We present here a straightforward, broadly applicable technique for real-time detection and measurement of protein conformational changes in solution. This method is based on tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers. We demonstrate our method by measuring the conformational changes that occur upon ligand binding with three well-characterized proteins labeled at lysine residues: calmodulin (CaM), maltose-binding protein (MBP), and dihydrofolate reductase (DHFR). We also create a single-site cysteine mutant of DHFR engineered within the Met20 catalytic loop region and study the protein’s structural motion at this site. Using published x-ray crystal structures, we show that the changes in the SHG signals upon ligand binding are the result of structural motions that occur at the labeled sites between the apo and ligand-bound forms of the proteins, which are easily distinguished from each other. In addition, we demonstrate that different magnitudes of the SHG signal changes are due to different and specific ligand-induced conformational changes. Taken together, these data illustrate the potential of the SHG approach for detecting and measuring protein conformational changes for a wide range of biological applications. The relationship between protein structure and function has long been recognized as fundamental for understanding biological mechanisms. Far from being static in structure, proteins are dynamic molecules that are capable of changing their shape, or conformation, in response to changes in their environment and upon ligand binding. By changing their conformation, proteins can carry out their functions and modulate the functions of other molecules. Conformational changes to the unliganded form of a protein provide the physical underpinning for allostery and signal transduction, and these changes are crucial for ubiquitous phenomena such as enzyme catalysis, protein-protein interactions, and motor protein movement . As our Second-harmonic generation (SHG) is a nonlinear optical technique  in whichEscherichia coli dihydrofolate reductase (DHFR). CaM is a messenger protein that transduces calcium-generated signals by binding calcium ions , maltose-binding protein (MBP), and ium ions . CaM is ium ions . Calciumβ-strand and are known to undergo large-scale motion from an open to a closed form upon maltose binding  and correlating the motions we observe at these sites upon ligand binding with those observed in the x-ray crystal structures.The Ni-NTA bilayer surface used in this work is available from Biodesy . Preparation of the bilayer surface on glass was performed according to the manufacturer’s instructions. A characterization of protein binding to the bilayer surface is available in the ecDHFR variants were constructed using the Stratagene QuikChange site-directed mutagenesis kit and the wild-type (WT) ecDHFR template as described previously . N-terminal poly-histidine-tagged human CaM was obtained from EMD Millipore . N-terminus His-tag (8 His) eviously . For theeviously . The choeviously . The pureviously ).The second-harmonic-active dyes SHG1-SE  and SHG2-maleimide  are available from Biodesy and were conjugated to each protein according to the manufacturer’s instructions.2O2, 70% H2SO4) at 100°C for 20 min. After cooling, the slides were washed five times with deionized water and dried with nitrogen gas. A custom 2-mm-thick silicone gasket template with adhesive backing  was applied to the Piranha-cleaned slides. Each silicone gasket template defined 16 wells, each with a total volume of ∼14 μL. The spacing and diameter of the wells were based on the standard 384-well-plate format. Small unilamellar vesicles (SUVs) were incubated with 1 mM of NiCl2  for 30 min, diluted fivefold in buffer, and added to each well. After bilayer formation, the wells were washed with buffer to remove unbound SUVs and imaged by fluorescence microscopy to determine that the bilayer was fluid and uniform across the surface (Glass slides (Fisher) were cleaned in Piranha (30% H surface . Protein2 (with or without 2 mM CaCl2). For DHFR, the optimized buffer was 50 mM sodium phosphate, pH 7.0, with 100 μM NADPH (Sigma). For conformational-change experiments, 9 μM MBP, 2 μM CaM, or 4 μM DHFR was incubated in each well for a minimum of 1 h. Stability experiments were carried out after overnight incubation in the wells at 4°C. For specificity experiments, each protein was incubated in 25 mM Tris pH 7.2 and 150 mM NaCl, with or without 300 mM imidazole. Excess protein and imidazole were removed by washing with 25 mM Tris pH 7.2, 150 mM NaCl before SHG data were acquired.Each protein was screened to determine an optimized binding buffer for bilayer attachment and SHG signal production. For MBP, the optimized buffer was determined to be 20 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween-20, and 1 mM dithiothreitol. For CaM, the optimized buffer was 25 mM MOPS pH 7, 150 mM KCl, 2 mM MgClμm. The second-harmonic light, which emerges from the prism nearly collinear to the incident beam and parallel to the ground, was collected by a lens, separated from the fundamental beam using a dichroic mirror and wavelength filters, and directed into a PMT module with a built-in preamplifier for photon counting, using a 1-s integration time for each measurement . No analyzer was used to select the polarization of the second-harmonic beam. A custom electronics board was used to digitize the signal, and the data were sent to a computer running customized control and data-collection software .Our instrument comprises a mode-locked Ti:Sapphire system, which provides the fundamental beam necessary to generate the second-harmonic signal (high peak power). For these experiments, we used a Mira 900 Ti:Sapphire ultrafast oscillator  pumped by a Millenia V DPSS laser . The fundamental beam was passed through a half-wave plate to select p-polarization  and focused into a Dove prism at an angle of 69°  to a spot size of ∼100 μm randomly addressable precision .For these experiments, the microscope slide with protein was coupled to a prism using BK7 index matching fluid , and the prism itself was secured onto a 1D translation stage capable of 1 Ligand addition was carried out while the SHG signal was monitored in real time. Once the baseline signal was established, buffer was injected into the well as a control. After 5–10 s to assess how the buffer injection changed the signal, the compound of interest was injected into the well to the desired final concentration. The SHG signal was monitored for several minutes after injection.SH), the second-harmonic intensity measured just before injection (It0) was subtracted from the second-harmonic intensity at tmax (Itmax) and then divided by the initial second-harmonic intensity (It0) according to the following equation:To calculate the percent change in SHG intensity .A, the addition of 2 μM of CBP in the presence of a calcium-containing buffer resulted in a positive change in the SHG signal of 18.1% ± 0.6%, whereas buffer injection resulted in a signal change of 0.1% ± 0.4% (N = 9). When calcium was omitted from the buffer, addition of the peptide alone resulted in a 3.1% ± 1.6% change in SHG intensity . As calcium-free CaM does not adopt a conformation capable of binding to peptide, these results confirm that the change observed upon peptide addition in the presence of calcium was due to the conformational change induced by peptide binding. Next, we evaluated the effect of adding calcium ions. When buffer containing 1 mM CaCl2 was injected into the system, a decrease in signal of −12.6% ± 1.4% resulted (C). Peptide was then added directly to this sample 2 min after calcium addition, producing a positive signal change of 8.7% ± 0.4%. The difference in the magnitude of the observed signal change between the two experiments in which peptide was added is due to the difference in the CaCl2 concentrations and incubation times used. In the first experiment (A), 2 mM of CaCl2 was incubated with CaM for 1 h before peptide addition, whereas in the second experiment, peptide was added 2 min after addition of 1 mM of CaCl2. The kinetics of calcium association and dissociation rates to and from the four binding sites on CaM were previously determined by rapid microfluidic mixing. Based on these measurements, and given that our measurements were performed in wells and kinetically limited by mass transport of the ligands to the surface-tethered protein, we would not expect the conditions of these two experiments to reach the same endpoint within 2 min. The magnitude of signal changes for each of these experiments at a 2-min endpoint is shown in D. As the magnitude of the SHG signal is proportional to the net, average orientation of the dye label relative to the surface normal, the different magnitudes of the SHG signal change upon binding CBP and calcium to unbound CaM confirm that these ligands bind to specific and different conformations of the protein. SHG signal changes can occur in either a positive or negative direction relative to baseline depending on whether the net, average orientation of the probe moves closer to or farther away from the surface normal, respectively.To explore the ability of our SHG bilayer system to detect and measure the conformational changes of a protein, we began by testing the system on CaM and MBP, two proteins previously studied by SHG . First, resulted C. Peptiperiment A, 2 mM E, all three residues undergo large structural changes upon binding both calcium and CBP. Taken together, the data demonstrate that conformational changes associated with both calcium and CBP binding to CaM can be resolved using SHG. Moreover, the peptide- and calcium-induced conformational changes are clearly different in both magnitude and directionality, illustrating SHG’s ability to discriminate the different conformations the protein adopts upon binding different ligands.We next sought to determine which residues were modified with our amine-reactive dye. MS of labeled CaM revealed complete labeling at K115 and less than complete modification at K13 and K94. As the degree of labeling for CaM was measured to be 1.0 (dye/protein ratio) by UV-Vis spectroscopy, most of the signal was likely due to labeling at K115. As can be seen from the overlay of the crystal structures of the apo, calcium-bound, and CBP-bound CaM in A). The MBP system offers an excellent control in lactose, a stereoisomer of maltose that does not bind to MBP. Because the addition of both 1 mM of lactose and buffer alone resulted in negligible changes in SHG intensity, the change in SHG intensity upon maltose addition is specific to ligand-induced conformational changes upon binding.We also performed a similar set of experiments with MBP. First, we monitored the SHG intensity of MBP labeled at pH 8.3 upon addition of buffer, lactose, or maltose. As can be seen in the real-time trace of the SHG signal, the addition of 1 mM of maltose resulted in a rapid decrease of 33.8% ± 1.0%, whereas the addition of either buffer or 2 mM of lactose resulted in a negligible change of 0.12% ± 0.61% or 0.44% ± 0.79%, respectively A. The MD, the side chains of the labeled residues show significant orientational differences, providing further validation that the observed change in SHG intensity is the result of ligand-induced binding of maltose to MBP.The MS analysis of MBP revealed that it is heterogeneously labeled at K15, K88, K127, K239, K256, K297, K326, and K362, with residues K15, K88, and K362 representing ∼90% of the total population of modified peptides. The degree of labeling for MBP was 1.3, confirming that more than one residue was labeled. We aligned the crystal structures of the apo and maltose-bound MBP and compared the positions of the labeled residues in these two structures. As can be seen in Ka. Based on inspection of the crystal structures, we hypothesized that when MBP is primarily labeled at K15 rather than K88, this could alter the directionality of SHG signal changes upon ligand addition relative to the reverse case, since K15 and K88 appear to rotate toward and away from the surface normal, respectively, assuming the protein is oriented with its N-terminus facing the bilayer. As can be seen in B, addition of 1 mM of maltose to pH 7.5-labeled MBP resulted in an increase in SHG intensity of 27.4% ± 0.61%. Consistent with previous results, the addition of lactose and buffer alone had no effect. These results are summarized in C.As MBP demonstrated a high degree of lysine modifications, we modified our labeling conditions to explore whether we could target specific lysines for labeling. Labeling MBP at pH 7.5 rather than pH 8.3 significantly increased the overall abundance of the K15 modification, with a corresponding decrease in the modification at K88: 70% of the peptides detected by MS showed labeling at K15 rather than K88. The converse trend, a preference for K88 over K15, was observed when the experimental conditions