with stimulus as required. Bradykinin (Sigma-Aldrich) was added to appropriate wells to a final concentration of 1nM, with photographs taken at regular intervals up to 1 hour. The surface area of each gel was measured at each time point using ImageJ (http://rsb.info.nih.gov/ij) by a blinded observer.Collagen gels , 37 μl of 10X DMEM (Invitrogen), 20 μl of sodium bicarbonate (Invitrogen) were impregnated with 0.25 × 10t-tests for parametric data, or Mann–Whitney tests for non-parametric data. Correlations were performed using either Pearson or Spearman of parametric and non-parametric data, respectively. A P value less than 0.05 was considered statistically significant.GraphPad Prism  and IBM SPSS version 20  were used to perform statistical analysis. Mean (standard error of the mean [S.E.M.]) was used to present parametric data, while median (interquartile ranges [IQR]) was used for non-parametric data. Comparisons between disease states used unpaired Student’s 2+]i response following bradykinin addition is shown in Figure 2+]i was determined for each donor used . Following addition of bradykinin (1 μM), the change in [Ca2+]i (ΔR) was not different between non-asthmatic and asthmatic donors . The area under the curve (AUC) values measured following bradykinin stimulation also did not significantly differ between health and disease . Finally, the rate of recovery following administration of bradykinin did not differ between ASM cells from non-asthma and asthma subjects . Example western blots showing total SERCA2 protein and β-actin in ASM cells derived from four asthma patients and three non-asthmatic donors are shown in Figure 1% predicted and FEV1/FVC% .The relative expression of SERCA2A/B/C mRNA was assessed in 10 non-asthmatic control subjects and 13 patients with asthma  were not significantly different between non-asthmatic and asthmatic smooth muscle donors (−log EC50 (M): 7.17 ± 0.11 and 7.22 ± 0.10, respectively; p = 0.74; Figure 1% predicted and FEV1/FVC% . The increase in IP3 accumulation  following stimulation was not significantly different between health and disease (∆IP3 (pmol mg−1 protein): 631 ± 68 and 647 ± 54, respectively; p = 0.86; Figure 3 accumulation stimulated by bradykinin did not correlate with FEV1% predicted and FEV1/FVC% .Concentration-dependent IP2+]i recovery rates following Ca2+ uncaging in the absence or presence of SERCA inhibition were made in ASM cells from patients with asthma or non-asthmatic controls : 3.31 ± 0.46 and 3.89 ± 0.58, respectively; p = 0.45; Figure +CPA (s−1): 1.43 ± 0.23 and 1.29 ± 0.15, respectively; p = 0.60; Figure -CPA: 3.80 ± 0.88 and 3.99 ± 0.83, respectively; p = 0.88) or presence  values in ASM cells from non-asthmatic and asthmatic patients did not reveal any differences , nor was there a difference between asthma GINA classification 1–3 and 4–5 .Comparisons of i kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation again did not demonstrate differences in rates of recovery of [Ca2+]i either with or without SERCA inhibition. However, we did observe correlations in the subjects with asthma between lung function and [Ca2+]i rate of recovery following flash photolysis with poorer lung function relating to an increased rate of recovery. Taken together this study does not support a fundamental difference in [Ca2+]i handling in ASM from subjects with asthma compared to non-asthmatic controls per se, but does not exclude a possible contribution in specific phenotypes, such as those with impaired lung function, as suggested here by subtle differences in [Ca2+]i recovery rates.Here we have studied [Ca2+]i conflicts with work published previously [2+]i response to the same concentration of bradykinin [et al. reported a reduced [Ca2+]i response to acetylcholine (10 μM) or histamine (10 μM) in ASM cells from asthmatic subjects [2+]i peak or rate of recovery with severity of disease defined by GINA treatment steps, nor with lung function. Our study had 80% power to observe a 0.05 difference between health and disease in [Ca2+]i (ΔR) following bradykinin activation. Mahn et al. reported [Ca2+]i (ΔR) of 0.07 and 0.12 between ASM from non-asthmaticsubjects versus those with either mild or moderate asthma, respectively [2+]i in response to agonist might be altered in some asthmatics. Therefore, whether altered [Ca2+]i handling is typically altered in asthma requires further study in a larger population.Our findings of no disease-related differences in agonist-induced evoked changes in i we were also unable to demonstrate differences in SERCA2 expression, either