Dietary intake of bioactive phytochemicals including the cruciferous vegetable derivative phenethyl isothiocyanate (PEITC) can reduce risk of human cancers, but possible epigenetic mechanisms of these effects are yet unknown. We therefore sought to identify the molecular basis of PEITC-mediated epigenetic tumor restriction. Colon cancer cells treated with low-dose PEITC for >1 month exhibited stable alterations in expression profile of epigenetic writers/erasers and chromatin-binding of histone deacetylases (HDACs) and Polycomb-group (PcG) proteins. Sustained PEITC exposure not only blocked HDAC binding to euchromatin but was also associated with hypomethylation of PcG target genes that are typically hypermethylated in cancer. Furthermore, PEITC treatment induced expression of pro-apoptotic genes in tumor cells, which was partially reversed by overexpression of PcG member BMI-1, suggesting opposing roles for PEITC and PcG proteins in control of tumor progression. These data demonstrate that PEITC regulates chromatin binding of key epigenetic writers/erasers and PcG complexes to restrict tumor development. Human cancers are highly diverse, exhibit complex aetiology, and remain extremely challenging to treat in the clinic. As the total burden of disease and associated healthcare costs continue to rise, attention is now increasingly turning to methods of cancer prevention rather than cure135CDKN2A, MLH1, CDH1, and VHL91112Rapid technological progress in recent decades has finally enabled investigators to begin to define the epigenetic characteristics of different cancers and identify the environmental factors that promote diseaseBrassica rapa varieties that are part of the human diet like Chinese cabbage, Pak Choi and turnips25Current epigenetic therapies for cancer include inhibitors of the enzymes HDACs and DNMTs that aim to restore a ‘healthy’ epigenetic state via the removal/reversal of cancer-associated epigenetic marks13151719202022In the current study, we investigated how PEITC exposure modifies epigenetic profiles in a tumor cell line model of colorectal cancer (CRC), which is the third most common type of cancer and the second leading cause of cancer-related death worldwide28in vitro  or 0.01% DMSO vehicle-only (SW620-CON). As shown in in vitro  and in a(in vivo . SW620-P2PEITC/CON ≥ 0.5) whereas 166 proteins had been redistributed to regions of heterochromatin (log2PEITC/CON ≤ −0.5). Next, the PEITC-modulated proteins (n = 366) were subjected to gene ontology (GO) analysis and functional classification as shown in We next sought to determine whether the ability of PEITC conditioning to restrict tumor growth was achieved via epigenetic modifications that influence gene expression. We therefore used a quantitative proteomic approach to assess whether PEITC treatment of SW620 cells alters the composition of the ‘chromatome’ i.e. the selection of proteins that are bound to regions of euchromatin or heterochromatin. To do this, we purified chromatin from either SW620-CON or SW620-PEITC cells and subjected this to partial digestion by micrococcal nuclease (MNase) which can access the relatively open structure of euchromatin but not the closed conformation of heterochromatin thereby sequentially extracting euchromatin- and heterochromatin-associated proteinsIn order to identify key mediators of chromatome modulation by PEITC, we next focused our attention on the distribution of major epigenetic regulators identified in the LC-MS/MS data. We observed that PEITC treatment modulated the chromatin association patterns of several major groups of epigenetic writers and erasersBAD, BIM, BLK, and BMF genes which encode pro-apoptotic BH3 domain proteins, as well as the multi-domain pro-apoptotic gene BAKWe next investigated the phenotypic and functional consequences of these PEITC-induced chromatome changes in terms of tumor cell apoptosis and cell cycle arrest using quantitative real-time PCR (qRT-PCR) and flow-cytometry. As shown in WAF1/CIP1 and p16INK4a 19As described before, properties of bioactive phytochemicals as potent inhibitors of DNA methylation were explained in reverse hypermethylation and activation of tumor suppressor genes p21in vitro and decreased tumorigenic potential in the mouse xenograft model in vivo, we next assessed whether PEITC-induced epigenetic changes could influence the methylation status of key oncogenes and/or tumor suppressor genes. When we analyzed the genes that were hypomethylated in PEITC-treated cells, we observed that these included several targets of polycomb repressive complexes (PRCs), consistent with our earlier proteomic data which revealed decreased PRC binding to heterochromatin in PEITC-treated cells. Indeed, when compared a list of 687 genes, which hypomethylated in SW620-PEITC cells, against those are either known targets of PRC1/PRC2 or frequently methylated/hypermethylated in cancer3738Since the PEITC-treated cancer cells displayed reduced growth in colony formation assays SPG20 (Spastic Paraplegia 20), PAK7 (p21 protein activated kinase 7), PCDH10 (Protocadherin 10), HNF4A (Hepatocyte Nuclear Factor 4), VWC2 (von Willebrand factor C domain containing 2), CDH6 (Cadherin 6), RASSF5 (Ras association domain-containing protein 5), and SOX3 (SRY-related HMG-box 3)  target genes are hypermethylated in SW620 cells but display reduced methylation after long-term PEITC exposure , we nextG-box 3) . The MeDG-box 3) . These dp = 0.0001, p < 0.0001). Since PEITC treatment up-regulated the expression of pro-apoptotic genes in SW620 cells and BMI-1 overexpression was able to rescue the viability of tumor cells undergoing PEITC treatment  in HCT116-Vec cells or HCT116-BMI-1 cells treated with 12.5 μM PEITC for 24 h  exhibited ~30% more growth over a 48 h period than did control HCT116 cells expressing GFP only (HCT116-Vec) , phenethyl isothiocyanate (PEITC) and indol-3-carbinol were studied in several cancer models64041Defects in epigenetic regulation of gene expression are increasingly being recognized as critical mediators of cancer development136445in vitro study47In order to better understand the epigenetic basis of BDC-mediated protection against tumors, we established a cell line model of habitual consumption of cruciferous vegetables by subjecting SW620 colon cancer cells to physiologically relevant concentration of PEITC for 6 weeks. Blood plasma concentrations of PEITC after consumption of glucosinolate-rich watercress or broccoli sprout isothiocyanates in human have been reported to be reached ~2.3 μM, which was comparable to those used in our Together, our data demonstrate that prolonged exposure to an archetypal BDC can induce stable changes in tumor cell expression of epigenetic regulators and pro-apoptotic genes. In particular, our results indicate that the chemopreventive effects of PEITC exposure are associated with stable changes in HDAC distribution and hypomethylation of known targets of the PcG complex and other genes that are frequently methylated in cancer.Much attention on chemopreventive effects of phytochemicals also bring the issue of interindividual differences in absorption, metabolism, and disposition of phytochemicals. It has been reported that genetic polymorphism of those key metabolizing enzymes including glutathione S-transferases, UDP-glucuronosyltransferases, and cytochrome P450 2E1 may partly account for variation in cancer risk49BMI-1 cDNA into the XhoI/BamHI sites of a pEGFP-C1 vector.Colorectal cancer cell lines SW620, SW480, and HCT116 were obtained from American Type Culture Collection. Cells were maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin and then cultured in the presence of 2.5 μM PEITC (or 0.01% DMSO vehicle-only) for a total duration of 6 weeks. In some experiments, the cells were subjected to various doses of PEITC (0–20 μM) for the indicated times. The pEGFP-BMI-1 plasmid was generated by insertion of the full-length human 3 cells/well seeded into 96-well plates and then incubated with various concentrations of PEITC for the indicated times. For colony formation assays, SW620 cells were treated with 2.5 μM PEITC (or DMSO only) for 6 weeks before being transferred into a 6-well plate and cultured for a further 21 days in the presence or absence of 2.5 μM of PEITC. Colony formation was then quantified by crystal violet staining and dye solubilization in DMSO prior to absorbance measurement using a microplate reader.Cell viability was determined by MTT assay using 5 × 106 SW620-CON (n = 6) or SW620-PEITC (n = 6) cancer cells into either the left or right flank of NCr nude mice and monitored tumor size on both sides of animals with caliper. The tumor size reached 1.5 cm in diameter, the mice were euthanized as per guideline.All animal studies were approved by an Institutional Animal Care and Use Committee  and were performed in accordance with approved guidelines and regulations of the Animal Facility Center of School of Biological Scienecs, Nanyang Technological University, Singapore. NCr nude mice were obtained from InVivos, Pte. Ltd. (Singapore). We performed subcutaneous (s.c.) injection of 1 × 10SW620-CON and SW620-PEITC cells were subjected to partial MNase treatment to enable differential extraction of euchromatin- and heterochromatin-associated proteins as described previouslyThe fractionated peptides were separated and analyzed using a Dionex Ultimate 3000 RSLCnano system coupled to a Q Exactive instrument . Separation was performed on a Dionex EASY-Spray 75 μm × 10 cm column packed with PepMap C18 3 μm, 100 Å (Thermo Fisher Scientific) using solvent A (0.1% formic acid in 5% ACN) and solvent B (0.1% formic acid in 90% ACN) at flow rate of 300 nL/min with a 60 min gradient. Peptides were then analyzed on a Q Exactive apparatus with an EASY nanospray source (Thermo Fisher Scientific) at an electrospray potential of 1.5 kV. A full MS scan  was acquired at a resolution of 70,000 at m/z 200 and a maximum ion accumulation time of 100 ms. Dynamic exclusion was set as 15 s. The resolution of the higher energy collisional dissociation (HCD) spectra was set to 17,500 at m/z 200. The automatic gain control (AGC) settings of the full MS scan and the MS2 scan were 3E6 and 2E5, respectively. The 10 most intense ions above the 2,000 count threshold were selected for fragmentation in HCD, with a maximum ion accumulation time of 100 ms. An isolation width of 2 was used for MS2. Single and unassigned charged ions were excluded from MS/MS. For HCD, the normalized collision energy was set to 28%. The underfill ratio was defined as 0.2%. Raw data files from the three technical replicates were processed and searched using Proteome Discoverer 1.2 (Thermo Fisher Scientific). The raw LC-MS/MS data files were loaded into Spectrum Files (default parameters set in Spectrum Selector) and TMT 6-plex was selected for the Reporter Ion Quantifier. The SEQUEST algorithm was then used for data searching to identify proteins using the following parameters; missed cleavage of two; dynamic modifications were oxidation (+15.995 Da) (M), deamidation (+0.984 Da) (N) and phosphorylation (+79.9966 Da) . The static modifications were TMT-6plex (+229.163 Da) (any N-terminus and K) and Carbamidomethyl (+57 Da) (C). The false discovery rate for protein identification was <1%. The ‘Normalize on Protein Median’ function was used for protein quantitation, and fold-change log2 value > |0.5| was defined as differential expression.Cells were washed in ice-cold PBS and lysed in modified RIPA buffer . Lysates were clarified by centrifugation  and subjected to western blotting using the indicated primary antibodies at 1:1000 dilution. Protein-antibody conjugates were visualized using a chemiluminescent detection kit (ThermoScientific). Antibodies against BMI-1, SUZ12, EZH2, Ring1A, Ring1B, and DNMT1 were purchased from Cell Signaling