were changed to pH 8.3. The degree of labeling of the conjugate labeled at pH 7.5 was 1.3, which suggests that the protein was primarily labeled at K15. This example shows that by varying the conditions of the conjugation reaction, one can bias the distribution of labeled lysines, most likely by exploiting the differences in each residue’s microenvironment and pE. coli DHFR, a protein previously uncharacterized by SHG. In particular, we focused on the protein’s response to two important pharmaceutical inhibitors: methotrexate (MTX) and trimethoprim (TMP)  for the buffer control. We next explored whether TMP addition by itself would result in a conformational change in DHFR. As can be seen in B, the addition of 100 μM of TMP resulted in a decrease in SHG intensity of −33.9% ± 5.31%. It is known that MTX and TMP bind competitively to the same site on DHFR. Subsequent addition of 1 μM of MTX after 100 μM of TMP resulted in a decrease in the SHG signal of −3.48% ± 1.31%, a negligible change. If the TMP-induced SHG signal change were due to loss of protein from the surface rather than conformational change, addition of MTX after TMP should have resulted in a further signal change, since ample baseline signal (and thus protein) remained after the TMP addition. However, MTX’s response was completely blocked after the TMP addition, demonstrating that the observed signal changes were due to a conformational change rather than loss of protein from the surface. The signal changes we observed for these experiments are summarized in C. MS analysis revealed that three residues, K76, K106, and K109, were modified by the amine-reactive SHG dye, with the modification at K76 being nearly complete. The degree of labeling of the amine-labeled DHFR conjugate was 1.1, suggesting that labeled K76 was the primary contributor to the observed signal. As can be seen in an overlay of the x-ray structures of the apo and MTX-bound DHFR holoenzyme (D), although there is relatively little motion at sites K106 and K109, there is a significant reorientation at site K76, corroborating our SHG measurements and the MS analysis.As a final demonstration of the technique, we studied im (TMP) . For theloenzyme D, althoA, the trends are similar to those observed with amine-labeled DHFR, although the magnitude of the observed change is different. Addition of MTX to cysteine-labeled DHFR on the bilayer resulted in a large change in SHG intensity of −81.03% ± 2.4%, compared with −0.6% ± 0.4% for buffer alone. The addition of TMP alone produced a similar decrease in SHG intensity of −88.05% ± 1.5%, and, as with amine-labeled DHFR, addition of 1 μM MTX in the presence of TMP resulted in an insignificant change in SHG intensity of −1.9% ± 1.0% (B). In addition to the antifolate compounds, we also tested the natural ligand DHF on the M20C-labeled DHFR. We found that the addition of 100 μM of DHF resulted in a signal change of −90.2% ± 0.4%. These results are summarized in C. In addition, we performed a dose-response experiment using TMP, and determined its EC50 for binding to DHFR as logEC50 of −8.05 ± 0.07 (EC50 = 8.9 nM) , which participates in the key catalytic steps of the enzyme. Complete labeling of the cysteine residue was confirmed by MS and the degree of labeling was 1.0, indicating that the labeling was site specific. The ΔCys M20C construct maintained the WT enzymatic activity described in the Materials and Methods section. As can be seen in % ± 1.0% B. In ad 8.9 nM) , which i 8.9 nM) . This ex 8.9 nM) . MoreoveE. coli DHFR). This approach is based on using SHG-labeled proteins tethered to an SLB membrane. Although most proteins are not intrinsically second-harmonic active, they can easily be made so by standard amine- and thiol-reactive chemistries. We identified the labeled sites by MS, which in combination with available x-ray crystal structures allowed us to inspect and correlate the structural motions we observed by SHG with the changes in orientation observed at the modified residues in both the apo and bound structures for all three proteins. Importantly, we found that the labeling site(s) determined by MS for both the amine- and cysteine-labeled conjugates for the three proteins studied here was remarkably consistent between preparations when the reaction conditions of the conjugation, including the pH, probe/protein molar ratios, and concentrations of the reactants, were held constant.In the work described here, we developed a broadly applicable and sensitive approach that uses SHG to detect and resolve ligand-induced protein conformational changes. We demonstrated the method using three different proteins  of MTX is ∼100 times greater than that of TMP, one might be able to establish an SAR by evaluating the Ki value against the magnitude of the SHG signal change and thus the different conformations produced upon binding of different ligands. In addition, one could establish other SAR correlations by correlating in vivo data with the conformational changes produced upon binding of different ligands. In general, once an SAR is established, the SHG platform offers a more convenient and efficient way to screen for potential drug candidates compared with current activity and kinetic-based assays, by classifying the conformational changes of compounds into groups that produce different functional outcomes.Furthermore, the technique is well suited for structure-activity relationship (SAR) experiments. The SHG signals are very reproducible, and the differences in signal produced by two different ligands—and thus conformations—can be easily distinguished. In particular, the ability to resolve different ligands by the distinct and specific conformational changes they produce, and to correlate these changes with activity measurements  offers a direct way to conduct SAR experiments to understand the connections between ligand structure, protein conformation, and function. Here, we show that In summary, our data demonstrate that the method presented here sensitively detects ligand-induced conformational changes that range in magnitude from the relatively small rotation of an amino acid side chain to the global motion of protein domains. Enabled by a biomimetic SLB as a two-dimensional platform for tethering and orienting proteins, the highly sensitive and generally applicable SHG structural technique described here can be used for many facets of research, such as probing the functional implications of protein structural rearrangements due to ligand, drug, or protein-protein interactions, as well as mutational screening.The instantaneous, real-time nature of SHG offers investigators the opportunity to obtain measurements on even faster timescales than are currently possible to study how the kinetics and dynamics of conformational changes engender protein function. We expect that such measurements will be a focus of future studies. Finally, this work lays the foundation for future polarization-dependent studies  in whichB.M., R.M., S.B., and J.S. designed the experiments. B.M., K.C., R.M., and T.L. performed experiments and analyzed data. B.M., K.C., T.L., S.B., and J.S. wrote the manuscript. S.B. and J.S. supervised the work.


In this review, drugs interfering with these targets  are evaluated and their future as clinical compounds is considered. These new targets prove to be interesting; however more insight into long-term drug effects is necessary before clinical applicability becomes reality.Sudden cardiac death due to ventricular arrhythmias is a major problem. Drug therapies to prevent SCD do not provide satisfying results, leading to the demand for new antiarrhythmic strategies. New targets include Ca Sudden cardiac death (SCD) due to arrhythmias is a major problem in the Western population . SCD occICa-L), are known to affect haemodynamics negatively.Besides the classical modulation of sarcolemmal ion channels by drugs (including both activation and block), new antiarrhythmic targets emerge that can be of great interest in pharmacological treatment of VT in cardiac disease. Especially arrhythmias that occur in hypertrophy and heart failure have our attention, because of the difficulty in treating these patients effectively. Besides disappointing data concerning antiarrhythmic efficacy, current therapies such as blockade of the late-type calcium current , the Na/Ca exchanger (NCX), the Ryanodine receptor  and the late component of the sodium current (INa-Late), all related to intracellular calcium (Ca2+) handling of the cardiac myocyte. With this development, emphasis of antiarrhythmic drugs seems to shift therapy more in the direction of focal arrhythmias, which can be caused by delayed (DAD) or early after depolarisations (EAD), a logical step knowing that triggered activity-related arrhythmias are becoming more and more common in hypertrophy and heart failure , while LTCC is inactivated by local [Ca2+]i via calmodulin (CaM) binding on the C-terminus of the channel. The initial Ca2+ influx via the LTCC leads to Ca2+ induced calcium release (CICR) from the sarcoplasmic reticulum (SR), which is mediated by the RyRs , protein kinase A , and sorcin (which connects RyRs and LTCCs) near the Ca2+ release complex. Subsequently, Ca2+ released from the SR binds to troponin to facilitate contraction of the sarcomere, the contractile element of the myocyte. Thus, Ca2+ links the electrical activation of cardiomyocytes to mechanical contraction: excitation-contraction coupling . When R2+ must decline to allow Ca2+ to dissociate from troponin leading to relaxation of the contractile element. This Ca2+ transport is facilitated by a Ca2+-ATPase (SERCA) on the SR which transports Ca2+ back into the SR and the NCX on the sarcolemma  and [Ca2+] across the sarcolemma as well as the membrane potential. Whether the current is in the forward or reversed mode depends on the driving force for NCX. High [Ca2+]i favours forward INa/Ca whereas high [Na+]i and positive membrane potential favours reversed INa/Ca . If. If2+ repatients .Fig. 4aINa-Late leads to higher [Na+]i, which pushes NCX in its reverse mode and Na+ is transported out and Ca2+ into the cell, also leading to increased [Ca]i i, but due to its capability of autophosphorylation is not solely dependent on the rise and fall in [Ca2+]i.. When phosphorylation of CaMKII has been accomplished, the enzyme becomes persistently active and therefore the natural beat-to-beat fall in [Ca2+]i will not immediately affect the enzymes’ activity. CaMKII has a central role in Ca2+ handling, influencing RyR, LTCC, and SERCA , activation of CaMKII is mainly facilitated by CaM, a small cytoplasmic protein. CaM needs to bind cytosolic Cactivated . For thaactivity –60. This50 of 370 nM i, and triggered arrhythmias.The total inward sodium current is composed of a peak and late component and is provided through voltage-gated Na channels in the sarcolemma, which are rapidly activated and inactivated. Under physiological conditions, the  cytosol . On its INa-Late is a topic of interest because it can possibly prevent EAD and DAD triggered activity. Ranolazine is a well-known inhibitor of INa-Late but its actions are not restricted to this channel as it evokes multiple other effects  [INa,late specifically (it affects peak INa and IKr only weakly at much higher doses). This new compound has been tested in animal models with positive findings [Recently, data have been published which suggest that sophocarpine also possesses r,ICa) . GS-967 findings , 88, whiOld antiarrhythmic strategies, though proven clinically effective, have important drawbacks such as negative haemodynamic effects and a small safety margin, thereby prohibiting the use of higher dosages . To imprSo far, blocking of NCX, using SEA-0400, appears to be neutral on inotropy due to the counteracting effects of 1) anticipated positive inotropy by NCX block alone and 2) negative haemodynamics by its additional inhibition of LTCC , 36. TakThe antiarrhythmic potential of RyR block remains debatable, as positive results of K201 are scarce and counteracted by negative results (Table In Table The use of ranolazine has also shown positive results: prevention and suppression of EADs, and VT/VF in several experimental approaches. Important aspect is that the drug is already registered and clinically applicable. Its antiarrhythmic efficacy is not complete, which opens the road for improvement using this approach. In this line of intervention, promising new drugs such as sophocarpine and GS-967 are being investigated.Overall, regardless of the current possibilities for pharmacological interventions, it is clear that new strategies will be welcome to prevent SCD due to arrhythmias. The new targets discussed here prove to be interesting strategies for the future and provide food for thought. Obviously, more research with respect to the long-term effects of chronically blocking each of the discussed targets is essential before research can proceed to a more clinical setting.