at the mRNA or protein levels. Mahn et al. found reduced expression of both mRNA and protein in primary ASM cells and reduced expression in ASM in vivo in tissue sections of endobronchial biopsies, in both moderate and severe asthmatics compared to non-asthmatic controls i handling to investigate the differences in ASM cells from subjects with asthma and non-asthmatic controls independent of agonist. [Ca2+]i kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation again did not demonstrate differences in rates of recovery of [Ca2+]i either with or without SERCA inhibition. Interestingly, we did observe correlations in the subjects with asthma between lung function and rates of recovery of [Ca2+]i following flash photolysis. Those asthmatics with poorer lung function had greater rates of [Ca2+]i recovery. This again conflicts with the report from Mahn et al., which suggests that rates of recovery are reduced in asthma [2+]i following flash photolysis requires further investigation. However, although the correlations reported here are intriguing, they are only observed in those ASM cells from subjects with asthma and there were no differences in uncaging [Ca2+]i rate of recovery between asthma and health, questioning whether these relationships are important.We report for the first time a comparison of the concentration dependency and magnitude of changes in the Can asthma ,15. Simin asthma . Whether2+]i handling in ASM cells from subjects with asthma, we and others have consistently found that primary ASM cells from asthmatics are hyper-contractile [2+]i originating from the sarcoplasmic reticulum stores, therefore, the hyper-contractility seen in asthma is likely to be due to abnormalities in [Ca2+]i handling, Ca2+ sensitivity or changes to the contractile machinery. Since we have not shown here any intrinsic abnormality in [Ca2+]i or SERCA2 in ASM cells derived from asthmatic patients when compared with non-asthmatic subjects, we must consider alternative mechanisms. Myosin light-chain kinase (MLCK) is a major player in this Ca2+/contraction pathway and indeed others have found expression of MLCK to be increased [2+-independent rhoA/rho kinase signalling pathway leads to inhibition of myosin light-chain phosphatase resulting in increased levels of phosphorylated myosin light-chains and increased ASM contraction force at a given increase in [Ca2+]i i handling remains important. Perhaps more critical is the heterogeneity of asthma and even though our study has sufficient power to observe large differences in [Ca2+]i handling we might have failed to observe small, albeit important, differences in specific phenotypes of asthma. For example, our intriguing observations of correlations between lung function and [Ca2+]i rates of recovery following NP-EGTA Ca2+ uncaging might reflect important associations in a sub-group of asthmatics.Our study has a number of potential limitations. We have used primary ASM cells and therefore whether important mechanisms present 2+ handling between ASM from subjects with and without asthma, in contrast to previous reports [2+]i homeostasis in asthma.In summary, we have not shown major fundamental differences in Ca reports ,16, we c


RNA-binding protein Musashi-2 (Msi2) is known to play a critical role in leukemogenesis and contributes to poor clinical prognosis in acute myeloid leukemia (AML). However, the effect of Msi2 silencing on treatment for AML still remains poorly understood. In this study, we used lentivirus-mediated RNA interference targeting Msi2 to investigate the resulting changes in cellular processes and the underlying mechanisms in AML cell lines as well as primary AML cells isolated from AML patients. We found that Msi2 was highly expressed in AML cells, and its depletion inhibited Ki-67 expression and resulted in decreased in vitro and in vivo proliferation. Msi2 silencing induced cell cycle arrest in G0/G1 phase, with decreased Cyclin D1 and increased p21 expression. Msi2 silencing induced apoptosis through down-regulation of Bcl-2 expression and up-regulation of Bax expression. Suppression of Akt, Erk1/2 and p38 phosphorylation also contributed to apoptosis mediated by Msi2 silencing. Finally, Msi2 silencing in AML cells also enhanced their chemosensitivity to daunorubicin. Conclusively, our data suggest that Msi2 is a promising target for gene therapy to optimize conventional chemotherapeutics in AML treatment. Acute myeloid leukemia (AML) is a group of clonal hematopoietic stem cell disorders characterized by failure to differentiation and