Technologies .RNA extraction was performed using Nucleospin RNA kits (MACHEREY-NAGEL GmbH & Co.) according to the manufacturer’s protocol. Quantitative real-time PCR was performed by CFX96 Real-Time PCR Detection System (Bio-Rad) with KAPA SYBR® FAST qPCR Master Mix. Actin or 18sRNA were used as internal controls. The primer sequences used for real-time PCR are provided in the 3 cells/well) were suspended in culture medium containing 0.3% agar  and plated over a lower layer of 0.7% agar-medium mixture. Viable colonies > 0.5 mm diameter were counted after 14 days culture.Colon cancer cells  according to manufacturer’s instructions and then hybridized to Illumina Infinium HumanMethylation450 (HM450) BeadChips . The chips were scanned with Illumina iScan and the data extracted using the methylumi package in R on Bioconductor. Methylation intensity was reported as β-values [β = intensity of the methylated allele/]. Probes were regarded as ‘failed’ when there was no significant difference from background noise after background correction (P > 0.01).Differentially methylated regions that overlapped with known PcG target genes were validated using MeDIP followed by PCR analysis. Enrichment of methylated DNA was carried out using anti-5-methylcytosine (5-mC) antibody . Briefly, genomic DNA was sheared by sonication to obtain fragments between 200–800 bp and the methylated DNA captured using a modified antibody immunoprecipitation protocol. Final eluates were obtained by re-suspending the protein A/G-coupled Sepharose beads in a 200 μL of TE buffer containing 2 μL proteinase K (20 mg/ml) and then subsequently incubated at 55 °C with 800 rpm agitation for 90 min. Methyl enrichment in the samples was confirmed by PCR analysis. Fully methylated DNA and completely unmethylated DNA samples  were used as experimental controls. After amplification, products were resolved on 2% agarose gels containing ethidium bromide and then visualized under UV transillumination.AP-1, CRE, and NFkB SEAP reporter vectors were purchased from Clontech . SW620-CON and SW620-PEITC cells were transfected with 200 ng of the indicated SEAP vector and incubated for 24 h. The culture supernatant was collected, heated at 65 °C for 30 min, and then assayed for alkaline phosphatase activity as follows; 30 μl of supernatant was incubated with 120 μl of assay buffer for 5 min, at which time CSPD substrate (1:20 dilution) was added and the samples were read on a TECAN microplate reader The DNA content of cultured cells was quantitated by staining with propidium iodide (PI) and analysed by flow cytometry . Briefly, cells were harvested with PBS and fixed with cold 70% ethanol at 4 °C for 30 min. The cells were washed with PBS and then resuspended in 500 μl of PI staining solution and incubated for 30 min at room temperature. Samples were then examined and analysed for cell cycle phase .Statistical analysis was done using SPSS18 statistical software . Statistical differences between variables were determined by student’s t-test and p < 0.05 was considered.How to cite this article: Park, J. E. et al. Dietary phytochemical PEITC restricts tumor development via modulation of epigenetic writers and erasers. Sci. Rep.7, 40569; doi: 10.1038/srep40569 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The properties of ferroelectric domain walls can significantly differ from those of their parent material. Elucidating their internal structure is essential for the design of advanced devices exploiting nanoscale ferroicity and such localized functional properties. Here, we probe the internal structure of 180° ferroelectric domain walls in lead zirconate titanate (PZT) thin films and lithium tantalate bulk crystals by means of second-harmonic generation microscopy. In both systems, we detect a pronounced second-harmonic signal at the walls. Local polarimetry analysis of this signal combined with numerical modelling reveals the existence of a planar polarization within the walls, with Néel and Bloch-like configurations in PZT and lithium tantalate, respectively. Moreover, we find domain wall chirality reversal at line defects crossing lithium tantalate crystals. Our results demonstrate a clear deviation from the ideal Ising configuration that is traditionally expected in uniaxial ferroelectrics, corroborating recent theoretical predictions of a more complex, often chiral structure. Understanding the structure of domain walls is an important step in developing nanoscale ferroelectric devices. Here, the authors use second harmonic imaging to verify predictions of Bloch and Néel, rather than simple Ising, domain wall structures in lead zirconium titanate and lithium tantalate. Ferroic domain walls (DWs) have recently been shown to present unexpectedly rich and diverse physical properties beyond those of their parent materials. These include the observation of polar walls in otherwise non-polar materials234911In ferroelectrics, the physical properties of DWs are closely related to their internal structure. More specifically, they depend on the local symmetry171819202122232425Theoretical predictions concerning the non-Ising character of ferroelectric DWs are abundant in the recent literature and are largely accepted in the community. Nonetheless, the experimental observation of these predicted Néel and Bloch-type walls remains elusive. A key difficulty is the detection of a contribution arising from an extremely narrow region, since the DW width typically extends only over a few atomic cells. Recently, high-resolution transmission electron microscopy (TEM) has determined the atomic arrangement within ferroelectric domainsNonlinear second-harmonic generation (SHG) microscopy offers a remarkable complementary approach, which not only allows for the non-perturbative observation of ferroelectric domains3334353 thin films (point group symmetry 4 mm). In addition, we investigate a trigonal LiTaO3 bulk crystal (point group symmetry 3 m) as an independent test of our approach. In spite of significant differences between these two systems, a deviation from the idealized Ising configuration is demonstrated in both cases. While we observe Néel-type DWs in tetragonal PbO3, periodically poled LiTaO3 develops mainly Bloch-type walls. Depth-resolved SHG measurements conducted in the LiTaO3 bulk crystal show that the Bloch-type walls are stable across the crystal depth. However, their chirality may change at line defects, in close analogy to Bloch lines in ferromagnetsHere, through local SHG polarimetry measurements and numerical simulations exploiting symmetry considerations, we demonstrate the existence of a planar polarization component within the wall regions separating c-domains in uniaxial ferroelectric systems. This study is conducted mainly on tetragonal PbOY-oriented walls in crystals of the lithium niobate familyet al.0.2Ti0.8O3. Moreover, PbZr0.2Ti0.8O3 is often used as a stand-in system for pure PbTiO3 in which Néel-type DWs are expected19To prove the general applicability of our approach and its ability to identify a possible deviation of ferroelectric DWs from the ideal Ising-type configuration, we focus on nominally uncharged 180° domain DWs in two fundamentally different crystals: the one is a tetragonal lead zirconate titanate thin film, the other a lithium tantalate bulk trigonal crystal. This choice is motivated by theoretical studies predicting the existence of non-Ising walls with an internal structure specific to each system. A Bloch-type configuration has been predicted for 0.2Ti0.8O3 (PZT) layer in which c-domains have been patterned by means of a PFM tip (see Methods section for more details), as can be seen in The internal structure of nominally uncharged 180° ferroelectric DWs is first investigated in a 50 nm-thick tetragonal PbZr3 DWs is not a spurious effect, but a result of the particular internal polarization structure of the DWs.Earlier studies based on observations by means of far field, near field and Cherenkov-type SHG434434464348dij elements of the SHG tensor). Assuming that the optical tensor is isomorphic to the piezoelectric tensor, the same transformation operations which hold for the piezoelectric tensor at a given symmetry also apply to the SHG tensor. Any new or modified tensor corresponding to an arbitrary coordinate system with a specific ferroelectric polarization of the underlying material can thus be deduced using the rotation transformation relations as they are commonly applied to piezoelectric tensorsd0 defined in the crystallographic reference frame of the parent phase. A detailed description of the transformation method as well as the resulting SHG tensors obtained in Néel and Bloch-type DWs is provided as α and ϕ) using the analytic form given in the Methods section , within the (zy)-plane in the case of Néel-type walls, and in the (zx)-plane in the case of Bloch-type walls. The SHG intensity is calculated at each rotation angle assuming a small amount of experimental noise, that is, random fluctuations of the polarization angle (±0.5 rad) around its equilibrium position. x-axis) and vertical (VDWs || y-axis) walls aligned with the cubic crystallographic axis of PZT  coincides with the crystallographic axes ). The SHG images are simulated at different settings of the analyzer and polarizer angles, in the case of Néel-type (α=0° in case of VDWs and α=90° in case of HDWs), while Bloch-type DWs are visible when the analyzer axis is along the walls  simulation of the SHG is obtained by subdividing the DWs into discrete regions, in which the ferroelectric polarization is allowed to rotate, for instance in horizontal DWs  bulk crystals. In this system, c-domains forming either micrometric stripes or hexagons have been produced by means of electric field poling (see Methods section for more details). To suppress spurious surface contributions to the SHG signal, the measurements were performed at a focal distance of 100–300 μm below the top surface. y) in LT, regardless of the fundamental wave polarization. Nevertheless, a small SHG signal is still measurable when the analyzer angle of the SHG polarization is perpendicular to the walls. This result is clarified in the following analysis of the SHG polarimetry based on symmetry arguments, in particular at oblique walls in hexagonal domains.For comparison, the polarimetry analysis of the SHG signal is similarly conducted in 500 μm-thick periodically poled nearly stoichiometric LiTaOY-crystallographic directions  when the SHG polarization is along y-axis (α=0°), while oblique walls (ODW1 and 2) show maximum SHG polarization at +60° with respect to y-axis in the case of ODW1, and at −60° with respect to y-axis in the case of ODW2. As previously explained in the case of PZT, the SHG response depends sensitively on the probed nonlinear optical coefficients of the system. Calculations accounting for the local symmetry at the walls and their orientation are provided for trigonal LT to better clarify the SHG polarimetry response  view in Bloch-type walls may exist in two variants since the ferroelectric polarization between c-domains of opposite sign can rotate either clockwise or counterclockwise across the wall. The absence of mirror symmetry makes these walls chiral. Their chirality combined with its possible reversal by moderate external fields propels Bloch walls to the first position among the most promising active nanofunctional components for memory devices53Nonlinear optical microscopy is used to probe the internal structure of ferroelectric domain boundaries. The non-Ising character of nominally neutral 180° DWs is demonstrated in two different systems. Néel-type DWs are observed in tetragonal PZT thin films and a dominant Bloch-type configuration is evidenced in quasi-stoichiometric LT crystals. This work shows that the combination of local polarimetry