XBP1 (XBP1s) is up-regulated in acute myeloid leukemia (AML) cell lines and AML patient samples. IRE1α RNase inhibitors  blocked XBP1 mRNA splicing and exhibited cytotoxicity against AML cells. IRE1α inhibition induced caspase-dependent apoptosis and G1 cell cycle arrest at least partially by regulation of Bcl-2 family proteins, G1 phase controlling proteins , as well as chaperone proteins. Xbp1 deleted murine bone marrow cells were resistant to growth inhibition by IRE1α inhibitors. Combination of HNA with either bortezomib or AS2O3 was synergistic in AML cytotoxicity associated with induction of p-JNK and reduction of p-PI3K and p-MAPK. Inhibition of IRE1α RNase activity increased expression of many miRs in AML cells including miR-34a. Inhibition of miR-34a conferred cellular resistance to HNA. Our results strongly suggest that targeting IRE1α driven pro-survival pathways represent an exciting therapeutic approach for the treatment of AML.Survival of cancer cells relies on the unfolded protein response (UPR) to resist stress triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The IRE1α-XBP1 pathway, a key branch of the UPR, is activated in many cancers. Here, we show that the expression of both mature and spliced forms of  Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by a small population of self-renewing leukemic stem cells (LSCs) giving rise to a large population of immature leukemic blasts –4. LSCs Hematopoietic cells, including LSCs, are exposed to low levels of oxygen in the bone marrow, which may cause accumulation of misfolded proteins in the endoplasmic reticulum (ER), thereby stimulating ER stress and activating the unfolded-protein-response (UPR) pathway , 10–12. Inositol-requiring enzyme 1 alpha (IRE1α) is one of major ER transmembrane sensors that activates the UPR . IRE1α iXBP1s, indicating activation of UPR and an increase of chaperone proteins [The functions of the ER and its associated stress pathways in AML have been studied including recent studies that found approximately 25% of AML samples had detectable proteins , 27. Manproteins –32. In tXBP1 was highly hypomethylated on its CpG island in AML cases . Interestingly, XBP1s was detectable in 85% (22 of 26) of the leukemia cell lines and 71% (17 of 24) of AML patient samples  as measured by QRT-PCR  subtypes in our own dataset (XBP1/XBP1s and FAB subtypes among the 24 AML samples (data not shown). We next performed similar statistical analysis using TCGA AML dataset. Since XBP1s was not discernable from total XBP1 in the dataset, we only tested total XBP1 level. Interestingly, XBP1 expression was significantly increased in FAB M3 subgroup compared with M0, M1 and M2 but significantly decreased in M4-M7 subgroup  retained the IRE1α RNase inhibitory activity [XBP1 splicing in many cells [XBP1s mRNA and decreased XBP1u  were observed in 293T and K562 myeloid leukemia cells  and AML patient samples  compared to untreated patient samples  of clonogenic growth in soft agar of AML cells from patients   was noted to have potent anti-proliferative activity in multiple myeloma (MM) . The comactivity . Tunicamny cells . Followiia cells . Compareia cells . STF-083ia cells . HNA dosia cells . HNA sigs Figure . In cont) Figure . We cond) Figure  and S2D.) Figure .cip1, p27kip1), and decreased level of cyclin D1 . After Cre activation, more than 92% of Xbp1 was confirmed to be deleted in the floxed cells as evidenced by QRT-PCR  [2O3, will enhance the inhibition of proliferation of AML cells. Indeed, various concentrations of either drug combined with HNA synergistically inhibited growth of both NB4 cells and an AML patient sample #19   and has ia (APL) , 48. Botia (APL) , 50. We  Figures . Also, b Figures .Recent studies discovered that IRE1α could cleave 4 anti-Casp2 pre-miRNAs , resulting in activation of CASP2 in mouse embryonic fibroblasts (MEFs) , 23. To Small RNA antagonist against miR-34a was transiently transfected into 3 AML cell lines , and cell viability was examined after treatment with HNA (12.5-100 μM). Knockdown efficiency of miR-34a by the antagonist was validated both at the pre- and mature miR level by QRT-PCR Figures ; Also inThe UPR is a defense mechanism activated by cells during stressful conditions in response to an accumulation of misfolded proteins in the ER . Cancer XBP1 and its spliced form XBP1s have been reported to be upregulated in several types of cancers, and blockage of the IRE1/XBP1 pathway is considered as a promising therapeutic option , 57–61. CDK4, c-Myc, Bcl-2 and Cyclin D1 and induction of cip1p21 and kip1p27 in AML cells. Silencing of miR-34a by small RNA antagonist significantly induced resistance of AML cells to IRE1α inhibitors and restored levels of miR-34a targeted oncogenes such as c-Myc and Cyclin D1. These results indicate that miR-34a plays an important role in IRE1α-dependent UPR in AML. Through cleavage of miR-34a, IRE1α decreases miR-34a induced apoptosis and helps AML cells to escape death.We particularly focused on miR34a. Recent studies indicate that miR-34a is a tumor suppressor . Down-rein vitro and in vivo [Bortezomib is a proteasome inhibitor which has been approved by FDA as the first example of UPR-modulating regimen for the treatment of Multiple Myeloma . Recentl in vivo , 55. Mec2O3 has unique favorable cure rates [2O3 has been reported to enhance UPR by increasing the expression of GRP78, CHOP, phosphorylated eIF2α and ATF4 [2O3 synergistically increased cell apoptosis of the APL cells NB4, which might be due to corroborative inhibition of different branches of the UPR pathway. In the previous clinical trials, QT interval prolongation and APL differentiation syndrome are the most serious side-effects of AS2O3 treatment [2O3 may improve the toxicity profile.Treatment of acute promyelocytic leukemia (APL) with ASre rates , and there rates . Notablyand ATF4 , 82. In reatment . The comXBP1 and its spliced form (XBP1s) are often increased in AML. Inhibition of IRE1α RNase activity by small molecules inhibited AML cell growth. This inhibition of proliferation probably occurred by inducing ER stress by blocking the compensatory pathways including the prevention of cleavage of tumor suppressor miRs (e.g. miR-34a) which may augment the anti-proliferative effect of IRE1α inhibitors. Therefore, compounds which inhibit activation of IRE1α represent a novel pathway for cell kill and may be a useful compliment to chemotherapy.In conclusion, we found that Information of reagents and vendors, and all methods are described in the


Why are spines so small? Here, we provide functional reasons; the size of spines is optimal for information coding. Spines code input timing information by the probability of Ca2+ increases, which makes robust and sensitive information coding possible. We created a stochastic simulation model of input timing-dependent Ca2+ increases in a cerebellar Purkinje cell's spine. Spines used probability coding of Ca2+ increases rather than amplitude coding for input timing detection via stochastic facilitation by utilizing the small number of molecules in a spine volume, where information per volume appeared optimal. Probability coding of Ca2+ increases in a spine volume was more robust against input fluctuation and more sensitive to input numbers than amplitude coding of Ca2+ increases in a cell volume. Thus, stochasticity is a strategy by which neurons robustly and sensitively code information.A dendritic spine is a very small structure (∼0.1 µm The CF input causes Ca2+ influx through VGCCs. The IP3 receptor, an intracellular Ca2+ channel, is activated only when it binds simultaneously with both IP3 and Ca2+. When the ternary complex of Ca2+·IP3·IP3 receptor is formed by the PF input, the Ca2+ influx via CF input triggers a positive feedback loop of Ca2+-dependent Ca2+ release that leads to a large Ca2+ increase from intracellular Ca2+ stores, such as the ER. This large Ca2+ increase induces a delayed negative feedback inhibition of Ca2+ release from the stores at higher levels of Ca2+. Therefore, PF and CF inputs-dependent Ca2+ increase is generated by positive- and delayed negative-feedback loops. This forms a single, large and transient Ca2+ increase, depending on the relative timing of the PF and CF inputs.We used the same kinetic framework and parameters of PF and CF inputs-dependent Ca2+ increase due to PF and CF inputs in a spine and in a cell volume . The volumes of spines and cells have been reported to be 0.1 µm333 as a spine volume and 5,000 µm3 as a cell volume. In response to coincident PF and CF inputs (Δt = 160 msec), a large Ca2+ increase was always observed in a cell volume  but not all cases , but not in many cases  rather than the amplitude of Ca2+ increase.We performed stochastic simulation of the Cantration . The pro2+ response in every 20 msec PF-CF intervals between −400 msec and 600 msec in a spine volume . We first measured the total input timing information coded by the distribution of the Ca2+ response by mutual information between the Ca2+ response and PF-CF interval  showed almost the same response as the previous deterministic model  Next, we examined the relative contribution of the probability or amplitude component to the total information Fig. 3F.2+ increase should be affected by the fluctuation of the amplitude of PF inputs (PF amplitude). We simulated the Ca2+ increase with the fluctuation of PF amplitude between trials, and asked whether the probability coding in spines are advantageous for robust information coding on input timing against fluctuation of PF amplitude (2+ response in a spine volume were similar regardless of the coefficient of variation (CV) of the PF amplitude . Based on the simulation results, we provided one of the predicted examples. With repetitive application of a glutamate pulse with the same concentration, a similar level of Ca2+ increase is always expected in a soma, whereas a similar number of spines with large Ca2+ increase is expected in spines . Importantly, the spines with large Ca2+ increases and the amplitudes of Ca2+ increases in each spine are different between trials. This result will support the idea that generation of large Ca2+ increases in spines is stochastic rather than deterministic. Upon repetitive application of glutamate pulses of various concentrations, the number of spines with large Ca2+ increase and the amplitude of Ca2+ response in soma are predicted to increase as the concentration of glutamate increases . The spines with large Ca2+ increases are different between trials, even at the same concentration of glutamate. Thus, repetitive application of glutamate pulses at various doses to a Purkinje cell will reveal the distinct characteristics of Ca2+ increases in spines and the soma. Importantly, at lower doses of glutamate, large Ca2+ increases were observed in spines but not in the soma, indicating that spines are more sensitive to glutamate than the soma . This will support our finding of increased sensitivity of spines to input  and delayed negative feedback . Blocking the interaction of IP3 with the IP3 receptor, which controls the feedback gain of the regenerative cycle of Ca2+, resulted in disappearance of the large Ca2+ increases above the threshold and decrease of the amplitude of Ca2+ below the threshold . Blocking the interaction of Ca2+ with the IP3 receptor, which triggers the regenerative cycle of Ca2+, resulted in disappearance of the large Ca2+ increases