Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1.DOI:http://dx.doi.org/10.7554/eLife.12856.001 A mammal’s sense of hearing and balance are helped by so-called hair cells within the inner ear. These cells are named after their long, hair-like protrusions called stereocilia, and mutations in the genes involved in stereocilia development and maintenance can lead to hearing loss in humans. Damage to the stereocilia caused by excessive exposure to loud noises can also have the same effect.Stereocilia are full of filaments made of a protein called actin. Other proteins called class III myosins and Espin are also both required for normal development of stereocilia. Mutations in the genes that encode these proteins can cause hereditary deafness in humans. However, it remains unclear exactly how these two proteins (myosins and Espin) interact with each other in stereocilia.Using biochemical and structural studies, Liu, Li et al. have now discovered that the so-called ‘tail’ part of the myosins contains a pair of repeated sequences that can each interact with an Espin protein called Espin1. This interaction allows each myosin to cross-link two Espin1 proteins. Espins assemble actin filaments into bundles, and further experiments showed that this cross-linking interaction between myosins and Espins helped this process, which is linked to stereocilia development and maintenance.Mammals actually have two related copies of class III myosins that play overlapping but slightly different roles. The next challenge will be to try to understand the differences between these related proteins, as well as to try to uncover the roles of other forms of Espin in stereocilia.DOI:http://dx.doi.org/10.7554/eLife.12856.002 Drosophila photoreceptors and named as NinaC  (Class III myosins (Myo3), together with class IX myosins, are two special groups of the myosin superfamily as these two sub-families of actin motors contain enzymatically active domains and thus are regarded as motorized signaling molecules . The firntial C) . There antial C) . It is bntial C) .Drosophila NinaC contains a PDZ binding motif at its very C-terminus capable of binding to a master scaffold protein called INAD   . Vertebrntial D)   . The THDntial D) . HoweverEspin. Later, shorter spliced isoforms of Espin gene products  were shown to be expressed in other F-actin rich structures such as brush border microvilli and Purkinje cell dendritic spines  motif; ARB1 in the next turn insert into the hydrophobic pocket in the N-terminal of Espin1-AR  insert into the negatively charged pocket (ARB2 and Lys1283ARB2) with alanines decreased its binding to Espin1-AR by ~10-fold   . Fully c~0.1 μM) . Given t~0.1 μM) , one migIt was shown that a synthetic peptide encompassing the Myo3a-ARB1 sequence identified here can stimulate the actin binding activity of xAB . Based oMyo3a is known to localize at the tip of filopodia when transfected in heterologous cells like HeLa or COS7 cells, whereas Myo3b alone cannot tip-localize as it lacks ABM . HoweverOur above structural and biochemical characterizations of the Myo3 and Espin1 interactions point to a likely regulatory role of Myo3 on Espin1’s actin binding and bundling activity. It can be deduced that binding of Myo3-ARBs can first release the auto-inhibited conformation of Espin1. Perhaps more importantly, formation of Myo3/Espin1 complex leads to two Espin1 molcules to be juxtaposed to each other, forming a Myo3 cross-linked Espin1 dimer. Due to the large space between the N-terminal AR and C-terminal ABD in Espin1 , this MyThe most important findings of this study are the structure-based discovery of two strong and independent Espin1 binding sites in the tail region of each class III myosins, and Myo3 binding-induced auto-inhibition release of Espin1. These discoveries, together with our cell-based filopodia formation and localization assay and microscopic-based actin bundling assay, provide compelling evidences showing that Myo3 and Espin1 work together to assemble and promote higher order parallel actin bundle formation in cellular processes such as stereocilia. We believe that our model reveals the probable underlying molecular mechanism of hearing loss development (stereocilia degeneration) in humans with mutations in Myo3a (DFNB30) . It is bjerker mice are thinner in their diameters likely due to loss of Espin1-mediated higher order actin fiber assembly, easier to form tapered structures and easier to degenerate presumably due to stability decrease of less bundled actin fibers in stereocilia  . Such lamyo3a or myo3b or defects in espin1 or espin-like may not always develop severe phenotypes in vertebrates. Moreover, we note that forked, the Drosophila ortholog of Espin, is expressed in bristle cells, a cell type that is enriched in bundled actin filaments  and