SHG and numerical simulations based on symmetry arguments constitutes a valuable tool for mapping polarization components at ferroelectric DWs.3 is determined by the competition between the depolarization and the flexoelectric fields.Little is known so far about the origin of the non-Ising structure of DWs. This deviation could be explained by localized electromechanical coupling. For example, lattice distortions caused by oxygen octahedron rotations in perovskite oxides are capable of exhibiting improper ferroelectricity  due to the coupling of the flexoelectric effect (polarization induced by strain gradient) and rotostriction51We hope that our study will stimulate further experimental and theoretical interest in chiral walls. In particular, the study of strain-gradient variations could reveal exciting patterns and distinct properties that could be useful for the design of nanoferroic devices based on domain walls.0.2Ti0.8O3 (50 nm) films (PZT) are epitaxially grown on SrRuO3(35 nm)-buffered SrTiO3(001) single crystals by means of off-axis radio-frequency magnetron sputtering, following the deposition route described in ref. −) is obtained with a uniform polarization along the z-axis, out of the plane of the film. Domains of opposite polarization separated by nominally neutral 180° DWs are prepared by applying a bias voltage (±10 V) between a conductive PFM tip and the SrRuO3 base electrode. The PFM tip is scanned over a selected area to pattern the desired domains.Tetragonal PbZr3 crystals. Hexagonal bulk domain structures with 180° DWs aligned with the Y-crystallographic axes were fabricated by electric field poling, through the application of 1.8 kV voltage pulses of 80 ms duration via patterned metallic (Ti) electrodes deposited on the +Z faceZ). The individual hexagonal domain shape matched the preferential domain wall orientations observed in the material 2O solution, followed by a thorough ultrasonic cleaning in organic solvents. The same method was used to fabricate 1D arrays of 2 mm-long stripe domains aligned to the Y-axis, with a periodicity of 25 μm along X.We used commercial (Oxide Corp) monodomain 0.5 mm-thick Z-cut 1 mol% MgO-doped nearly stoichiometric LiTaO2O3 laser (Millenium-Tsunami combination), which generates 100 fs pulses with a repetition rate of 80 MHz and a wavelength centreed at 800 nm. The laser beam is directed at normal incidence to the sample, and focused with a × 40 magnification objective lens . The SHG images are obtained by scanning the sample with respect to the incoming beam using computer-controlled stepping motors with a minimum step of 100 nm, and recording the SHG signal at each scan step with a typical exposure time is of 20–40 ms per step at a power of 100–150 mW. The output intensity was spectrally filtered and collected into a photomultiplier box. Polarimetry measurements are performed by recording the images at different polarizer and analyzer angles . See Local SHG measurements are conducted by means of a scanning confocal microscope. The fundamental wave is provided by a Spectra Physics Ti:AlP2ω oscillating with the double frequency. The leading term in the frequency doubling process is the second-order nonlinear optical susceptibility tensor χ(2). The elements of the susceptibility tensor are usually replaced by a contracted d-tensor, following the Voigt notation . P2ω depends on the optical susceptibility tensor (dij elements) and on the electric field of the fundamental wave E as follows:The SHG emission involves the coupling of two incident photons at frequency ω to produce a dipole characterized by a polarization ϕ is the polarization angle of the fundamental wave. The complete polarimetry representation of the SHG polarization as a function of the polarizer and analyzer angles ϕ and α, respectively, is obtained using the Jones formalism:where SHG≈|P2ω|2, provided that the symmetry of the material  is known, and vice versa. By choosing the set of polarizations (ϕ and α angles) properly aligned with respect to the crystal orientation, the elements of the χ(2) tensor can be directly accessed through polarimetry measurements (polar plots). Quantitative evaluations tend to be difficult as they require relative measurements with respect to a reference system  and specific measurement geometry. Nevertheless, the qualitative probe of the χ(2) elements provides valuable information on the symmetry and on the ferroic domain structure.ϕ constant) using the analytic model deduced from SHG polarimetry data (polar plots) were fitted at a given polarization of the fundamental wave (χ(2) SHG tensor is calculated at each rotation angle. The result is then inserted as input parameter in d31=−11.09 pm V–1, d15=−11.09 pm V–1 and d33=−18.34 pm V–1 for tetragonal PZTd31=1.54 pm V–1, d22=0.46 pm V–1 and d33=12.9 pm V–1 in trigonal LTɛ0E0=1 and it is presented in arbitrary units.The 2D simulations of the SHG images were obtained by subdividing the DWs into discrete regions, in which the ferroelectric polarization is allowed to rotate. The The data that support the findings of this study are available from the corresponding author upon reasonable request.How to cite this article: Cherifi-Hertel, S. et al. Non-Ising and chiral ferroelectric domain walls revealed by nonlinear optical microscopy. Nat. Commun.8, 15768 doi: 10.1038/ncomms15768 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes, Supplementary Discussions and Supplementary References3D Simulation of polarimetry SHG in tetragonal PZT3D Simulation of polarimetry SHG in periodically poled lithium tantalate


Here we report that a heavy-load strength trainingbout greatly alters the structure of the membrane network inside the fibres, the tubular(t-) system, causing the loss of its predominantly transverse organization and an increasein vacuolation of its longitudinal tubules across adjacent sarcomeres. The transversetubules and vacuoles displayed distinct Ca2+-handling properties. Botht-system components could take up Ca2+ from the cytoplasm but onlytransverse tubules supported store-operated Ca2+ entry. The retention ofsignificant amounts of Ca2+ within vacuoles provides an effectivemechanism to reduce the total content of Ca2+ within the fibrecytoplasm. We propose this ability can reduce or limit resistance exercise-induced,Ca2+-dependent damage to the fibre by the reduction of[Ca2+]cyto to help maintain fibre viabilityduring the period associated with delayed onset muscle soreness.High-force eccentric exercise results in sustained increases in cytoplasmicCa 2+ that can damage muscles. Here the authors show that the t-tubulesystem remodels into vacuoles that can sequester calcium from the cytoplasm and are notresponsive to store-operated Ca2+ entry, thereby potentially protectingmuscles against elevated [Ca2+].Heavy-load eccentric exercise causes an increase in cytoplasmicCa These types of exercise involve eccentriccontractions, where the muscles lengthen while tension is developed. An eccentric workload cancause muscle damage and induce soreness in the days following exercise, commonly referred toas delayed onset muscle soreness (DOMS). The type of damage observed is structural damage tosarcomeres, increased permeability of the plasmalemma and reduced efficiency of theCa2+ entry into the muscle, which increases the basal level ofcytoplasmic [Ca2+]([Ca2+]cyto) to activate calpains232+ may enter the muscle through non-specificpathways in the permeant plasmalemma, an event that occurs presumably post-exercise. Duringexercise, Ca2+ entry is excitation-dependent. Gissel and Clausen52+ imaging experiments have confirmed that there is an actionpotential-activated Ca2+ current, which is tightly associated withindividual action potentialsA major contributor to the damage seen in muscle fibres following eccentric contractions isdue to CaIn human muscle, eccentric contraction causes a significant increase in the muscle calciumcontent, depending upon the exercise and the duration of the exercise89112+in response to action potentials to raise [Ca2+]cytoseveral-fold. Transverse tubules also exchange Ca2+ with the cytoplasm viaNa+–Ca2+ exchangers (NCX) and the plasmamembrane CaATPase (PMCA) to support Ca2+ uptake from the cytoplasm2+ entry  muscleactions, which produce muscle damage and DOMS. Muscle soreness and increases in blood creatinekinase activity in the days post-exercise was reported2+ from the cytoplasm and therefore lower the calcium content of themuscle during the period of time associated with DOMSTo determine whether vacuolization of the t-system provides a ‘safety net’ thatsequesters calcium while the muscle recovers from a bout of eccentric contractions, weemployed recently developed techniques to reconstruct the three-dimensional (3D) structure oft-system networkvastus lateralis to describe the structure of the t-system before and afterheavy-load resistance exercise. We also determine the Ca2+-handlingproperties of the t-system in human skeletal muscle fibres, delineating the properties ofthe transverse tubules and vacuoles. It is accepted that a muscle biopsy from a small regionof the large v. lateralis muscle is representative of that muscle for biochemical andphysiological determinations  properties ofthe transverse tubules and vacuoles of the t-system of human skeletal muscle, we conducteda series of multi-compartment fast Ca2+ tracking experiments withfluorescence optical sectioning. For this, resting muscle biopsies were obtained fromhealthy, recreationally active men and women (n=7). Rhod-5 N trappedin the t-system of mechanically skinned fibres from biopsies was continuously imaged inxyt mode during changes in internal bathing solutions that induced a unidirectional fluxof Ca2+ across the t-system membrane. A unidirectional flux wasgenerated by either depleting the SR of Ca2+ with caffeine (to induceSOCE) or applying a known [Ca2+]cyto underotherwise resting ionic conditions (to induce Ca2+ uptake by theCa2+-depleted t-system). The t-system rhod-5N signal (t) wasconverted to [Ca2+]t-sys (t) using arecently established method2+]t-sys (t) during application ofcaffeine or [Ca2+]cyto is displayed in 2+]t-sys corresponding to points within thetransient in a are shown in b. The spatial resolution of the image is significantly lowerthan the images in 2+]t-sys. Fromthe seven biopsies, a total of 27 fibres were analysed and for each fibre displaying aregular t-system , the steady state[Ca2+]t-sys, peak t-systemCa2+ uptake flux, peak store-dependent Ca2+ fluxand myosin heavy chain isoform were determined .However, the application of [Ca2+]cyto at1.3 μM caused the t-system to change its structure over the course of the next∼100 s  the vacuoles cannot conduct SOCE; and (ii) the luminal connectionbetween vacuoles and transverse tubules must be significantly restricted as vacuoleCa2+ is not lost to the cytoplasm via diffusion into the transversetubules and exit through the chronically open store-dependent channels.The response of the partially vacuolated t-system to store-depletion is shown in theimage marked 587 s in 2+-dependent fluorescence signal from the t-systemprogressively increases over the ∼200 s in 1.3 μM[Ca2+]cyto and the restricted access betweenthe lumen of the transverse tubules and vacuoles indicates that Ca2+was being continuously sequestered from the cytoplasm across the vacuole membrane. We canconclude from these observations that the absolute amount of Ca2+ heldby the t-system in the presence of vacuoles is significantly increased, probably by afactor close to the increase in volume of the t-system under vacuolation  during SRCa2+ release and during exposure of the fibres to different[Ca2+]cyto to assess theCa2+-handling properties of vacuoles. In largely vacuolated fibresthe transverse tubules could not be discriminated in confocal images . 