above the threshold . Blocking Ca2+ influx through the IP3 receptor, which is responsible for regenerated increase of Ca2+, resulted in the disappearance of the large Ca2+ increases above the threshold and decrease in the amplitude of Ca2+ below the threshold . Thus, the regenerative positive feedback loop in which gain is controlled by IP3 appeared to be an essential mechanism for probability coding.The large Cafeedback Fig. 1B 2+ increase in a cell volume.The IICR system is an excitable system, but its mechanism is different from that of conventional excitable systems, such as the relaxation oscillator 2+ increases even in a cell volume 2+ increase may arise from stochastic individual Ca2+ release through Ca2+ channels in microdomains It has recently been shown that the IICR system may use probability coding of CaNoise-dependent enhancement of the detection of weak information-carrying signals in a threshold system is known as stochastic resonance. Stochastic resonance in a narrow sense, or classical stochastic resonance, is a phenomenon in which detection of weak periodic signals by nonlinear threshold systems is enhanced by exogenous additive white noise 2+ increases based on the deterministic kinetic model of Ca2+ increases in cerebellar Purkinje cells 1k, −1k, and catk, and 1k was assumed to be four times larger than catk2+ pulse was applied to the cytosol to achieve a Ca2+ influx as a result of VGCC opening, because the period of VGCC opening is known to be very short, less than Δt = 20 msec 2+ influx (83.3 µM/msec for 2 msec) as Ca2+ influx through VGCCs. The 5 PF inputs give 5 pulses of glutamate influx (5 µM/msec for 1 msec for each pulse) and 5 pulses of Ca2+ influx (25 µM/msec for 1 msec for each pulse) at 100 Hz, the latter of which were regarded as Ca2+ influx through VGCCs activated by AMPARs.We created a stochastic simulation model of input timing-dependent CaIn the deterministic model, each reaction was represented as an ordinary differential equation (ODE) as was conducted in the previous model 2+ increase did not occur any more at any PF-CF intervals . In order to uncover the simple influences of the smallness of a spine and the number of molecules, it is required that the effect of the stimulation for a cell on the cytosolic molecules through the membrane protein is transferred as well as that for a spine. Therefore, we assumed that the number of membrane proteins is proportional to the volume, i.e. the cube of length, throughout this study.In general, surface area of the membrane is proportional to the order of the square of length, whereas cell volume is proportional to the cube of that, which means that, as a system size increases, the increase rate of number of membrane molecules becomes smaller than that in cytoplasm. Hence, in the cases of larger systems than the spine, the number of membrane molecules that can activate the cytosolic molecules is so small that most of substrates are not activated by the stimulation. Actually, when a volume was 8- or 125-fold larger than a spine volume, large CaAnother reason for this assumption is that, if the number of membrane proteins is to be set proportional to the surface area, the unknown parameters whose values were determined by fitting the simulation outputs to the experimental results in the previous deterministic model et al.). We repeated the same simulation with different random seeds. The number of simulation trials were as follows: 8,000 trials in the volume of 0.05 µm3, 2,000 trials in the volume of 0.1 µm3, 1,000 trials in the 0.5 µm3, 0.8 µm3, and 1 µm3 volumes, 100 trials in the 10 µm3, 12.5 µm3, and 100 µm3 volumes, and 20 trials in the 1,000 µm3 and 5,000 µm3 volumes. The simulation for the testable prediction was conducted 100 times. The numbers of trials were confirmed to be sufficient by resampling the simulated data . The simulation result in a cell volume was nearly identical to that in the previous deterministic model, supporting the validity of the stochastic model.To solve the stochastic model numerically, we utilized the modified tau-leaping method, which is an approximation of the stochastic simulation algorithm (SSA) 2+ increase, resCa, by logarithm of temporally integrated Ca2+ subtracted by the basal Ca2+ concentration:2+](t) and [Ca2+]basal denote intracellular Ca2+ concentration at time t and basal Ca2+ concentration, respectively. We used logarithmic scale for Ca2+ concentration assuming that downstream molecules of Ca2+ respond linearly to the log-scaled stimulus. This assumption is justified by the fact that serially diluted solution is often used as stimulus in experiments. The probability distribution of resCa over trials was expressed by t denotes the PF-CF interval and the time difference between the first PF and CF inputs. The marginal distribution over PF-CF interval, We measured the response of CaresCa into the amplitude and probability components:s = 1 represents that resCa exceeded the threshold, and s = 0 otherwise  between the resCa and PF-CF interval:Δt denotes the PF-CF interval . We used the uniform distribution for Fig. S8). In the calculation of the MI, we divided the entire range of resCa from minimum to maximum into 50 bins, where the intervals were determined based on the method described in Cheong et al. [ref], who estimated the MI taking the limit as the sample number approaches infinity. Note that for the probability density function, the MI  divergence) is invariant under the any transformation of random variables. In this study, we estimated the MI by the abovementioned method, therefore, we could obtain almost the same value of MI for any transformation of resCa .We measured the input timing information coded by the CaresCa without the probability component, which was calculated by marginalizing We also measured the information coded by the probability component by use of the KL divergence:resCa without the amplitude component, which was calculated by marginalizing Further, we measured the information coded by the amplitude component:The relative contributions of the probability and amplitude components to the input timing information were defined by 2+ pulses fluctuated between trials under the fixed mean concentration (5 µM). The fluctuation of PF input amplitudes follows the zero-truncated normal distribution with a given coefficient of variation where the PF input amplitude must be over zero.For fluctuation of PF input amplitudes, the concentration of 5 glutamate pulses and 5 CaFigure S13) and Ca2+ in the endoplasmic reticulum (ER).Time courses of concentrations of inositol trisphosphate  and Δt = −400 msec (B) in the stochastic model in a spine volume  and in a cell volume , respectively. C, D, Time courses of concentrations of Ca2+ in the ER in response to PF and CF inputs with Δt = 160 msec (C) and Δt = −400 msec (D) in the stochastic model in a spine volume  and in a cell volume , respectively.(TIF)Click here for additional data file.Figure S22+ response and that without the probability and/or amplitude components.Distribution of CaA, Distribution of Ca2+ response. B, C, Distribution of Ca2+ response without probability (B) or amplitude (C) component. D, Distribution of Ca2+ response without both components. Input timing information coded by the probability and amplitude component was calculated by the Kullback–Leibler (KL) divergence of the distribution of B and C from that of A, respectively. Input timing information coded by the distribution of Ca2+ response was calculated by the mutual information between Ca2+ response and PF-CF interval, which is equal to the KL divergence of the distribution of D from that of A, and also equal to the sum of the input timing information coded by probability and amplitude component.(TIF)Click here for additional data file.Figure S3Mechanism of probability coding.A, Schematic representation of the pathway deleted in the following figures. B–D, Distribution of Ca2+ response along the PF-CF interval in a spine volume with blocking of the interaction of IP3 with the IP3 receptor (B), with blocking of the interaction of Ca2+ with the IP3 receptor (C), and with blocking of Ca2+ influx through the IP3 receptor (D) in the stochastic simulation. E, Distribution of Ca2+ response without deletion (control).(TIF)Click here for additional data file.Figure S4Possible experimental tests of probability coding in spines and amplitude coding in a soma.A, Ca2+ increase in response to repetitive addition of glutamate pulses with the same concentration in the stochastic model (spines) and in the deterministic model (soma). Colours code the concentration of Ca2+. Darker colours indicate higher concentrations of Ca2+. The time course of Ca2+ increase in the deterministic model and stochastic model are shown. B, Ca2+ increases in response to glutamate pulses of various concentrations in the stochastic model (spines) and the deterministic model (soma). Darker colours indicate higher concentrations of Ca2+. The time course of Ca2+ increase in the deterministic model and stochastic model are shown. C, Glutamate dose-responses curves of the fraction of spines with large Ca2+ increase above the threshold in the stochastic model (solid lines) and Ca2+ response in the deterministic model (dashed lines). Note glutamate inputs with amplitude CVs of 0.1 were used.(TIF)Click here for additional data file.Figure S5Validation of the numerical simulation.A, B, Ca2+ increase due to stimulation of PF and CF inputs with Δt = 160 msec (A) and Δt = −400 msec (B) by the stochastic simulation algorithm (SSA) . C, Distributions of the Ca2+ response in a spine volume with Δt = 160 msec (solid lines) and Δt = −400 msec (dashed line) by the modified tau-leaping method Click here for additional data file.Figure S6Input timing informations calculated in normal scale. Here we calculated the input timing informations in normal scale of Ca2+ response, and compared them with the input timing information calculated in logarithmic scale , by the probability component (red), and by the amplitude component (blue). B, Volume-dependency of the input timing information per volume. C, Relative contribution of the probability (red) and amplitude (blue) component to the input timing information. D, Input timing information per volume, coded by the Ca2+ response, in a spine (red) and in a cell (black). E, Input timing information per volume in a spine (red) and in a cell (black). Fig. S6A, B, and C correspond to Fig. S6D and E correspond to (TIF)Click here for additional data file.Figure S7Results of simulation under the condition that the numbers of membrane molecules were proportional to the surface area. In this study, we assumed that the numbers of membrane molecules were proportional to the volume of the system. Here, to check the results of the simulation with the numbers of membrane molecules proportional to the surface area, we changed the model and performed the stochastic simulation. In this model, we altered the following three types of parameters: (i) initial numbers of the membrane molecules, (ii) membrane permeability coefficients, and (iii) constants for the propensity functions of the bimolecular and trimolecular reactions occurring on the membrane. (i) Initial numbers of the membrane molecules were set proportional to the surface area, thus they were multiplied by the surface-to-volume ratio . (ii) Membrane permeability coefficients were also set proportional to the surface