overproliferation in the stem cell compartment leading to accumulation of myeloblasts. Refinements of supportive treatment have contributed to improved outcomes of AML patients in the past 30 years. However, more than half of young adult patients and approximately 90% of older patients still succumb to their diseases . One majThe Musashi (Msi) family is an evolutionarily conserved group of RNA-binding proteins containing two RNA recognition motifs and plays a critical role in cell fate determination, asymmetric stem cell division and stem cell function regulation –5. In maAlthough Msi2 has been regarded as a new prognostic marker in leukemia, Msi2 directly interacts with and retains efficient translation of crucial transcription factors and epigenetic modulators including HOXA9, IKZF2, and MYC so as to directly maintain the mixed-lineage leukemia self-renewal program . Msi2 exIn the present study, we demonstrated that Msi2 silencing decreased proliferation, induced cell cycle arrest in G0/G1 phase, and increased apoptosis in AML cell line Dami, HL-60, and primary AML cells from patients with AML, which were attributed to suppression of Akt, Erk1/2 and p38 phosphorylation. Msi2 silencing in AML cells also increased their chemosensitivity to daunorubicin.Human AML cell lines HL-60, NB4, U937, HEL, and Dami and CML cell line K562 were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum . Primary AML cells were obtained from bone marrow aspirates of 8 newly diagnosed and untreated patients with AML  according to our previously described methods , which wMsi2 silencing was carried out with four separate short hairpin RNA (shRNA) constructs targeting Msi2 . One scramble sequence was used as a negative control. The shMsi2 sequences and the scramble sequence see 3/well in 96-well plates for 1–3 days, and cell viability was assessed using Cell Counting Kit-8 (CCK-8) . The absorbance was read at 450 nm using an ELISA reader .AML cells untreated and treated with scramble or shMsi2 lentivirus were seeded at a density of 4 × 10AML cells infected with scramble or shMsi2 lentivirus were treated or untreated with insulin-like growth factor-1 (IGF-1) or thrombopoietin  or daunorubin . Then, cells were harvested and analyzed for apoptosis using APC-Annexin V and propidium iodide  according to the manufacture’s protocols. Data acquisition and analysis were performed using CellQuest software on a flow cytometry .AML cells infected with scramble or shMsi2 lentivirus were harvested and stained with PI using a Cycletest™ plus DNA reagent kit  according to the manufacturer’s instructions.Quantitative real-time PCR was used to analyze the expression of CCND1, p21, Cdk2, Bcl-2, Bax, and a reference gene GAPDH according to our previously reported method . The seq® Mammalian protein extraction reagent  supplemented with Halt protease and phosphatase inhibitor cocktail (Pierce). The protein was subjected to western blot analysis with antibodies against Msi2 , Cyclin D1, p21, Bcl-2, Bax, PARP, p-Akt (Ser473), Akt, p-Erk1/2 (Thr202/Tyr204), Erk1/2, p-p38 (Thr180/Tyr182), p38, or GAPDH  according to our previously described method [After infection with scramble or shMsi2 lentivirus, AML cells were collected and lysed immediately using M-PERd method ,20. The 7 HL-60 cells expressing shMsi2-3 or scramble control were injected into the tail vein of each mouse. After transplantation, the mice were observed until they died of infiltration of HL-60 cells and the time of death was recorded. Animal procedures were carried out in accordance with institutional guidelines after Wenzhou Medical University and Fudan University Animal Care and Use Committee approved the study protocol.NOD/SCID mice , 6 weeks old, were maintained throughout in specific pathogen-free environment and divided into two groups of eight mice per group. A total of 1 × 10P values less than 0.05 was considered statistically significant.Data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 5.0 . Msi2 expression was analyzed in five AML cell lines  and primary AML cells from AML patients as well as a CML cell line K562. In contrast to HL-60 cells, high expression of MSI2 in HEL, Dami, and K562 cells, low expression of Msi2 in NB4 and U937 cells, and similar expression of Msi2 in primary AML cells are shown . To idenTo determine whether Msi2 affects the proliferation of AML cells, cell viability assay was performed using CCK-8. The proliferation of AML cells including Dami cells, HL-60 cells, and primary AML cells were decreased in shMsi2-3 group compared to the scramble control