2+]vac (t) during:Ca2+ release from SR; and exposure to different[Ca2+]cyto. The release ofCa2+ from the SR induced a spike in[Ca2+]vac (t). The amplitude of thespike was greater following the period where the fibre was bathed in higher[Ca2+]cyto  was lowered by theactivation of SOCE following chronic SR Ca2+ release (2+]vac (t) couldslowly increase under resting conditions when the[Ca2+]cyto was raised above normal restinglevels  can be estimated as the Bt-sys ×[Ca2+]t-sys × t-sysVol  the vacuoles depolarized, causing a more favourable electrochemical gradientfor the vacuolar accumulation of Ca2+.The absolute amount of Cat-sysVol , where Be volume . The ince volume  was rest2+]cyto. The absence of change in the% fibre volume occupied by the vacuoles between 24 and 48 h post-exercise the transverse tubules canrespond to store-depletion and increases in[Ca2+]cyto va from eccentric exercise2+-handling proteins of the vacuole membrane are likely to be theNCX and PMCA that have previously been described to translocate Ca2+into the skeletal muscle t-system4546In addition to the apparent passive redistribution of Caobserved . Vacuole release , even wh release . We note2+-uptake ability ofvacuoles causes a net shift of Ca2+ from the intracellular environmentof the fibres to the vacuolar lumen. Our estimation of an increase in total calcium contentof the t-system upon vacuolation may be a low one because it does not include thepossibility that diffusible Ca2+-buffers may occupy the vacuoles toincrease the Ca2+-buffering power of the t-system (2+ accumulation. The vacuolated t-systemsequesters (conservatively) 8–9% of the total fibre calcium  from the oxidative fibre types (type I and IIa) of rodents18The change in Caeriphery . Thevact-system  at the etraction , 3, 4, 5yto ref. or the mrst time , 4, 5. Wtraditional for strength training with respect to containing multiple sets,combining concentric and eccentric contractions of the lower body  andcreatine kinase activity≤48 h post-exercise , suggesting muscle damage occurredn=5, 18–42 years old) and women . The fibres collected from this group were used to assess theCa2+-handling properties of the t-system. All procedures wereapproved by the University of Queensland Human Ethics Committee and informed consent wasreceived from all participants prior to commencement of their involvement in thisstudy.All participants who undertook exercise bouts were men between the ages of 22 and 26years with over 12 months of strength training experience. Exercise bouts were−1 with adrenalin,5 μg ml−1) from the mid-portion of the vastuslateralis muscle, using a 6-mm Bergstrom biopsy needle modified for manualsuctionMuscle biopsies were collected under local anaesthesia , 0.05;and HEPES, 10 (pH adjusted to 7.4 with NaOH). Fibres were allowed>10 min toequilibrate with the physiological solution and then individual fibres were isolated andmechanically skinned. Skinned fibres with t-system-trapped fluorescent dye were mounted ona custom-made chamber that used a coverslip as a base and bathed in a standard internalsolution, which contained (mM): EGTA, 50; Hepes, 10; K+, 126;Na+, 36; ATP; 8; Mg2+, 1; creatine phosphate,10; Ca2+, 6.7 × 10−4.[Ca2+] in the standard internal solution was varied in therange 28 nM to 1.3 μM. Ca2+ was released from the SRusing a similar solution in the nominal absence of Ca2+,0.01 mM Mg2+ and 30 mM caffeine. Fibres with t-systemtrapped rhod-5N, rhod-5N fluorescence and[Ca2+]t-sys were calibrated by permeabilizingthe t-system to Ca2+ with ionophore and consecutively introducingsolutions containing 5 mM and 0 Ca2+ to remove blood and external fluid. The muscle tissue was then placed in a Petri dishunder a layer of paraffin oil54a2+ ref. . Fibres 2+ movements across the t-system membrane images were continuousrecorded in xyt mode with an aspect ratio of 256 × 512, with the long aspect of theimage parallel with that of the preparation. Temporal resolution of imaging in this modewhere the fluorescence signal emanated from within the borders of the fibre was0.8 s.Mounted skinned fibres were imaged using an Olympus FV1000 confocal microscope equippedwith an Olympus 1.3NA 63x Plan-Apochromat objective. Fluo-5N and rhod-5N dyes were excitedwith a 488 nm Ar-ion laser and 543 nm HeNe laser, respectively, and theemission was filtered using the Olympus spectra detector. In 3D imaging for reconstructingthe fibre structure, the pinhole was adjusted to 0.7 airy units and each confocal sectionwas averaged over 2–4 sweeps. Both the zoom and z-stepping were set to achieve avoxel size finer than 90 nm in x and y and 150 nm in z. All images wererecorded onto 640 × 640 pixel 16-bit TIFF format images. For trackingCaImage analysis for 3D imaging. Confocal z-stacks were subject to a Richardson-Lucymaximum-likelihood iterative deconvolution with the primary objective of reducing noise inthe image but with an additional benefit of improved contrast and the effective resolutionas detailed previously17Image analysis for Ca measurements. t-system rhod-5N fluorescence (t)(F (t)) was collected during continuous xyt imaging during multipleinternal solution changes. At the end of the experiment each fibre was exposed toionophore and 5 mM Ca2+, followed by 0 Ca2+to obtain the fluorescence maximum (Fmax) and minimum (Fmin), respectively.These values were used in conjunction with the previously determined KDof rhod-5N in the t-system of 0.8 mM =kD,Ca ×(F(t)−Fmin)/(Fmax−F(t)). mM ref.  todeter2+-handling wasperformed blinded, that is without knowledge of type of exercise or fibre type.Note that image analysis for t-system structure and CaData are presented as mean±s.e.m. Statistical analysis was performed with GraphPad Prism. All data are available from the authors, on request.The data that support the findings of this study are available from the correspondingauthor upon reasonable request.How to cite this article: Cully, T. R. et al. Human skeletal muscleplasmalemma alters its structure to change its Ca2+-handling followingheavy-load resistance exercise. Nat. Commun.8, 14266 doi: 10.1038/ncomms14266 (2017).Publisher’s note: Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Methods and SupplementaryReferences


Based on recent observations, we propose that the Ca2+ crosstalk between mitochondria, endoplasmic reticulum and cytoplasm may form a proportional–integral–derivative (PID) controller. This PID mechanism (which is well known in engineering) could be responsible for computing cellular decisions. In addition, we point out the importance of analogue and digital signal processing in T cell life and implication of mitochondrial Ca2+ transport in this process.In this article, we review how mitochondrial Ca Initiated by the triggering of T cell receptor (TCR) at the plasma membrane, activation propagates into the nucleus, resulting in transcriptional changes. Obviously, the amplitude and duration of T cell activation should be tightly controlled owing to undesirable outcomes of under- or overactivation, such as immunodeficiency or autoimmunity, respectively. Furthermore, T lymphocytes should respond adequately to the type of pathogen  and to its dose. Finally, T cell memory serves to fight the repeated pathogen encounters and needs remodelling of metabolic processes to meet re-activation requirements.Studies of the last 10–15 years shed light on metabolic reprogramming in T cells. Currently, it is well accepted that resting T cells mostly use oxidative phosphorylation and beta-oxidation to retrieve energy from nutrients. However, upon activation, T cells switch from respiration to more active glucose and glutamine uptake, as well as aerobic glycolysis . The lat2+ uptake/uniport (mtCU). Although the existence of a process of Ca2+ inward transport  was known for at least 50 years, the pore-forming and the Ca2+ sensor regulatory subunits of the mtCU, mitochondrial Ca2+ uniporter (MCUa) and mitochondrial Ca2+ uptake 1 (MICU1), respectively, have been described for the first time only a few years ago [+/Ca2+ exchanger 1) was identified as a protein molecule responsible for Na+/Ca2+ exchange, a process that balances mtCU-induced Ca2+ accumulation and prevents matrix Ca2+ overload, which could potentially lead to apoptosis [2+ influx and efflux in and out of mitochondria, other pathways have been reported to play a role in these exchange processes [Recent data in mitochondrial biology allow us to consider mitochondria simply as cellular energetic stations, but also as important signalling hubs. Indeed, first results in this field came from findings that mitochondria produce reactive oxygen species (ROS) and communicate with the nucleus via oxidative modification of proteins including transcription factors –8. Anothears ago –12. Currears ago –17. Finapoptosis ,19. Althrocesses .2+ transfer between the endoplasmic reticulum (ER) and mitochondria that is mediated by molecular bridges (2+ accumulation: mtCU has an intrinsic low affinity (i.e. mitochondria import Ca2+ only after significant Ca2+ elevations around 3–10 µM), whereas most of global cellular Ca2+ spikes do not reach this level [2+ domains with ion concentration of 10 µm and above [2+ elevations are transmitted to mitochondria [Further studies in mitochondrial biology progressed towards the understanding of interorganelle interactions, in particular Ca bridges . Major pis level ,22–24. Tnd above –27. Thuschondria ,29 , acetate, bicarbonate, etc. [2+ until reaching the buffer capacity; after this point, Ca2+ phosphates trigger mPTP opening [2+ increases. If rapid and submicromolar concentrations of cytosolic Ca2+ elevations induce fast accumulation of free matrix Ca2+ (mCU1 mode), which upregulates electron-transport chain , then sustained mtCU activation (mCU2) leads to the expansion of the compartment of matrix Ca2+ sequestered by phosphate buffer [Mitochondria are able to accumulate large amounts of Cate, etc. ,31. Mito opening . mtCU an2+, as this ion stimulates the tricarbon acid (TCA) cycle in the mitochondrial matrix, leading to an increase in proton pumping, ATP synthesis and ROS production [2+ elevation and ROS production for mitochondria–nucleus communication [2+ transport and mitochondria could also play a key role in the central regulation of T cell fate. In addition, we try to understand why activated T lymphocytes use mitochondria for cell decisions, in particular from the perspective of Ca2+ transport.How might all these molecular mechanisms be related to T cell functions, activation and fate? The most obvious hypothesis is that mitochondria participate in the supply of ATP in activated T cells, and in turn in ATP-dependent transport processes in immune synapse, where the majority of signalling events are triggered ,34. Secooduction ,43 , IP3R and Orai1 channel subunits mediating store-operated calcium entry (SOCE), are coordinately associated with MCU expression; downregulation of these proteins in activated T cells leads to a compromised upregulation of the MCU mediated by the CREB transcriptional factor [2+ turnover has been shown for the Jurkat T cell line: dissipation of mitochondrial membrane potential (ΔΨm), the driving force for mtCU, causes early Ca2+ release-activated channel (CRAC) inactivation (discussed below), Ca2+ signal reduction and decreased nuclear import of NFAT [2+ response after their activation, which is proposed to be triggered by upregulated nitric oxide-dependent mitochondrial biogenesis [2+ uptake and affecting T cell activation and death [+ T cells lacking mitochondrial tumour suppressor Fus1/Tusc2 with a potential Ca2+-binding domain , upregulation of NFAT- and NFκB-driven genes after activation was diminished [2+ stimulate protein kinase D, which in turn activates DRAK2, thus negatively regulating the influx of external Ca2+ [2+ crosstalk, new linkers with profound effect on T cell responses