area, and thus they were multiplied by the surface-to-volume ratio. (iii) Originally, in the tau-leaping method, constants for the propensity functions of bimolecular reactions are proportional to the inverse of the system volume. Thus, in this model, to set them proportional to the inverse of the surface area, constants for the propensity functions of the bimolecular reactions occurring on the membrane were divided by the surface-to-volume ratio. Likewise, the constants for the propensity functions of the trimolecular reactions were divided by the square of the surface-to-volume ratio. Surface molecules in this model are metabotropic glutamate receptor (mGluR), IP3 receptor (IP3R), plasma membrane Ca2+-ATPase (PMCA), sacro- and endoplasmic reticulum Ca2+-ATPase (SERCA), Na+/Ca2+ exchangers (NCX), and their complexes with other moleucles. Reactions occurring on the membrane were all reactions which involve the surface molecules. As a result, if the numbers of the membrane molecules are set proportional to the surface area, large Ca2+ increase did not occur at any PF-CF intervals. A, B, Time courses of the concentrations of Ca2+ in the volume of 0.8 µm3 (A) and 12.5 µm3 (B) in response to PF and CF inputs with Δt = 160 msec. Results of 20 trials of simulation with the numbers of membrane proteins set proportional to the surface area (red lines) and to the cytosolic volume (black lines) were shown. C, D, Distribution of Ca2+ response in 0.8 µm3 (C) and 12.5 µm3 (D) under the condition that the numbers of membrane proteins were set proportional to the surface area. The numbers of membrane proteins were set 4/8 = 0.5 times in 0.8 µm3 and 25/125 = 0.2 times in 12.5 µm3 as large as those in (E) and (F). E, F, Distribution of Ca2+ response in 0.8 µm3 (E) and 12.5 µm3 (F) under the condition that the numbers of membrane proteins were set proportional to the cytosolic volume.(TIF)Click here for additional data file.Figure S8Input timing informations calculated on the assumption that the distribution of PF-CF interval follows Gaussian distribution. Here we calculated the input timing informations on the assumption that the distribution of PF-CF interval follows Gaussian distribution, and compared them with the input timing information calculated on the assumption that the distribution of PF-CF interval follows uniform distribution. Their similarity demonstrates that the results were qualitatively not affected by the distribution of PF-CF interval. PF-CF intervals were assumed to follow Gaussian distribution with mean of 0 ms and half-width of 300 ms (dashed lines with triangles), Gaussian distribution with mean of 200 ms and half-width of 300 ms (dotted lines with squares), and uniform distribution (solid lines with circles). A, Volume-dependency of the input timing information coded by the total distribution of the Ca2+ response. B, Volume-dependency of the input timing information per volume. C, Relative contribution of the probability component to the input timing information. D, Input timing information per volume, coded by the Ca2+ response, in a spine (red) and in a cell (black). E, Input timing information per volume in a spine (red) and in a cell (black). Fig. S8A, B, and C correspond to Fig. S8D and E correspond to (TIF)Click here for additional data file.Table S1Molecules and their initial number. This model consists of 56 molecules. Each of them is assumed to exist in one of the following compartment: cytosol, postsynaptic density (PSD), endoplasmic reticulum (ER), or extracellular space, with volumes of 0.1 µm3, 0.02 µm3, 0.002 µm3, and 10 µm3 in a spine respectively. The initial number of the molecules and the volumes of the compartments are the same as the previous deterministic model.(PDF)Click here for additional data file.Table S2Reactions and their parameters. This model consists of 43 reactions, one decay (decay of glutamate), one channel permeation (Ca2+ permeation through IP3R), two diffusions (Ca2+ and IP3 diffusions between cytosol and PSD), and two membrane permeations (Ca2+ permeation through the membrane of the cell and that through the membrane of the ER). In the stochastic model, decay of glutamate, two diffusions, and two membrane permeations were implemented as unimolecular reactions. Ca2+ permeation through IP3R was implemented as bimolecular reaction between Ca2+ and IP3R. All parameters are the same as the previous deterministic model.(PDF)Click here for additional data file.


Second harmonic generation (SHG) imaging can be used to visualize unique biological phenomena, but currently available dyes limit its application owing to the strong fluorescent signals that they generate together with SHG. Here we report the first non-fluorescent and membrane potential-sensitive SHG-active organic dye Ap3. Ap3 is photostable and generates SH signals at the plasma membrane with virtually no fluorescent signals, in sharp contrast to the previously used fluorescent dye FM4-64. When tested in neurons, Ap3-SHG shows linear membrane potential sensitivity and fast responses to action potentials, and also shows significantly reduced photodamage compared with FM4-64. The SHG-specific nature of Ap3 allows simultaneous and completely independent imaging of SHG signals and fluorescent signals from various reporter molecules, including markers of cellular organelles and intracellular calcium. Therefore, this SHG-specific dye enables true multimodal two-photon imaging in biological samples. Current dyes for second harmonic generation (SHG) imaging strongly fluoresce, limiting their application. Here the authors develop a SHG-specific dye, Ap3, that partitions into cell membranes, displays sensitivity to membrane potential and has virtually no fluorescence emission at SHG imaging wavelengths. Previous studies have utilized pre-existing or newly synthesized molecules9121617181920212223trans–cis isomerization and is devoid of undesirable photochemical processes31In previous studies, hyperpolarizable amphiphilic dyes were successfully used for SHG imaging of plasma membranesP<5 × 10−9, Mann–Whitney test). SHG signals remain stable for tens of minutes under this experimental condition, and then exhibit a gradual decrease over time, presumably due to the flip–flop movement of Ap3 from the outer to inner leaflet of the lipid bilayer, resulting in disruption of the non-centrosymmetric distribution of Ap3 molecules at the plasma membrane  cells, with FM4-64 dye as a reference, using a multi-photon microscopy system . These d33membrane  (ref. 35R=0.99 for both FM4-64 and Ap3). Next, SHG signals were measured while inducing action potentials in neurons under a current-clamp protocol. Ap3-SHG signals changed quickly on membrane potential changes and showed minimal delays (∼0.3 ms) when the peak time was compared, similar to FM4-64 . These results further raise the possibility that the photostable nature of Ap3 may circumvent the unintended photochemical reactions that then become toxic to neurons. To test this possibility, neurons were imaged in frame-scan mode with a long pixel dwell time  to intentionally induce photodamage. Indeed, this imaging protocol induced time-dependent membrane potential depolarization in neurons loaded with FM4-64, as reported previouslyP<2 × 10−3, Mann–Whitney test). Taken together, these results demonstrate that Ap3 is a photostable and less phototoxic membrane potential-sensitive SHG-specific dye that can be applied to cultured cells as well as neurons in brain slices.Previous studies have reported that SHG signals from various molecules are linearly sensitive to membrane potential changes11121618222626282926o FM4-64 . During o FM4-64 , presumao FM4-64 , the SHGHaving confirmed the SHG-specific nature of Ap3, we explored the possibility of multimodal two-photon imaging by combining it with other fluorescent molecules. First, considering the wide use of FM4-64 as a plasma membrane marker, simultaneous imaging of SHG with other fluorescent structural markers was examined. CHO cells were loaded with various types of fluorescent cell marker molecules and visualized by two-photon microscopy before and after the application of SHG dyes . The addTaking advantage of the multimodal two-photon imaging ability of Ap3, we then attempted to simultaneously monitor changes in membrane potential and calcium concentration in the cytoplasm. To this end, neurons in acute brain slices were patch-clamped and loaded with the fluorescent calcium indicator Rhod-2 together with Ap3, as well as Alexa Fluor 488, which was used as a morphology marker. Simultaneous imaging reveal that two-photon fluorescent signals in green (Alexa Fluor 488) and red (Rhod-2) channels are strictly restricted to inside the cell and do not contain those from the plasma membrane, while SHG signals in the blue channel arise only from the plasma membrane . ImportaIn this study, we successfully developed an SHG-specific dye, Ap3, and applied it to multimodal two-photon imaging in cultured cells as well as neurons in acute brain slices. Ap3 is distinct from other organic dyes currently available for cellular imaging, including FM4-64, owing to its SHG specificity and serves as a prototype for further SHG dye development.The repertoire of fluorescent reporters of various biological phenomena is continually expanding both for small synthetic dyes and proteins37394041131415In this study, we utilized Ap3 as a membrane marker that allows multimodal two-photon imaging with other fluorescent markers and applied it to cultured CHO cells and neurons in acute brain slices. Previous studies have shown that the juxtamembrane region has specific biochemical reactions including calcium microdomains44To improve organic dye-based SHG imaging, it is important to consider some theoretical aspects. As Ap3 is expected to undergo rapid non-radiative deactivation from the excited state to the ground state311248N,N-dihexylaniline and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate) were purchased from Wako Pure Chemical Industries. Ethyl acetate was purchased from Kanto Chemical. FM4-64 was obtained from Biotium (SynaptoRed C2). All other reagents were purchased from Tokyo Chemical Industry or Sigma Aldrich, unless otherwise noted.All reagents and solvents used for the synthesis and spectroscopic measurements were of the highest commercial quality and were used without purification. Tetrahydrofuran (THF), dimethyl sulfoxide, methanol, distilled water, acetic acid, 4-aminopyridine, tetrafluoroboric acid, sodium nitrite, 1H NMR at 270 MHz; 13C at 67.8 MHz) or AV-400FT NMR . δ values are given in parts per million relative to the peak for tetramethylsilane. Silica gel column chromatography was performed using silica gel 60N  or Wakogel 50NH2 . High-resolution mass spectra (HRMS) were measured on a JEOL AccuTOF CS. Absorption spectra were measured on a Shimadzu UV-1600. All absorption measurements were carried out at room temperature under air. See NMR spectra were recorded on a JNM-EX270  and 42% tetrafluoroboric acid (5 g) was cooled to 0 °C in an ice bath and stirred to give a milky solution. Sodium nitrite  dissolved in 5 ml of water was added slowly to the mixture under efficient stirring with at a constant temperature of 0 °C. N,N-dihexylaniline  in 3 ml of acetic acid was slowly added to the mixture under efficient stirring. Then, 5 ml of THF was added to the mixture for homogeneous mixing and was stirred for 3 h while slowly raising the temperature to room temperature. After the reaction was completed, 50 ml of 2 M sodium hydroxide was added to the reaction mixture and was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered and evaporated. The residue was purified with column chromatography , which provided a red oil as the product  with 30% yield.1H NMR δ 0.91 . 