group . Msi2 siWe next investigate whether Msi2 silencing affects the cell cycle progression of AML cells. As shown in We then examined whether Msi2 silencing increases apoptosis in AML cells using flow cytometry analysis. Compared with the scramble control, apoptosis was significantly increased in shMsi2-3 group in Dami cells, HL-60 cells, and primary AML cells , suggestTo further determine the mechanisms regulated by Msi2 in the induction of apoptosis in AML cells, the phosphorylation of Akt, Erk1/2 and p38 were examined. Msi2 silencing decreased the phosphorylation of Akt in Dami cells, HL-60 cells, and primary AML cells , inconsiBased on abovementioned findings, we then investigated whether AML cells with reduced Msi2 expression are more sensitive to daunorubicin, an anthracycline in combination with cytarabine as the standard induction therapy for AML patients of all subtypes except M3 . As showIncreasing evidences have shown that Msi2 overexpression is a common characteristic of leukemic cells, where up-regulation of Msi2 was negatively associated with rapid progression and poor prognosis. However, the biological roles and the underlying mechanisms mediated by Msi2 in AML are not well understood. Here we found that Msi2 silencing inhibited proliferation and induced apoptosis and enhanced chemosensitivity to daunorubicin in AML cells. In addition, we also found that Akt, Erk1/2 and p38 signaling were involved in apoptosis induced by Msi2 silencing in AML cells.We measured the expression of Msi2 in five AML cell lines and a CML cell line K562 as well as primary AML cells isolated from AML patients. High Msi2 levels were observed in HEL and Dami cells, whereas low Msi2 levels were observed in NB4 and U937 cells among five AML cell lines, similar to a previous report . The anaCell viability assay demonstrated that knockdown of Msi2 inhibited the proliferation of Dami cells, HL-60 cells, and primary AML cells. Consistent with our results, Msi2 silencing also resulted in decreased proliferation in other AML cell lines and a CML cell line ,25. The In addition to cell cycle arrest, we also found that Msi2 silencing in AML cells markedly increased apoptotic cells compared to the scramble controls. Western blot analysis demonstrated that Msi2 silencing significantly decreased anti-apoptotic protein Bcl-2 and increased pro-apoptotic protein Bax expression, implying the role of Msi2 as a regulator of apoptosis. The PI3K/Akt pathway plays important roles in leukemic cell proliferation, growth, and survival ,31. We fAnthracycline antibiotics, such as daunorubicin, are still key chemotherapeutic drugs used in AML treatment . In thisIn conclusion, this study demonstrated that Msi2 silencing exerted potent activity against AML and enhanced the cytotoxicity of daunorubicin. Therefore, Msi2 may be a promising therapeutic target for AML.S1 ARRIVE Checklist(PDF)Click here for additional data file.


Thoracotomy is a major thoracic surgery procedure that has various indications and can be marred with various complications and occasionally dismal outcome especially in resource poor environment like ours.To determine the demographic pattern, indications, complications and challenges of open thoracotomy in a new cardiothoracic unit in sub-Saharan Africa.It is a prospective study of patient admitted into the unit since its inception between January 2012 and May 2015. Patient's demographic data, clinical presentation, indications for and outcome of thoracotomy were analysed.A total of 40 patients were admitted and treated during the period with a male to female ratio of 2.6:1. The mean age was 29.5 years. The most common indication for thoracotomy in our series is chronic empyema thoracis, which accounted for 16 (40%) of all the thoracotomies, chest trauma was an indication in 12 (30%) of cases. Eight patients out of 12 (66.7%) had emergency thoracotomy. One patient (2.5%) had thoracotomy and bronchotomy for removal of a long-standing foreign body in the right lower lobe bronchus. Three patients (7.5%) died, 2 died intraoperative and one died in the intensive care unit after emergency thoracotomy following penetrating chest trauma.Chronic empyema thoracic is currently the most common indication for thoracotomy in our setting. This can be attributed to the fact that we are still battling with various forms of pulmonary and pleural infections that are poorly treated and most of these patients present late to the hospital. The outcome of thoracotomy is good despite the fact that it is a new unit and our resources are limited.