have been identified. In T cells, transglutaminase 2 (TG2) binds RAP1, the guanine nucleotides exchange factor of small GTPases, which enhances ER Ca2+ release and induces mitochondrial Ca2+ accumulation [+ T cell activation and memory cell formation in vivo [in vitro [2+ concentration and to maintain membrane Ca2+ currents via SOCE, thus favouring T cell survival [2+ accumulation triggers ATP synthesis and secretion via panx1 transporter in the activated Jurkat and human CD4+ T cells, whereas secreted ATP activates P2 purinergic receptors involved in the maintenance of intracellular Ca2+ elevation [2+ in T cell life and fate.The importance of the mtCU in physiology and pathology of T cells is demonstrated by several different examples. Components of Cal factor . The sig of NFAT . Apparenogenesis ,47. Protnd death . Furtherminished . An intenal Ca2+ . Considemulation . The abs in vivo ; in Jurkin vitro . Anotherin vitro . Mitochosurvival . Finallylevation . These e2+/calmodulin (CaM) kinase I alpha phosphorylates DRP1, leading to its binding with Fis1 and mitochondrial fission [2+-dependent proteins, Miro 1 and 2 possessing Ca2+-binding domains called EF-hands, promote mitochondrial fragmentation after interaction with Ca2+ ions [2+ should affect the processes of mitochondrial dynamics similar to other Ca2+-dependent processes such as activation of PKB/Akt [However, the significance of mitochondria in T cell activation and homeostasis will not be fully appreciated without quoting mitochondrial dynamics, a process of mitochondrial remodelling via fusion, fission, autophagy and movement . These p fission . Other Ca2+ ions . In T cea2+ ions . Upon sta2+ ions . It is na2+ ions . Obvious PKB/Akt  and NADP PKB/Akt .2+-dependent phosphatase calcineurin (CaN) [2+ signals including mtCU would coordinate mitochondrial dynamics with metabolic demands and execute metabolic reprogramming.Interesting connections between mitochondrial dynamics, respiration and metabolic reprograming have been recently described for T cells facing the choice between effector (Te) and memory (Tm) phenotypes. In particular, it was demonstrated that downregulation of DRP1 function leads to mitochondrial fusion-favouring activation of respiration over aerobic glycolysis in Tm cells, whereas the opposite effect was observed in Te lymphocytes . It is nin (CaN) . In this2+ dynamics in T cells after their activation. Upon identical stimulation, Th1 lymphocytes display higher mtCU activity compared with Th2 [2+ clearance [2+ dynamics have been found. Th1 cells display high-amplitude elevations and multiple oscillations after TCR activation, whereas Th2 cells exhibit only a few post-stimulation oscillations and fast recovery to baseline, albeit a significant initial rise in Ca2+ level; Th17 lymphocytes show an intermediate pattern with Ca2+ response amplitude higher than Th2 but lower than Th1 and Th1-type oscillations [2+ and NCX are required to sustain late NFAT accumulation during activation of CD4+ T cells (see below for more details) [Th polarization is another process that correlates with Cawith Th2 . Accordilearance . Finallyllations . During details) .3.2+ transport should be tightly associated with its biochemical and biophysical properties. As mentioned above, the mtCU has a rather low affinity for Ca2+ ions, thus setting a high threshold for Ca2+ signals in mitochondria [2+ signals are not transmitted to—nor affected by—mitochondria [figure 2a). From a molecular perspective, this threshold is related to the expression of the MICU1 protein as in its absence mitochondria accumulate Ca2+ already at submicromolar content [2+ uptake at higher Ca2+ concentration by preventing MCU self-inhibition [figure 2b). De la Fuente et al. [2+ response in mitochondria in time-consuming mode may result in their unresponsiveness to small amplitude and fast Ca2+ changes; downregulation of MICU1 would ‘give a green light’ for Ca2+ signals with these characteristics to be transmitted to mitochondria [2+ dependence of mtCU is biphasic: Ca2+ release from ER via IP3R triggers mtCU activation, whereas sustained cytosolic Ca2+ response suppresses mtCU [2+-binding EF-hand motif similar to MICU1 protein [2+ levels than wild-type cells [figure 2b).Obviously, the physiological and pathological function of mitochondrial Cachondria ,22,24,36chondria   mechanisms to orchestrate Ca2+ oscillations [2+ release [2+ sensor called stromal interaction molecule 1 (STIM1) containing EF-hand domain facing intraluminal space. The Ca2+ drop in the ER causes conformational alterations in STIM1 and its oligomerization, resulting in its interaction with the plasmalemmal Orai1 protein, the pore-forming subunit of CRAC, followed by the opening of Orai1 and Ca2+ influx into the cell [2+ release from ER store: after reaching the threshold level, locally increased Ca2+ sensitizes IP3R and RyR to opening and propagation of Ca2+ waves along the ER [2+ domains with high Ca2+ concentration, which can autoinhibit channels [2+ concentration [2+ ATPases contribute to decay phase of oscillations and terminate Ca2+ signal; lower affinity of Ca2+ ATPases to Ca2+ and slower rates, compared with IP3R and RyR, allow them to enter the process of Ca2+ signal propagation later than Ca2+ channels, thus allowing Ca2+ response to develop [To better understand how mitochondria shape intracellular Callations –79. Liga release  .A mathematical model shows that the optimal distance between the ER and mitochondria should be 30–85 nm; when it is less, mitochondria prevent positive feedback of low Ca current ,87. Finaing CICR ,89. It illations ,89,90. F B cells . Collect2+ translate into transcriptional changes based on frequency, amplitude and spatial decoding of Ca2+ signals [2+ oscillations: NFAT requires more frequent oscillations (approx. every 6 min) to maintain a transcriptionally active state, whereas NFκB needs less frequent fluctuations (every 30 min) [2+ activates the CaM-dependent phosphatase CaN, leading to dephosphorylation of inactive NFAT followed by its translocation into the nucleus, whereas re-phosphorylation of NFAT promotes its return into the cytosolic compartment. Thus, rapid periodic oscillations are required for NFAT activation state via maintenance of its dephosphorylated state [2+-induced proteolysis of its inhibitor, IkB, and relocation of NFκB from the nucleus into the cytosol, which requires binding of NFκB with IkB. Re-synthesis of IkB is counterbalanced by its Ca2+-triggered proteolysis; thereby, Ca2+ fluctuations with a period of 30 min are able to maintain NFκB in the activated state [Oscillations in cytosolic Ca signals ,92,93. C 30 min) . This ised state . At the ed state .2+ signalling, we assume a simple concept formulated in the next way: if mitochondria are used for this process, it means that they meet requirements/criteria for the computational tasks of signal processing they are provided with. However, how do T cells decode the information transferred from the APC to T cells' nuclei, and help in computing cell decisions? To respond to this question, we have to take into consideration specificity of T cell activation (i.e. how T lymphocytes distinguish specific antigenic peptides from non-specific at the levels of information processing and decision-making). Altan-Bonnet & Germain [2+ signals has been demonstrated in another work focused on the signal discrimination by autocorrelation of detrended time series [2+ concentration leading to filtering out non-functional noise from functional data [Coming back to the initial question of why T cells use mitochondria to make decisions involving Ca Germain  showed t Germain . Negativ Germain . This ime series . Autocore series  proposednal data ,97.in vivo for tumour rejection: prolonged activation of TCR leads to sustained nuclear NFAT accumulation and transcription of IFNg gene, whereas short-term transitory stimulation is accompanied by only transitory NFAT nuclear translocation and Egr2 gene upregulation inducing CD8+ T cell tolerance [Thus, cellular systems involved in signal reading should demonstrate specific features such as filtering properties (thresholding) and memory in signal dynamics to compute difference between two time points (series). The importance of signal memory has been demonstrated olerance .2+ signalling from the perspective of information processing. Mitochondria have (i) a threshold for Ca2+ signals  [2+ elevations, which allow to process the incoming information (input) by analysing both the amplitude and the duration of Ca2+ responses (MICU1-mediated block) [2+ dynamics in mitochondria induced in large part by the large Ca2+ buffering capacity of this organelle. Also, (iii) MICU1 removes self-inhibition (negative feedback loop) of mtCU [2+ removal from mitochondria through the mNCX triggers Ca2+ oscillations, after the opening of mtCU and accumulation of mitochondrial Ca2+ (positive feedback loop) [2+ signalling is supported by the fact that MICU1 and Fus1 are both involved in the control of mNCX besides their effect on mtCU [2+ [2+ might be resolved if one considers that mitochondrial Ca2+ transport is a part of a molecular proportional−integral−derivative (PID) controller often used in engineering applications. In the following, we analyse mitochondria from this prospective.At this point, we can already draw some conclusions about the involvement of mitochondria in Ca1, Fus1) ,73–76, ad block) ,99. This of mtCU , and (ivck loop) ,90. The  on mtCU ,73,76. TmtCU [2+ . In turnmtCU [2+ . Such a 4.u(t) defined asu(t) is the controller output, Kpe(t) is the proportional function, e(t) represents the difference (e= SP − PV) between a well-defined set point (SP) and the current value of a given variable . The PID controller computes the output signal based on the sum of the proportional (P), integral (I) and derivative (D) terms (a). The proportional term helps to adapt the process variable proportionally to the error. The derivative term responds to the error depending on how fast the error is approaching zero. Finally, the integral term operates based on the accumulated error and thus set PV proportionally to past errors (a). If the PID controller overshoots the SP, the output signal would oscillate around the SP with increasing, decreasing or constant amplitude, thereby reflecting the system stability [PID controllers are sensitive to the magnitude of input signals and correct it in the course of time according to a controller function ntroller . Error eD) terms a. The prt errors a. If thetability .Figure 2 concentration acts as the reference SP, whereas the signal error (e(t)) is erythropoietin (Epo) secretion induced via HIF1/HIF2 activation by low oxygen content [in vivo. However, what is an integrative component is still not defined although it might be a self-renewal process [2. The difference between current O2 concentration and optimal (set) O2 content defines the amount of Epo secretion [PID controllers are widely used in industry for regulation of temperature, speed, pressure, flow, etc. . The ery content . Early e process . The resecretion . On a moecretion . At the ecretion . Undoubt2+. The PV would be a given value of cytosolic Ca2+, which is continuously adjusted to the SP by the appropriate Ca2+ fluxes between the cytosol and the mitochondria. Which are the processes that might be considered as proportional, integral and derivative terms? Most probably, transmitochondrial Ca2+ transport (mtCU + mNCX) represents the proportional element of PID controller. Mitochondria proportionally increase Ca2+ uptake after an increase in cell Ca2+ load [2+ increase is apparently based on the mtCU cooperativity determined by the presence of two Ca2+-binding sites on the MICU1 protein, the regulatory subunit of MCU [2+ buffers (e.g. Pi) in mitochondrial matrix [2+ uptake, thereby