1.28–1.43 , 1.55–1.71 , 3.37 , 6.68 , 7.62 , 7.87 , 8.70 ; 13C NMR δ 14.0, 22.6, 26.7, 27.3, 31.6, 51.3, 111.1, 116.0, 126.2, 143.0, 150.0, 151.6, 158.1; HRMS (ESI) m z−1 calculated for C23H34N4 [M+H]+: 367.2856, found 367.2856.Ap3. A mixture of compound 1  and (3-bromopropyl)triethylammonium bromide  in acetonitrile was stirred at 80 °C under nitrogen atmosphere. The solution turned dark purple during the reaction. After the reaction proceeded for 24 h, the solvent was removed on a rotary evaporator. The dark purple residual oil was washed with hexane and ether. Purification by column chromatography (SiO2-amino), followed by elution with a gradient solvent system of dichloromethane:methanol  was performed three times, yielding 1.0 mg (2%) of Ap3 as a deep purple oil. The resulting Ap3 dye was dissolved in the internal solution containing 10 mM NaCl, 10 mM KCl, 135 mM KMeSO4, 2.5 mM MgATP, 0.3 mM NaGTP and 10 mM HEPES (pH 7.3) at a concentration of 10 mM.1H NMR δ 0.92 . 1.25–1.42 , 1.50 , 1.60–1.77 , 2.83–2.98 , 3.43-3.50 , 3.83 , 5.18 , 6.74 , 7.94 , 8.01 , 9.88 ; 13C NMR 8.4, 14.0, 22.6, 25.9, 26.7, 27.6, 31.5, 51.8, 54.2, 54.7, 55.7, 112.3, 118.9, 144.7, 146.2, 154.5, 162.4; HRMS (ESI) m z1− calculated forC32H55N5 [M]2+: 254.7223, found 254.7216.The absorption spectra of Ap3 and FM4-64 in phosphate-buffered saline  containing 10 mM CHAPS, THF, dimethyl sulfoxide, methanol and distilled water were measured at a 3.5-μM concentration using a Shimadzu UV-1600 ultraviolet–visible spectrophotometer. All absorption measurements were carried out in a 1-cm path length quartz cuvette at room temperature under ambient atmosphere.Fluorescence spectra of Ap3 and FM4-64 in PBS (pH 7.4) containing 10 mM CHAPS were measured using a Hitachi F-7000 fluorescence spectrophotometer at a 3.5-μM concentration with excitation and emission slit widths both set to 10 nm. The excitation wavelengths for Ap3 and FM4-64 were 490 and 500 nm, respectively. The absorbance values at the excitation wavelengths were the same for Ap3 and FM4-64 under this condition. All fluorescence measurements were carried out in a 1-cm path length quartz cuvette at room temperature under ambient atmosphere.3 in a Pylex NMR tube were irradiated at 25 °C for 0, 10, 60, 120 and 360 min using a 150-W xenon lamp (Ushio UXL-159) with a monochromator. The irradiation wavelengths were set to 570 and 500 nm for Ap3 and FM4-64, respectively, which are the absorption maxima of these dyes in chloroform.The NMR solutions of Ap3 or FM4-64 in CDClIn addition to NMR, Ap3 was also analysed by high-performance liquid chromatography using methyl orange as an internal standard. Ap3 solution was irradiated at 600 nm, which is absorbed by Ap3 but not by methyl orange, and detected at 500 nm light, which is absorbed both by Ap3 and methyl orange. The eluent was acetonitrile:water (60:40) containing 0.2% (v/v) trifluoroacetic acid.2 incubator at 37 °C until use. For imaging, CHO cells were plated on poly-L-lysine -coated coverslips  in 35-mm culture dishes  or glass-bottom dishes  coated in the same manner at a density of 5 × 104 cells per 35-mm dish.CHO cells  were maintained in culture media  in a humidified 5% CO2 and 2 mM CaCl2, pH 7.3) and applied to the cells at a final concentration of 10–20 μM. Both dyes were readily adsorbed to the membrane and reached equilibrium within minutes, without any obvious cellular toxicity during the imaging period (−1), Calcein Red-Orange , Tubulin Tracker Green , ER-Tracker Red  and Calcein  were applied to cells in the imaging buffer at the indicated concentration and incubated at 37 °C for 30 min–1 h before imaging. For multimodal imaging with EGFP and DsRed, a total of 1.5–2.0 μg of pEGFP-N1 and pDsRed-N1 empty vectors was transfected into CHO cells using 3.0–4.0 μl of Lipofectamine 2000 (Life Technologies) following the manufacturer's protocol, and imaging was performed 24–60 h after transfection.Stock solutions of Ap3 and FM4-64 were diluted in the imaging buffer  on an FV1000MPE multiphoton microscopy system (Olympus) equipped with MaitaiHP Ti:sapphire mode-locked femtosecond laser working at repetition rates of 80 MHz (Newport) and a LUMPlanFL60 × WIR2 objective lens , bubbled with 95% O2/5% CO2. In the voltage-clamp experiments, 1 mM NiCl2 and 1 μM tetrodotoxin were included in the artificial cerebrospinal fluid to block voltage-gated calcium channels and sodium channels, respectively. Glass pipettes  were filled with 100–400 μM Ap3 or 100–200 μM FM4-64 together with 100 μM Alexa Fluor 488 hydrazide (Life Technologies) in the internal solution containing 10 mM NaCl, 10 mM KCl, 135 mM KMeSO4, 2.5 mM MgATP, 0.3 mM NaGTP and 10 mM HEPES (pH 7.3), and neurons were loaded under the whole-cell patch-clamp configuration using MultiClamp 700B (Molecular Devices). In the multimodal two-photon imaging with a calcium indicator, Rhod-2 tripotassium salt  was also included in the internal solution. All recordings were performed at room temperature.All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals of Keio University. The 300-μm-thick cortical slices were prepared from 2- to 3-week-old C57/BL 6J mice of either sex. Neurons were visualized and patch clamped under IR-DIC using a micromanipulator  in a recording chamber that was perfused with artificial cerebrospinal fluid containing 126 mM NaCl, 3 mM KCl, 1.14 mM NaHNeurons loaded with Ap3 or FM4-64 were illuminated using a 950-nm laser, and TPF and SHG signals were simultaneously collected. In the frame-scan mode, the membrane potential of neurons was manipulated frame by frame in a sequential manner under current clamp  using cuPhototoxicity assays of Ap3 and FM4-64 in neurons were performed in accordance with a previous reportQuantification of the TPF and SHG signal intensities from CHO cells was performed using ImageJ software (NIH) by calculating mean signal intensities at the plasma membrane from each cell. Sensitivity of the SHG signal to the membrane potential of neurons was analysed using FV10 software and ImageJ; the average SHG signals were obtained from the pixels inside the regions of interest that were manually selected to cover the plasma of the soma . SHG and TPF intensity profiles before and after the application of SHG chromophores were generated using the ImageJ ‘plot profile' function. In all figures describing differences between groups , images taken under two different conditions are displayed on the same linear scale for comparison. In the point-scan experiments, average data from control and stimulated groups were normalized using the initial 10-ms period, when no stimulation was delivered to compensate for any systematic changes, and changes in current, voltage and SHG signals were calculated using custom software (MATLAB). To calculate voltage sensitivity, average SHG signal changes at 5–15 ms after the onset of voltage pulses were used. SHG signal intensity data under control conditions in point-scan mode were used to calculate the extent of photobleaching. Statistical analyses were performed using Origin Pro (OriginLab) using non-parametric Mann–Whitney tests because data points were not normally distributed when assessed by the Shapiro–Wilk test.How to cite this article: Nuriya, M. et al. Multimodal two-photon imaging using a second harmonic generation-specific dye. Nat. Commun. 7:11557 doi: 10.1038/ncomms11557 (2016).Supplementary Figures 1-12


Ca2+ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca2+ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC4 evokes a series of cytoplasmic Ca2+ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca2+ entry into the matrix, prevents the mitochondrial Ca2+ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca2+ oscillations appeared to be a consequence of enhanced Ca2+-dependent inactivation of InsP3 receptors, which arose from the loss of mitochondrial Ca2+ buffering. Ca2+ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca2+ release, mitochondria also sequestrated Ca2+ entry through store-operated Ca2+ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca2+.Ca Ca2+ uptake across the latter is accomplished through the mitochondrial Ca2+ uniporter (MCU). Although this transporter has been known to exist for several decades, only recently has it been identified at a molecular level. MCU comprises a membrane-spanning 40 kDa protein that forms a low conductance Ca2+-selective channel pore 2+ transporters that extrude Ca2+ from the matrix have also been characterised recently, including the mitochondrial Na+-Ca2+ exchanger 2+-H+ exchanger.Mitochondrial Ca2+ uptake by MCU is determined by both the large voltage across the inner membrane that results from proton pumping by the respiratory chain, and the Ca2+ concentration gradient between the cytoplasm and matrix 2+ that followed a cytoplasmic Ca2+ increase 2+ signalling.Ca3 and diacylglycerol 2+ oscillations that arise from regenerative Ca2+ release from InsP3-sensitive Ca2+ stores followed by Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, which refills the stores in readiness for the next oscillatory cycle. Information is encoded in the amplitude, frequency and spatial profile of the oscillation 4 evokes a series of Ca2+ oscillations 2+ entry through CRAC channels during the oscillatory responses activates the transcription factors NFAT Stimulation of cell-surface receptors that couple to phospholipase C generates the second messengers InsP2+ signals and subsequent activation of gene expression following cysLT1 receptor stimulation. We find that MCU is essential for supporting these responses, reinforcing its role as a key regulator of physiological Ca2+ signals. We also find that MCU, by buffering Ca2+ influx, is important for sustaining CRAC channel activity.In this study, we have investigated whether MCU regulates the pattern of oscillatory Ca4 evoked a series of cytoplasmic Ca2+ oscillations  of LTCllations  that decllations  and sizellations  due to r quickly , disappe quickly . The amp quickly . Similar rapidly , and at  rapidly .2+ oscillations, we compared the effects of FCCP on both mitochondrial membrane potential and the number of Ca2+ oscillations that were produced over a 600 seconds recording period. Increasing the concentration of FCCP led to a progressive depolarisation of the mitochondrial membrane potential, measured with TMRE . This is not unexpected, because inactivation of the InsP3 receptor can develop slowly, over tens of seconds 2+ transients to LTC4 were also unaffected by FCCP/oligomycin or MCU knockdown  to clamp the cell membrane potential close to 0 mV, and thus eliminate potential changes in electrical gradient for Ca2+ entry following MCU knockdown. Under these conditions the rate of cytoplasmic Ca2+ rise following readmission of external Ca2+ (5 mM) to MCU-deficient cells treated with thapsigargin in Ca2+-free solution was 28.9±4% that of controls .Previous work has established that impaired mitochondrial Cachannels . Ratiomechondria . Knockdo2+ entry . Mitochonockdown . The ratCRAC channel activation in response to thapsigargin induced robust c-fos expression and this was