helping mitochondria to take up Ca2+ ions. It is important to note that the rate of dynamic change in ΔΨm is proportional to the rate of gain in Ca2+ uptake [Although speculative, it seems that mitochondria may act as part of a PID controller for T cell activation through the regulation of cellular Caa2+ load . The abit of MCU , and on l matrix ,106. Thel matrix ; in the l matrix . Pi buff2+ concentration). The cytosolic Ca2+ response including Ca2+ oscillations indeed relies on the process of STIM1-dependent oligomerization and activation of the SOCE Ca2+ pathway. In turn, STIM1 oligomerization is proportional to the depletion of the endoplasmic Ca2+ store, which is indirectly controlled by the mtCU through both the MCU and UCP2 [2+ responses directly correlate [2+ are proportional. This effect depends on mtCU activity. Finally, gradual dissipation of ΔΨm, the driving force for Ca2+ exchanges between the cytosol and mitochondria, proportionally diminishes Ca2+ oscillations in mast cell line RBL-1 [b). Indeed, the significance of mNCX for cytosolic Ca2+ oscillations is well documented and based on the ability of the mNCX to fuel the ER store with the amount of Ca2+ ions necessary to trigger CICR and the accompanying Ca2+ oscillations [There is another, more indirect way by which the mtCU produces an output value that is proportional to the current error value . As mentioned above, Ca2+ activates the TCA cycle and production of NAD(P)H [2+ signal triggering the changes in NAD(P)H production [2+ signals leads to a larger NAD(P) reduction even if the decline in NADP(H) from a previous signal still propagates, thereby summarizing responses from few inputs [2+ can leave mitochondria via the mPTP in its low-conductance mode, a process that also shows the characteristics of an integrative element in physiological conditions. Fast rates of repeated Ca2+ stimulation favour low-conductance flickering of the mPTP (permeability < 300 Da) and consequent Ca2+ release [2+ accumulation after repeated cytosolic Ca2+ stimuli.Another term of the mitochondrial PID controller, chondria ,108 (fig NAD(P)H . The redoduction , althougoduction –111. Furw inputs . Thereby release . MPTP op release ,113, twoderivative element of the PID controller anticipates the system behaviour based on the calculation of the slope of the error with respect to time. We propose here that mitochondrial ROS production might represent the D term of the mitochondrial PID controller. Treatment of cells with mitochondria-targeted antioxidants abolishes cytosolic Ca2+ oscillations induced by physiological stimulation [2+ release [2+ release channels is dual: RyR opens at moderate concentrations of ROS but becomes inhibited at high ROS concentration [2+ leading to an increase in the electron flux of the ETC [2+ sparks in a time/history-dependent manner. During the first phase, ROS-sensitized ER channels release Ca2+ (a). Emptying Ca2+ ER store is accompanied by expansion of mitochondrial ROS production. In the second phase, high concentrations of ROS significantly depress Ca2+ sparks [b). Thus, the level of Ca2+ is dynamically regulated by the rate of the mitochondrial ROS changes. Besides, the buffering of ROS in mitochondria is tightly connected to the mitochondrial NADH pool as alterations of only one parameter in the antioxidant system (e.g. superoxide dismutase concentration) can significantly change the amplitude and the frequency of mitochondrial oscillations  [b).The mulation –116. Thi release ,115. It ntration . ROS accntration . Product the ETC . It has ase Ca2+ a. Emptyiillator) ,120,121 2+ signalling, ETC activation and metabolic reprograming  in a manner that depends on the temporal changes in ROS production associated with T cell development.ROS also play a role in the dynamic and concentration-dependent regulation of glucose metabolism that couples glycolysis and ETC fuelling with reducing equivalents. Indeed, short exposure of cells to ROS triggers glucose uptake, its conversion into pyruvate and hyperpolarization of the inner mitochondrial membrane. By contrast, prolonged presentation of ROS leads to downregulation of glucose transport in order to prevent excessive feeding of ETC with electron-donor molecules . Thus, R2+ signal [PID controllers have an important limitation: they have a low capability to minimize noise. In engineering, to remove noise in the system using PID-based control system, low-pass filters  for feedback response are used . In the + signal .2+ signals with high amplitude, then we could expect that mitochondria will be proportionally accumulating Ca2+ with equal emptying of the ER store as the mNCX would provide the IP3R and RyR with enough Ca2+ ions to trigger their opening. That would favour CRAC channels in an active state. Concurrently, Ca2+ response will be tuned out by the amount of ROS and availability of antioxidant buffers. Thus, the resulting response will be directed by the summation of PID controller terms. If the value of any parameter H concentration) is changed, then the PID controller outcome will be adjusted according to these modifications. In our case, the resulting process might involve Ca2+ signalling and Ca2+-dependent signal transduction systems such as NFAT cytosol/nuclear shuttling. For example, during Ca2+ responses, the treatment of cells with pro-oxidant  leads to the CaN inhibition and the dampening of Ca2+-induced NFAT nuclear translocation, whereas the prevention of oxidative stress H production) suppresses this effect [After performing the analogue computational procedures, the PID controller combines outcomes from all three terms  and sends a final (summarized) decision to elements of the driving process . How does effect .2+ oscillations [2+ threshold for mtCU activation might serve as an SP for Ca2+ oscillations. Indeed, Ca2+ oscillations would be maintained until the error would reach zero; in other words, when SP = PV and e = SP − PV = 0. However, while PV is greater than SP, the Ca2+ system will be oscillating owing to the difference between the cytosolic Ca2+ concentration and the threshold for mtCU opening. It is noteworthy that these two parameters are coupled via the mtCU-dependence of STIM1 oligomerization and further interaction with CRAC1, the pore unit of SOCE currents [2+ from the ER store, thus emptying it and allowing the ER Ca2+ level to drop below the Kd for Ca2+ binding to the EF-hand of STIM1, thereby leading to its oligomerization [Before further discussion, we return to the properties of the mtCU and remind that (i) the mtCU acts as a low-pass filter, which has a high threshold ,22–24,99llations . We propcurrents . Mitochorization ,124.2+ oscillations, mitochondrial oscillations, etc.) arise at special points called Hopf bifurcations [e = SP − PV = 0 would be analogous to the bifurcation point between these two states.As mentioned above, if the PID controller overshoots the SP, the output signal will oscillate around the SP . In the rcations ,125,126.e are flexible as cells can use different strategies to shape cellular Ca2+ signals through post-translation or transcriptional regulation of proteins involved in ER/mitochondria Ca2+ crosstalk:2+ currents by depolarization of cellular membrane potential, downregulation of IP3R or RyR, modified efflux of Ca2+ by Ca2+ ATPase (PMCA or SERCA);(1) reduction of Ca2+ , mitochondrial Ca2+ export or expression of its components (e.g. NCLX1), ΔΨm increase or decrease; and(2) changes in sensitivity of the mtCU to Ca(3) alterations in the distance between the ER and mitochondria .How can T cells use these strategies in order to regulate their fate? IP3R-mediated Ca2+ release is important for the initial production of cytokines [+ T cells [2+ exchange [+ channels: voltage-gated Kv1.3 and Ca2+/calmodulin(CaM)-regulated KCa3.1. The Kv1.3 channel has a voltage sensor located in the fourth transmembrane domain containing four arginine residues, whereas the C-terminus of KCa3.1 channel bears intracellular CaM-binding motif and senses Ca2+ elevations [2+ currents owing to a decrease in the driving force for Ca2+ [2+ currents in activated T cells is accompanied by an upregulation of KCa3.1 channels, which hyperpolarizes the membrane potential and maintains CRAC influx [2+ currents compared with Th2, which is translated into stronger and sustained Ca2+ signals in Th1 cells [2+-dependent Na+ channel; opening of Trpm4 leads to depolarization of the membrane potential and reduced Ca2+ oscillation patterns [+, Ca2+ or Na+ channels pursue the goal of fine-tuning Ca2+ responses according to a programme dictated by different steps in T cell life. The role of mitochondria in these processes might be to set the parameters responsible for the spatio-temporal Ca2+ patterns such as oscillations.The SP, PV and ytokines  but not  T cells  where miexchange . It is wevations . Naive Tevations . There ifor Ca2+ . An incrC influx –134. Theh1 cells . In contpatterns . Thus, c5.2+ mobilization  after specific stimulation display incremental characteristics when increasing the amount of MHC/peptide molecules [+ T cell and that additional pMHC molecules do not increase the secretion of effector cytokines (such as IL-2 or TNFa), demonstrating an ‘all or none’ type of response [2+ domains, which cause CaN activation if they reach the threshold Ca2+ concentration; CaN-dependent dephosphorylation of NFAT overcomes another threshold set by the highly phosphorylated state of inactive NFAT [Another important aspect of T lymphocyte life is the transduction of stimuli with different intensity into analogue  or digital (binary) outcomes in order to adequately respond to environmental signals. An early study demonstrated that in T cells, parameters of Caolecules . Howeverresponse . The folresponse . It is nresponse . Moreoveive NFAT . As descive NFAT . At the ive NFAT . In addiive NFAT  and digiive NFAT  modes afa, stimulus I). In the case of EGF, this mechanism is lacking as only a negative feedback loop is present, which implies that the strength of signal transduction is determined exclusively by the amount of ligand  [figure 6b, top). Ras nanocluster formation is digital in its nature, because GTPase molecules start clustering after reaching a given threshold. Below this threshold, Ras signalling does not provide any significant output. The amount of clusters, however, depends on the ligand concentration and displays analogue characteristics when Ras is above the threshold. The signal output then rises proportionally to the input. This process is called signal integration and is necessary for high-fidelity signal transduction when the output (ERK activation) should precisely reflect the input (ligand concentration) signal  [The importance of digital and gradual signals has been demonstrated for the MAPK module activation in PC-12 cells. Endothelial (EGF) and neuronal (NGF) growth factors both activate Erk1/2 MAPK, but with different modalities: gradual for EGF and digital for NGF . Functiofigure 6ulus II) . Anotherulus II) . Ras is  graphs) ,145.Fig2+ waves into the nucleus (most probably from perinuclear mitochondria) leads to strong association of both proteins, preventing NFAT from re-phosphorylation and further Crm1-mediated nuclear export [2+ response, thereby exhibiting graded mode. This inter-relationship is determined by the ability of mitochondria to slowly release Ca2+ via the mNCX after its accumulation during cytosolic elevations in sensory neurons [+ T cells [2+ concentration [2+ response dependence is very steep and close to a switch-like response [2+ responses is very important for its biological effects, because NFAT can trigger different transcription programmes depending on the nuclear lifetime (e.g. activation versus tolerance) [2+ events [2+ release quanta gives graded response depending on agonist concentration at submaximal doses [2+ and release it during the plateau phase following the peak  . The pro+ events ,155 . At the same time, if the level of Z is below threshold parameters for X2, then stimulus S will favour activation of sensor X1 via mediator Z, resulting in activation of pathway Y1 (a). Applying this assumption to mitochondria, mtCU opening and Ca2+ uptake can limit availability of Ca2+ ions to other Ca2+-dependent targets. Thus, Akt/PKB-mediated survival requires the release of mitochondrial Ca2+, and in the absence of mNCX, the apoptotic programme is triggered [2+ responses, which correlate with the requirements for Th1 differentiation [2+ transport would lead to different patterns of Ca2+ signalling, including lack of regenerative Ca2+ oscillations [2+-dependent PKC activation [b). Thus, the threshold for mtCU opening might serve as a switch for rewiring between signalling pathways responsible for different T cell programmes.We now briefly review possible mechanisms related to the involvement of mitochondria in the rewiring of the signalling pathways, in order to switch between the various T cell programmes. To achieve this aim, we need to imagine a hypothetical stimulus S, which in a simple assumption activates two signalling pathways Y1 and Y2 via competing sensors X1 and X2, respectively. Both sensors are specific to the mediator Z, but possess different activation parameters . If the level of Z, reaches the threshold parameters for X2, then the latter will buffer mediator Z and occlude it from interaction with X1, thus keeping pathway Y1 inactive a. At thethway Y1 a. Applyiriggered . Short-triggered . Sustainriggered . As descriggered . It is kriggered –162. Comntiation ,70. At tllations ,90. Indetivation . In turntivation   would change the cellular output. Translating this hypothesis to T cells, we expect multiple remodelling processes in the Ca2+ signalling network during activation, differentiation, apoptosis, etc. Multiple cytokines are crucial players in the regulation of the T cell states and we believe that their role is to provide cells with correct instructions, including how to remodel Ca2+ signalling patterns, in order to respond adequately to modifications of the environment. This view is confirmed by observations that T cell activation is associated with coordinated expression of RyR, IP3R, Orai1 together with MCU expression [2+ signalling in T cells is subjected to remodelling in terms of Ca2+ sources depending on the stage of activation [In this article, we have attempted to characterize mitochondrial Capression , while Ctivation .2+ fluxes as key mediators of mitochondrial control of T cell activation. It does not provide a complete description of the molecular pathways responsible for T cell activation, which involve many other important factors such as nitric oxide, mitochondrial mass, mTOR signalling, etc. The model could be applied to other cell types owing to similarity in the fundamental mechanisms and principles of signalling regulation among different cell types. As an example, decoding of Ca2+ oscillations by cardiomyocytes and hypertrophic response are executed by the Ca2+-sensitive NFAT/CaN system, which can act as an integrator [2+ concentration results in primary salivary gland epithelial cells acquiring an acinar-like phenotype. This process is associated with the remodelling of the Ca2+ signalling machinery: upregulation of Orai1, STIM1, STIM2 and NFAT1, accompanied by an elevation in SOCE [2+ response and the parameters of output of the PID controller that result in the realization of specific programmes. Finally, these conclusions might have not only fundamental interest, but also therapeutic implications, because they would allow us to predict which spots in cellular Ca2+ signalling have the most crucial effect in different pathological states.The present model focuses on the roles of Categrator  in the stegrator . Further in SOCE . Accordi


Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment.Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca For example, numerous experiments have shown that fluoxetine induces an increase in [Ca2+]cyt in immune cells cyt cyt induced by fluoxetine is due to a Ca2+ entry cyt triggered by fluoxetine.Fluoxetine and SSRIs also have reported effects on cytosolic calcium concentration . The initial calcium leak is produced by a direct or indirect inhibition of SERCA activity, since fluoxetine impairs ATP production by inhibiting the respiratory chain. The rise in [Ca2+]cyt resulted in a mitochondrial Ca2+ overload leading to cell death, mainly by necrosis.The purpose of this study was thus to determine the signaling pathway triggered by fluoxetine, leading to a [Ca2+]cyt  channels, constituted of Orai1, a plasma membrane protein, and STIM1, a calcium sensor located in the ER membrane. When Jurkat cells  channels are involved in fluoxetine-induced Ca2+ entry.To determine how extracellular Cas Figure  and 2B, s Figure  and S1B s Figure  and S1D e Figure  and 2D. e Figure  and 2F. 2+ stores. In line with this, depletion of ER calcium with thapsigargin (TG) abolished fluoxetine-induced Ca2+ signals in HeLa cells  domain of PLCδ and eGFP, which is able to bind PIP2 and IP3 and hence allows the monitoring of IP3 localization after PIP2 cleavage, regardless of the PLC isoform . Using dantrolene (an inhibitor of RyR1 and RyR3 isoforms), we ruled out the potential implication of these receptors in fluoxetine-induced increase in [Ca2+]cyt  inhibitor, Ned19. Cells treated with Ned19 did not show significant modification of the fluoxetine-induced Ca2+ increase . When Ps Figure  and 4B. % Figure  and 4D, 2+]cyt increase after a fluoxetine addition, we took a closer look at the mitochondria, in order to determine if Ca2+ could be stored in this compartment. For that purpose, we performed confocal imaging experiments with HeLa cells, using the mitochondrial marker and Ca2+ sensing probe Rhod2, in association with the Mitotracker Green mitochondrial probe. The addition of fluoxetine led to an increase in red fluorescence, with the overlap with the Mitotracker Green signal yielding a yellow color, demonstrating the increase in mitochondrial Ca2+ concentration , we used Ru360, a specific inhibitor of the uniporter. We show that in the presence of Ru360, the fluoxetine-induced increase in [Ca2+]mt was abolished in HeLa cells , one of the most biologically relevant member of the Reactive Oxygen Species  and propidium iodide (PI+) were late apoptotic cells . The original Annexin V / PI plots are shown in We next sought to determine which mechanism was responsible for fluoxetine-induced cell death. Cell death was also induced by i) CD95L, a death ligand known to initiate the death-inducing signaling complex (DISC) upon binding to its receptor, that culminate in the induction of apoptosis ; ii) hydies (ROS ) generaties  are able to induce a rise in intracellular Caphocytes ), in add2+ entry , 34, 35]phocytes . By usin studies , 20, 34]ch as TG , 20 or ALa cells . This efanslocon . In agrechondria . Since foduction .2+]cytincrease involving translocon correlates with cell death in PBLs and Jurkat cells. It is noteworthy that the Jurkat cell line was found to be sensitive to this fluoxetine-induced cell death, contrary to what Gordon's team reported i was measured ratiometrically in Indo-1–loaded cells using a Hitachi F2500 spectrophotometer, as described previously [2+]i was recorded as the F340nm/F380nm fluorescence ratio. The values of the emitted fluorescence for each cell (F) were normalized to the starting fluorescence (F0) and reported as F/F0 (relative [Ca2+]cyt). Each experimental condition was repeated independently at least three times; values are reported as mean ± SD.In cell populations, [Caeviously . Cells b2+]i imaging was performed ratiometrically as described previously [Single-cell [Caeviously . Cells weviously . Imaging2+-free medium, consisting of the HBSS described above in which CaCl2 was omitted and 100μM EGTA was added in order to chelate residual Ca2+ ions. This medium was added to the cells just before recording to avoid intracellular calcium stores leaking.In some experiments, cells were placed in a CaJurkat, PBLs and HeLa cells were loaded with Rhod-2 AM fluorescent probe . Cells were grown on glass coverslips and placed in an Attofluor observation chamber for confocal microscopy . Helium/Neon laser (543 nm) was used for excitation, and emitted fluorescence was recorded through a low-pass filter (560nm). Analysis was made with Zeiss software (Axiovision). Mitochondria were stained using Mitotracker green .2 and a higher affinity for IP3 than for PIP2. It translocates thus from membrane to cytosol with IP3. This system enables thus real time IP3 production in living cells, with migration of green fluorescence (GFP) from the membrane to cytoplasm [A fusion protein between PH domain (Pleckstrin Homology domain) of PLCγ1 and GFP (GFP-PH) was used. This PH domain has a high selectivity for PIPytoplasm . Constru4Na2/K, pH 7.2 and supplemented with inhibitor cocktail (Sigma), 2mM EDTA, 1mM sodium orthovanadate (New England Biolabs), phosphatase inhibitor (Thermo Fisher) and 5mM PMSF . Benzonase nuclease (Santa Cruz) was added to reduce viscosity in protein extracts. Protein content was measured using BCA method (Biorad). Each sample was dissolved in Laemmli 5X buffer, and then denatured at 100°C for 10min. After SDS-PAGE and transfer onto nitrocellulose membrane , membranes were incubated with goat polyclonal primary antibodies against p-AMPk  or AMPk  overnight at 4°C. Membranes were incubated with anti-goat peroxidase-conjugated anti-IgG secondary antibody , developed using ECL substrate solution, exposed to the Fusion Fx7 (Thermo Fisher) and analyzed using Quantity One software (Biorad).Cells were lysed into lysis buffer containing 1% Na deoxycholate (Sigma), 150mM NaCl, 10mM PO2) were determined from the slope of a plot of O2 concentration versus time, and normalized by million cells. Respiration assays were performed in the growth medium supplemented with HEPES 10mM, pH 7.2.Respiration assays were performed using an Oroboros Oxygraph-2k. The oxygen consumption was measured polarographically at 37°C using a Clark oxygen electrode in a thermostatically controlled chamber. Respiratory rates  and images were collected using 440nm excitation and alternate 485/535 nm emission on an Axiovert S100 TV microscope through a ×40, 1.3 NA oil-immersion objective  equipped with a 16-bit CCD camera, Xenon lamp and filter-based wavelength switcher . Minimum FRET emission ratios were obtained by washing cells in Ringer's were glucose was replaced by 2-deoxyglucose and contained 10μg/ml oligomycin. Relative changes in mitochondrial ATP are reported as background-subtracted FRET ratios normalized to baseline and minimum FRET emission ratios where Relative Mito-ATeam FRET = ((F335/485 - Fmin) / Fbaseline). The loss of ATP is reported as Loss ATP = (1 - Relative Mito-ATeam FRET) at t=10min after stimulation. Bars show mean ± SEM of 3 independent experiments.Mitochondrial ATP levels were measured by transfecting HeLa cells with the mitochondrially targeted ATP-sensitive FRET-based probe (Mito-ATeam), as previously described . BrieflyCells were processed using annexin V – propidium-iodide (PI) apoptosis assay kit according to manufacturer's protocol (Life Technologies). A total of 10000 events were analyzed by flow cytometry.The results are expressed as the mean ± standard error of the mean (SEM) of the indicated number of experiments . Statistical analysis was performed using the Student's t-test, a non-parametric Mann–Whitney test, or an ANOVA statistical test (Sigmastat). Differences between the values were considered significant when p < 0.05. * and ** mean p-values smaller than 0.05 and 0.01, respectively.