significantly reduced following MCU knockdown . Stimula2+ signals has been documented in numerous cell types  Is the MCU important for regulating cytoplasmic Ca2+ signals in response to physiological levels of stimulation? (ii) Through its ability to transport physiological Ca2+ pulses, does the MCU influence downstream Ca2+-dependent responses such as gene expression? (iii) Is the MCU required to sustain store-operated Ca2+ influx?The ability of mitochondria to shape the pattern of intracellular Ca2+ oscillations to modest stimulation of cysLT1 receptors are faithfully propagated into mitochondria to generate oscillatory Ca2+ signals within the matrix. Knockdown of MCU or a reduction in the electrical gradient for Ca2+ flux through the MCU accelerated the run down of these oscillations. Hence the MCU is important for mitochondrial Ca2+ uptake in response to physiological levels of cell stimulation. These new findings support and extend our previous patch clamp studies that demonstrated a central role for mitochondria in sustaining CRAC channel activity in the presence of physiological levels of intracellular Ca2+ buffering 2+ uptake is essential for STIM1 aggregation on the ER membrane following store depletion in response to an increase in InsP3. According to Deak et al., Ca2+ release through InsP3 receptors inhibits STIM1 aggregation unless mitochondria are able to buffer the released Ca2+. This mechanism could contribute to the accelerated rundown of Ca2+ oscillations evoked by LTC4 that we have found in the presence of external Ca2+ following knockdown of MCU or mitochondrial depolarisation. However, rundown was also prominent after knockdown of MCU in the absence of external Ca2+  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, as described RBL-1 cells were purchased from ATCC (via UK supplier LGC) and were cultured at 37°C with 5% CO2+ measurements were carried out at room temperature using the IMAGO charge-coupled device camera-based system from TILL Photonics, as described previously 2, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4, with NaOH. Cells were left for 15 min to allow further de-esterification. Ca2+-free solution had the following composition: 145 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, 0.1 mM EGTA, pH 7.4, with NaOH. Ca2+-free solution containing La3+ had the following composition: 145 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, 1 mM LaCl3, pH 7.4, with NaOH. Ca2+ signals are plotted as R, which denotes the 356/380 nm ratio. Rmin was 0.42 and Rmax was 2.1. LTC4 was bought from Cayman Chemicals.Cytosolic CaCells expressing the mitochondrial ratiometric pericam were analyzed 24 hours after transfection by videoimaging using the TiLL Photonics system. Cells were illuminated alternately at 430 and 488 nm (20 msec exposures) and the emitted light was filtered at >510 nm.Cells were loaded with TMRE (50 nM) in standard external solution for 30 minutes in the dark, followed by three washes in external solution. Cells were excited at 545 nm and emitted light was collected at >560 nm.4 and the % of cells expressing EGFP measured subsequently (∼24 hours later). Gene expression was defined as fluorescence 3xSD> cell autofluorescence, measured in non-transfected cells, as described 24–36 hours following transfection with the EGFP-based reporter plasmid that contained an NFAT promoter , cells were stimulated with LTCCells were transfected with the Amaxa system, as described. siRNA against MCU was from Origene (Cat No.: SR508660).Total RNA was extracted from RBL cells by using an RNeasy Mini Kit (Qiagen), as described


Plasmodium falciparum PfMDR1 have been implicated to play a significant role in the emergence of worldwide drug resistance, yet the molecular and biochemical mechanisms of this transporter are not well understood. Although it is generally accepted that drug resistance in P. falciparum is partly associated with PfMDR1 transport activity situated in the membrane of the digestive vacuole, direct estimates of the pump rate of this transport process in the natural environment of the intact host–parasite system have never been analysed. The fluorochrome Fluo-4 is a well-documented surrogate substrate of PfMDR1 and has been found to accumulate by actively being transported into the digestive vacuole of several parasitic strains. In the present study, we designed an approach to use Fluo-4 fluorescence uptake as a measure of compartmental Fluo-4 concentration accumulation in the different compartments of the host–parasite system. We performed a ‘reverse Fluo-4 imaging' approach to relate fluorescence intensity to changes in dye concentration rather than Ca2+ fluctuations and were able to calculate the overall rate of transport for PfMDR1 in Dd2 parasites. With this assay, we provide a powerful method to selectively measure the effect of PfMDR1 mutations on substrate transport kinetics. This will be of high significance for future compound screening to test for new drugs in resistant P. falciparum strains.Mutations in the multidrug resistance transporter of  Plasmodium falciparum parasites have severely compromised the ongoing global efforts to control the disease tot) according to the equationConfocal laser scanning fluorescence microscopy was performed using a Zeiss LSM710  equipped with visible laser lines and an Axiovert 200M microscope. Fluo-4 was excited at 488 nm with 1% transmission , and emission signals were detected in the range of 505–560 nm. Single images were obtained using a 63× objective  and a fivefold software zoom, 512 × 512 pixel. Fluo-4 fluorescence was assessed using cordings . Our app2+] does not change during loading conditions free) was given. The time course of fluorescence, F(t), in the respective compartments was normalized to the initial background condition, F(0), at time-point zero (immediately after adding Fluo-4), to compare relative fluorescence between strains and batches and to avoid artefacts from fluctuating background conditions. For the time course of normalized F(t), the equilibrium described by Eq. (i.e. ∼7.2 in the parasite cytoplasm and ∼5.2 in the DV). pH effects on F(t) cannot be calculated from Eq. (t) to steady-state fluorescence for a given total [Fluo-4]tot and [Ca2+]free at a given pH, an in vitro calibration was performed as described below, which served as input to convert F(t) values from the live-cell recordings to [Fluo-4]tot concentrations. This conversion enabled us to calculate kinetics of absolute Fluo-4 dye accumulation characteristics in the compartments in situ.because intracellular  values was performed. Intracellular solutions were based on ‘high Ca2+’ and ‘Ca2+ free’-solutions . HA consists of 30 mM Hepes, 30 mM EGTA, 30 mM CaCO3, 7.46 mM Mg(OH)2, 10 mM Na2-creatine phosphate, 8 mM Na2ATP; HR is the same as HA except with 8.1 mM Mg(OH)2 and omitting CaCO3. The pH was adjusted either to 5.2 or 7.2 using KOH or HCl. Mixtures of HA:HR were pipetted at ratios ranging between 0 and 0.9 in 96-well plates in a matrix, where rows represented HA:HR ratios and columns were supplemented with the respective Fluo-4 salt concentrations. Plates were prepared at least in duplicates for pH 5.2 and pH 7.2, respectively. After ∼30 min. incubation on a rocker shaker, Fluo-4 fluorescence in each well was assessed using a Synergy H4 plate reader . Each well corresponded to a Fluo-4 value at a given pH, [Fluo-4]tot and total [Ca2+]. To convert total [Ca2+], ([Ca2+]tot) (which was the same irrespective of pH) to free [Ca2+] concentrations [Ca2+]free , the chelating program React II was used . This program calculates [Ca2+]free from known solution compositions of Ca2+-buffered solutions with known [Ca2+]tot and pH using published critical stability constants. Using this conversion, Fluo-4 fluorescence values at a given pH and [Ca2+]free, normalized to pCa 9 (background), could be compared with the normalized F(t) values from the live-cell P. falciparum-infected erythrocyte recordings to calculate absolute [Fluo-4]tot concentrations, after establishing calibration curves for each [Fluo-4]tot concentration over the complete [Ca2+]free range.To convert normalized Fluo-4 fluorescence, F/F(0), in the compartments to absolute dye concentrations at any given time, and to account for the pH differences affecting the dye buffer properties , an P. falciparum-infected erythrocytes were readily identified by eye. Using the Zen software (Carl Zeiss), a region of interest within the host–parasite system, e.g. the DV, the cytoplasm of the parasite, the cytosol of the infected RBC or the uninfected RBC, was defined. The average pixel intensity of the region of interest was calculated. The measured fluorescence was further analysed using Excel , Sigma Plot , and ImageJ 1.47g . Fluorescence values were normalized to the initial condition, i.e. time-point zero after Fluo-4 incubation, F(t) = F/F(0), for in situ time series of Fluo-4 uptake. Images were processed using Corel Draw and Corel Photopaint .pfmdr1 was verified in our laboratory HB3 and Dd2 strains. Parasite strains were grown to ≥5% parasitaemia and DNA extracted using the QIAamp DNA Blood Mini Kit  according to the manufacturer's instructions. The DNA was amplified in overlapping PCR fractions using HotStarTaq DNA polymerase (Qiagen Inc.). To account for the AT-rich nucleotide content in the P. falciparum genome, dNTPs  were mixed at 75% AT and 25% GC. For PCR optimization, 2 mM MgCl2, 300 μM dNTPs and 300 nM primers were used for the reaction. For each reaction mix, a total of 20 ng genomic DNA was used. PCR reactions consisted of an initial activation step of 94°C for 3 min. followed by 35 cycles of 94°C for 60 sec., 49–61°C (adjusted for each primer pair) for 30 sec. and 72°C for 1 min. Primers used for pfmdr1 gene sequencing were: pfmdr1.1_F: 5′-AT ATATGTGTACATAGCTTATTTCA, pfmdr1.1_R: 5′-TTGTACTAAACCTATAGA TACTAATGA, pfmdr1.2_F: 5′-GTTTAAATGTTTACCTGCACAACATAGAAAA, pfmdr1.2_R: 5′-CTCCACAATAACTTGCAACAGTTCTTA, pfmdr1.3_F: 5′-ATGCACGTTTGACTTTATGTATTAC, pfmdr1.3_R: 5′-GGATCTTGACTAACAACTCCAA, pfmdr1.4_F: 5′-CTTTATGATCCAACCGAAGGAGATA, pfmdr1.4_R: 5′-GTTATCCGATCCATTATCATTTCCA, pfmdr1.5_F: 5′-ACAAGGTACACATGATAGTCTTATGA, pfmdr1.5_R: 5′-GGATTTCATAAAGTCATCAACTAATATAG, pfmdr1.6_F: 5′-CTGTAATTTGATAGAAAAAGCTATTGATTA, pfmdr1.6_R: 5′-CCATATGGTCCAACATTTGTATC, pfmdr1.7_F: 5′-CAATAGTTAGTCAAGAACCCATGTTA, pfmdr1.7_R: 5′-TCAATATAACGGACAAGAGTTGATAC, pfmdr1.7.1_F: 5′-ACCAATCTGGATCTGCAGAAGA, pfmdr1.7.1_R: 5′-TCTCAGAATTGGAATCAAGTGATGA. If DNA amplification using pfmdr1.7 primers was unsuccessful, the PCR was repeated with the alternative primers pfmdr1.7.1. Samples were sent for sequencing to Genome Quebec, Canada and analysed using the BioEdit software free-[Fluo-4]tot landscape of the in vitro calibration is shown in Figure Kd values of the Fluo-4–Ca2+ buffer reaction for all [Fluo-4]tot investigated at each pH . The evaluated Kd values for the Ca2+–Fluo-4 system indicate a roughly 40 times lower affinity for Fluo-4 Ca2+ binding at pH 5.2  from past Fura-Red studies , Eq. (compartment) curves , Eq.  was usedves Fig. .bulk and internal total [Fluo-4]compartment for each compartment using the steady-state values  fitted well with a ligand-binding model containing a one-site saturation plus a non-specific binding site of the equationUsing the kinetics time course data Fig. , we sougues Fig. A. All covia PfMDR1  of this transporter is required. Rather than expressing PfMDR1 in an artificial expression system, which could introduce artefacts to its mode of action, our approach deduces PfMDR1 pump rates directly from intact host–parasite Fluo-4 fluorescence uptake data. Calculating the overall transport rate of PfMDR1 required the conversion of Fluo-4 uptake fluorescence into absolute Fluo-4 concentrations. For this, several parameters were quantified. First, the apparent steady-state Kd was calculated in the respective compartments of live parasites, as these values are profoundly influenced by environmental parameters such as pH, temperature and ionic strength  were assumed as fixed for each compartment and taken from our previous work, where we used Fura-Red to measure resting Ca2+ concentrations in Dd2 and HB3 parasites [i.e. 5.2 and 7.2) and varying known free Ca2+ concentrations was required to extract the absolute and normalized Kd values for all Fluo-4 concentrations evaluated at pH 5.2 and 7.2 in this study. With this information, absolute Fluo-4 concentrations for the various compartments of the host–parasite system were obtained for the first time.To better understand the role of PfMDR1 in multidrug resistance, a more comprehensive biochemical characterization  import kinetics. The methodology can be implemented to monitor drug resistance associated with distinct mutations in the transporter and will be used to screen small molecule libraries available in our laboratories. We are now able to identify which PfMDR1 amino acid mutations can more effectively modulate the transport of any given drug or substrate into the DV. In turn, this will allow for a better understanding of the transporter's mode of action and possibly reveal its natural substrates to us.In conclusion, this is the first direct quantification of


Previous reports established that homozygotic murine RyR2-P2328S (RyR2S/S) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2S/S hearts to connexin-43 (Cx43) and Nav1.5 expression and Na+ current (INa). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2S/S hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2S/S than WT, but comparable changes in AP durations (APD90) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2S/S compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced INa in RyR2S/S ventricles. We thus attribute arrhythmogenesis in RyR2S/S ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing INa, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak INa of the AP but nonlinear relationships between peak INa and maximum Na+ permeability.Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca Additionally, the Ca2+ channel blocker verapamil and the Ca2+ ionophore calcimycin increased and decreased Nav1.5 mRNA and Nav1.5 protein expression respectively [2+], by chronic treatment with high extracellular [Ca2+] and [K+] or BAPTA-AM, decreased and increased Na+ current densities, respectively [2+] were capable of reducing both Na+ current density and (dV/dt)max.The atrial and ventricular phenotypes are replicated by the izations , 22. Howpression , connexipression , 23, or pression . Interespression , 11, 42 pression  to be moectively . More rev1.5 by cytosolic [Ca2+] identifies both direct and indirect Ca2+ binding sites on Nav1.5. Direct Ca2+ binding to Nav1.5 is mediated at an EF hand motif resident at the carboxy-terminal region of Nav1.5  [2+] would result in increased sodium-calcium exchanger (NCX) activity whose electrogenic actions would result in triggering events including delayed after-depolarizations leading to ectopic APs that could potentially initiate ventricular arrhythmia. However, these initial studies did not explore for the presence or otherwise for arrhythmic substrate that could sustain the resulting arrhythmia.The murine genotype , 40. RyReostasis  thought s [Ca2+] . The conRyR2 are also associated with AF phenotypes . In the long term, chronic elevations in cytosolic [Ca2+] can result in a downregulation of either Nav1.5 expression or activity, thereby reducing action potential conduction and resulting in arrhythmic substrate. In such a situation, short-term triggering events could potentially form a means for initiating electrical events then perpetuated by the pre-existing arrhythmic substrate. These findings may have broader implications for the mode of therapeutic intervention in a variety of Ca2+ dependent, and potentially some Nav1.5 dependent, arrhythmia.Our findings therefore suggest that the arrhythmic substrate results from reduced expression of Na


BCR–ABL fusion oncogene. The development of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of CML, as TKI therapy leads to inhibition of BCR–ABL activity, suppression of the BCR–ABL-containing clone and restoration of normal hematopoiesis in the vast majority of cases.Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome, an acquired clonal abnormality resulting from translocation of chromosomes 9 and 22, and the generation of the BCR–ABL-containing clone in CML, it has been associated with the development of non-BCR–ABL rearranged (Ph−) clonal cytogenetic abnormalities (CCA).4 CCA have been observed in 3–10% of cases after complete cytogenetic resolution of the BCR–ABL fusion oncogene, occurring 6–18 months after initiation of TKI therapy.3 These abnormalities—suggestive of clonal hematopoiesis—are often transient and not associated with CML progression. However, as routine cytogenetic monitoring during TKI therapy is rarely performed, the occurrence of CCA may be underestimated.4Although TKI therapy is remarkably effective in clearing the − MDS/AML (myelodysplastic syndrome/acute myeloid leukemia) following TKI therapy has been reported to occur infrequently and, in contrast to Ph− CCA, is associated with poor outcomes.6 Analysis of two CML patient cohorts treated with TKI therapy reported 2/985 and 3/1701 patients subsequently developed MDS/AML.5 Both studies were published with relatively short follow-up, raising the possibility that they may also underestimate the prevalence of Ph− AML.Development of PhBCR–ABL.7 This scenario raises the possibility of a 'multi-hit' mechanism of CML development in which an early mutation(s) occurs and predisposes the premalignant clone to acquisition (or tolerance) of BCR–ABL. A high frequency of somatic mutations was reported in genes associated with myeloid malignancies in Ph− cells of CML patients treated with TKI.7 Reports published prior to the TKI era suggested that multiple events are required for CML pathogenesis .4 In this scenario, one could hypothesize that the early mutation(s) that cooperates with BCR–ABL could also cooperate with mutations that lead to AML. Thus, a premalignant clone could increase susceptibility to both leukemias. Alternatively, it is possible that some CML patients harbor an abnormal bone marrow stroma that predisposes them to both the acquisition of BCR–ABL and other genetic aberrations (that could lead to MDS or AML), which could arise in distinct founding clones.The relationship between CML and other clonal abnormalities that arise after TKI therapy is unclear, but they have been theorized to result from the unmasking of a premalignant clone that existed before acquisition of − AML (Case Synopses). Both patients achieved durable complete molecular remissions before being referred to our center with AML. Consistent with the multi-hit model of leukemogenesis, we hypothesized that both malignancies were clonally related, having arisen from the same premalignant clone, and thus expected the presence of shared variants between the CML and AML.Here we report two patients with CML treated with TKIs who achieved complete molecular remission but subsequently developed Ph8 Sequence analysis was performed using the Genome Modeling System.9 Mean coverage for filtered variants was over 100 × for all samples with a required minimum coverage of at least 30 ×. For validation, Ion Ampliseq custom panels  were constructed containing each variant (n=85). Unfortunately, validation of case 1 variants failed for most amplicons and further analysis could not be performed due to exhaustion of the sample. All case 2 exome variants validated in this manner were found to be present with variant allele frequencies (VAFs) that correlated well with the exome sequencing results  with a median depth of 2264 ×  development. To test whether it was present in non-malignant hematopoietic cells, we sequenced DNA from sorted peripheral blood T lymphocytes and neutrophils obtained after AML therapy . By Sanger sequencing, the EEF1A1 deletion was observed in the AML and CML samples but not the T cells .14EEF1A1 was not detected above background (VAF 0.15%) in this cell population. Although the presence of the EEF1A1 variant in both the AML and CML samples suggests a common clonal origin, the large number of unique variants in each sample, the absence of this variant in the post treatment neutrophils and the lack of other shared variants suggest that each disease arose from a non-malignant clone that diverged early in development. Therefore, it is possible, but unlikely, that the EEF1A1 deletion, which has not been described previously, contributed to leukemogenesis.The number of filtered variants found in each sample was comparable with that reported previously. T cells ; however T cells . We readTET2, a previously reported missense mutation in PRPF8 and a highly recurrent frameshift deletion in NPM1 , suggesting these variants may act as driver mutations in this case , a gene found mutated in two other AML cases in the TCGA data set.13 It is unclear what role mutations in this gene may have in AML pathogenesis, and there were no other mutations in genes that have been identified as recurrently mutated in AML in this sample. In our CML samples, there were no mutations in genes considered likely drivers of myeloid disease. Still, we cannot rule out the possibility that some of the somatic variants in the CML clones may cooperate with BCR–ABL to promote disease.Among the somatic variants in the case 1 AML, we observed an in-frame deletion in his case .13, 14 An=12–28) were observed in each sample. Although both CML and AML are relatively rare diseases, it is possible that in our patients they each arose independently by chance. Alternatively, environmental, stromal or epigenetic factors may predispose some patients to separate hematologic malignancies that arise from distinct cell clones. We also cannot rule out the possibility of TKI-induced secondary malignancy.3Our findings suggest that CML and AML arose independently in both patients. It is unlikely that the absence of common variants was the result of limiting sequencing analysis to the 'exome' (in contrast to whole-genome sequencing), as numerous unique variants (+ clone does not lead to restoration of normal hematopoiesis. Schmidt et al.7 reported mutations in leukemia-associated genes shared between Ph+ and Ph− clones in a subset of CML patients with CCA, indicating that the Ph+ and Ph− clones were derived from a common progenitor that predated the acquisition of BCR–ABL. Although the low incidence of secondary malignancy prevents a definitive conclusion that patients being treated for CML are not at increased risk for subsequent MDS/AML, we did not identify the anticipated clear clonal relationship or shared leukemia-associated mutations between the initial CML and subsequent Ph− AML in our patients.In summary, in a subset of CML cases, regression of the Ph