This study was to assess the accuracy of measuring the tibial and talar components position and to investigate the outlier rate of each component and predisposing factors related to component malalignment after total ankle arthroplasty (TAA) using a conventional method.One hundred fifty consecutive primary total ankle arthroplasty were performed using the three-component HINTEGRA prosthesis for ankle end-stage osteoarthritis. Radiographic analysis for the accuracy of component position in coronal and sagittal plane was conducted at postoperative 6 months. Additionally, the accuracy of component position was evaluated according to presence of preoperative deformity or joint incongruency.The mean postoperative coronal angles of the tibial and talar components (α and γ) were 91.9° and 91.3°. The mean postoperative sagittal angles of the tibial and talar components (β and δ angle) were 84.6° and 91.7°. In the coronal plane, 16 (10.7%) tibial components and 15 (10.0%) talar components showed outliers greater than 5°. In sagittal plane, 15 (10.0%) tibial components and 29 (19.3%) talar components showed outliers greater than 5°. There was no meaningful increase of the outlier rate regarding presence of preoperative deformity or joint incongruency.In conventional method of TAA, the outlier rate of the tibial and talar components was about 10 to 20%, especially, the outlier rate of talar component in sagittal plane was up to 20%. Therefore, careful attention should be paid to implant the talar component in conventional TAA. Total ankle arthroplasty (TAA) has been widely used for the treatment of end-stage ankle arthritis in recent years and satisfactory results have been reported –6. HowevThe success of joint arthroplasty depends on many factors, including patient selection, prosthetic design, soft tissue balancing, severity of joint deformity, and component position –17. AmonPrevious studies have reported radiographic outcomes and predisposing factors that increase the risk of postoperative malalignment after hip or knee joint arthroplasty. The malalignment of component has been reported ranges from 4 to 23% after primary total hip arthroplasty. Severe varus deformity and retroversion of the femur can cause incorrect insertion of the stem , 26. In There are several studies about the accuracy assessment of component position following TAA. The rate of malalignment was varied from 0 to 35% after TAA. However, in these studies, the criteria for acceptable alignment of component was not standardized, and there are no comprehensive studies conducted regarding the outlier rates of the tibial and talar components or predisposing factors of incorrect component position following TAA. Therefore, the purpose of the present study was to evaluate the accuracy of component position after conventional TAA and to analyze the predisposing factors that affect outliers by using radiographic analysis.This study was approved by our institutional review board, and informed consent was obtained from all patients. Between January 2005 and December 2011, 153 consecutive TAAs were performed by a single surgeon in 146 patients (153 ankles) with symptomatic end-stage ankle osteoarthritis using cementless mobile-bearing HINTEGRA prosthesis . Three patients (3 ankles) were excluded because plain radiographs were not available to measure radiographic parameter. The study cohort consisted of 150 ankles  with a mean age of 61.6 years (42 to 82). Ninety (60%) ankles were diagnosed with posttraumatic ankle arthritis, and 60 (40%) ankles were diagnosed with primary ankle osteoarthritis.All patients received total ankle arthroplasty by a single surgeon using a longitudinal anterior approach between the anterior tibial tendon and the extensor hallucis longus with the patient in the supine position. After removal of anterior capsular synovial tissue and osteophytes, the tibial cut was made perpendicular to the mechanical axis of tibia in coronal plane and had a posterior slope of 6° in sagittal plane while sparing as much subchondral bone as possible. The talar cut was made parallel to the tibial cut in coronal plane and parallel to the sole, then the medial, lateral, and finally posterior talar cuts were made. After the selected implants were inserted, the alignment, stability, and joint motion were checked clinically, while component position was checked by image intensification. The wound was closed in a standard fashion and a closed suction drain was inserted.All patients followed the same postoperative protocol. In order to keep the foot in a neural position, a short leg splint and non-weight bearing were necessary for the first 2 weeks after surgery. For the next 4 weeks, patient was instructed to start gentle range of motion (ROM) exercise. Progressive weight-bearing ambulation was initiated with ankle-foot orthosis at 6 weeks after surgery. The full weight-bearing ambulation and rehabilitation program, which included stretching of the triceps surae, calf strengthening, and proprioceptive exercise were allowed to start at 8 to 10 weeks after the surgery. The period of immobilization may differ a little whether additional procedures were performed or not.Radiographic examinations including anteroposterior and lateral radiographs of the ankle taken preoperatively, immediate postoperatively, at 3 and 6 months postoperatively, and annually thereafter. With preoperative radiographic analysis, we conducted radiographic analysis after 6 months of operation when the patients were able to perform weight-bearing exercises. For the radiographic analysis, we measured the following by using PACS (Picture Archiving and Communication Systems: Marotech 5.4).We evaluated the accuracy of component position depending on the type of preoperative coronal deformity: varus, neutral, or valgus. Neutral position in coronal plane was defined as within 5° of varus or valgus on anteroposterior radiograph. On preoperative radiograph, 80 (53.3%) ankles showed coronal varus tilting above 5°, 28 (18.7%) ankles showed valgus tilting above 5°, and 42 (28%) ankles showed coronal tilting below 5° of varus or valgus. Coronal positions of the tibial and talar components were assessed using α and γ angles Fig. .Fig. 1ThAs done with coronal plane analysis, we evaluated the accuracy of component position depending on preoperative sagittal deformity: extension, neutral, and plantar flexion. The aimed value of the anterior distal tibial angle was 84°. Therefore, we defined, a neutral position in sagittal plane as being in the rage of 79° to 89° of the anterior distal tibial angle. On the sagittal plane, 85 (56.7%) ankles showed extension of the distal tibial slope  above 5°, 7 (4.7%) ankles showed plantar flexion of the distal tibial slope  above 5°, and 58 (38.7%) ankles showed sagittal tilting below 5°. Sagittal angular positions of the tibial and talar components were assessed using β and δ angles on postoperative radiographs Fig. .In addition, we evaluated the congruency of the ankle joint. An ankle was defined as congruent when the difference between the tibial and talar articular surface was less than 5°. Accordingly, four categories of deformity were defined: varus-congruent, varus-incongruent, valgus-congruent, and valgus-incongruent.Outcomes were defined as “excellent” when the difference between the postoperative alignment and aimed value was within less than 3°, “acceptable” when more than 3° and less than 5°, and “outlier” when more than 5° from aimed value in the coronal-tibial(α), coronal-talar(γ), sagittal-tibia(β), and sagittal-talar(δ) angle.To avoid potential bias, plain radiographs were evaluated by two independent observers who were not involved in the surgical treatment of the patients and who were blinded to the intention of this study.p value of <0.05 was considered significant, and all aspects of the statistical analysis were reviewed by a statistician.Descriptive statistics  were calculated with use of standard formulas. The one-way analysis of variance (ANOVA) test was used to analyze differences in continuous variables such as α, β, γ, and δ angles among three groups. Tukey’s honestly significant differences (HSD) was performed was used for post-hoc comparisons. The Fisher’s exact test was used to analyze differences in outcomes such as “excellent”, “acceptable,” and “outlier”. A The mean coronal angle of the tibial and talar components  were 91.9° and 91.3° at 6 months postoperatively  were 84.6° and 91.7° at 6 months postoperatively  and 15 (17.7%) ankles in the extension group, 5 (8.6%) and 11 (19.0%) in the neutral group, and 0 (0.0%) and 3 (42.8%) in the flexion group, respectively. However, there were no meaningful differences between the 3 groups in the accuracy of component position according to preoperative deformity in coronal and sagittal plane.In addition, presence of joint incongruency did not increase the outlier rate Table .Table 3AThe most important finding of the present study is that the outlier rate of component was higher than we expected, and the outlier rate of each component in coronal plane is about 10%, but the talar component position in sagittal plane showed the outlier rate up to 20%. Our results highlight a common problem in TAA, which is the lack of repeatability in implantation position, particularly the talar component. Malalignment of component following TAA has been reported in about 0 to 35% of patients, even though components were thought to be appropriately placed at the time of the operation , 30. TheHowever, there was no comprehensive study concerning the outlier rate of each component or predisposing factors by assessing the accuracy of component after TAA. The present study indicate that outliers rate of the tibial, talar component in coronal plane, and tibial component in sagittal plane showed only about 10.7, 10, and 10%, of cases, but outliers rate of the talar component in sagittal plane occurred in 19.3%. This result suggests that the talar component position intraoperatively is much more difficult than the tibial component position.There are several factors which can affect alignment of the talar component. Firstly, in operative techniques, the component position in the sagittal plane is a very subjective approach and it concerns different types of ankle prosthesis. In this study, distal tibia cutting block was used as a guide for resection of talar dome. So, the talar component position was affected by distal tibial cutting plane. Such problems may be solved by applying separate talar cutting block without considering the tibia in the process, but this is not easy intraoperatively because there is no distinct landmark and anatomy of talus varies much individually. In addition, stabilizing the foot during talar cutting can only be done manually. Therefore, the talar component position tends to vary and easy to be influenced depending upon foot position, particularly in sagittal alignment. Barg and Lundberg et al. , 35 alsoBased on our results concerning component position according to the preoperative coronal deformity, varus ankles showed a lower rate of excellent outcomes and a higher rate of outlier than neutral or valgus ankles. Previous studies have reported that for varus ankles with ligament imbalance and asymmetric joint loading. Wood and Haskel , 36 repoOur study has some limitations. First, the sample size in neutral and valgus group was relatively small. This may limit our ability to assess outcomes and may evaluate risk factors. Second, we retrospectively reviewed preoperative radiographs, and thus, radiographs were not precisely controlled. It is possible that slight variability of ankle position and rotation influenced assessments the alignment of implant. Finally, this study did not evaluate functional outcomes, which may have been influenced by the component position. Therefore, further studies involving these functional outcomes are required.In present study, the outlier rate of components was high as 10 to 20%, especially, the talar component position in sagittal plane showed the outlier rate up to 20% and wider variation. However, presence of preoperative deformity or joint incongruency did not increase the outlier rate of component. Therefore, careful attention should be paid when implanting the talar component in sagittal plane and other reliable instrumentation system is necessary to improve the accuracy of component.