Espin1-AR  were PCR amplified from mouse cDNA library. The full-length human Myo3a and Espin1 plasmids have been described earlier . The mouEscherichia coli BL21 (DE3) except for Myo3a-ARB12 and Myo3a-ARB2 which were expressed in Escherichia coli Rosetta (DE3). The N-terminal thioredoxin-His6-tagged or His6-tagged proteins were purified with a Ni Sepharose 6 Fast Flow column and subsequent Superdex-200 prep grade size-exclusion chromatography.All proteins were expressed in Protein samples  was injected into an AKTA FPLC system with a Superose-12 10/300 GL column  using the column buffer of 50 mM Tris-HCl (pH 7.8), 1 mM DTT, 1 mM EDTA, and 100 mM NaCl. The chromatography system was coupled to a multi-angle light scattering system equipped with a 18 angles static light scattering detector  and a differential refractive index detector . The elution profiles were analyzed using the ASTRA 6 software (Wyatt).Crystals of the Espin1-AR/Myo3b-ARB1 complex and Espin1-AR/Myo3b-ARB2 complex  were obtained by sitting drop vapor diffusion methods at 16°C. The crystals of the Espin1-AR/Myo3b-ARB1 complex were grown in buffer containing 0.2 M lithium acetate and 20% w/v PEG3350 and soaked in crystallization solution containing additional 25% glycerol for cryoprotection. The crystals of Espin1-AR/Myo3b-ARB2 complex were grown in buffer containing 1.6 M ammonium sulfate, 0.1 M Tris pH 8.0 and soaked in crystallization solution containing higher concentration of ammonium sulfate for cryoprotection. Diffraction data were collected at the Shanghai Synchrotron Radiation Facility BL17U at 100 K. Data were processed and scaled using HKL2000 .http://www.pymol.org). The coordinates of the structures reported in this work have been deposited to PDB under the access codes of 5ET1 and 5ET0 for the Espin1-AR/Myo3b-ARB1 and Espin1-AR/Myo3b-ARB2 structures, respectively.Structure of the Espin1-AR/Myo3b-ARB1 complex was solved by molecular replacement with the model of short ANK repeats (1N0R) using PHASER . Structu200 at 25°C, except for the two endothermic titrations which were performed at 16°C. Titration buffer contained 50 mM Tris-HCl pH 7.8, 1 mM DTT, 1 mM EDTA and 200 mM NaCl. Each titration point was performed by injecting a 2 μL aliquot of a protein sample from a syringe into a protein sample in the cell at a time interval of 120 s to ensure that the titration peak returned to the baseline. The titration data were analyzed by Origin7.0 .Isothermal titration calorimetry (ITC) measurements were carried out on a MicroCal iTC2 in air. For transfections, 30–40 × 103 cells were plated on acid washed 22 mm square 1.5# glass coverslips and allowed to adhere over-night. 24 hr later, cells were transiently transfected using FUGENE HD transfection reagent (Promega) as per manufacturer’s protocol and imaged after ~20–30 hr.COS7 cells were cultured in Dulbecco's Modified Eagle Medium (Corning) supplemented with 1 mM Sodium Pyruvate, 4 mM L-glutamine, 4.5 g/L D-Glucose, 10% fetal bovine serum (FBS) (Gemini), and 100 units of penicillin-streptomycin (Corning). Cultured COS7 cells were maintained at 37°C with 5% COt test.For live cell imaging, the coverslips with transfected cells were placed in rose chambers filled with Opti-MEM media without phenol red (Life Technologies) and supplemented with 5% FBS (Gemini) and 100 units of Penicillin-streptomycin. Images were acquired by using a TE2000-PFS fluorescence microscope (Nikon Instruments) with a 60x/1.4 N.A. phase objective. Image acquisition was managed by NIS-Elements AR (Nikon Instruments) and the tip to call body measurements were done using ImageJ (ND2 plugin), as described previously . Data ar2, 0.2 mM ATP, 0.5 mM DTT, pH 8.0, on ice for 1 hr and centrifuged at 150,000 g for 10 min at 4°C. Actin concentration in supernatant was determined by NanoDrop. Actin was polymerized at room temperature for 1 hr after adding one-tenth volume of 10×polymerizing buffer . Bundles were prepared by mixing 5 µM F-actin with 1.25 µM Espin1 and incubating at room temperature for 1 hr, with or without 1.25 µM Myo3a-ARB1/2 or 0.625 µM Myo3a-ARB12 added in the mixtures. For fluorescence microscopy, F-actin were labeled with Alexa Fluor 555 Phalloidin. Aliquots (5 µl) were delivered onto microscope slides. The cover slips were then placed over the drop of samples gently. All the samples were imaged using a Fixed Stage Upright Microscope (Olympus). Samples for TEM (FEI Tecnai 20) were adsorbed to glow-discharged, carbon-coated formvar films on copper grids for 1 min and negatively stained with 0.75% (m/v) uranium formate for 30 s.Rabbit skeletal muscle actin (Cytoskeleton) were hydrated in 5 mM Tris-HCl, 0.2 mM CaCl In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor) and three reviewers, one of whom, Cynthia Wolberger, is a member of our Board of Reviewing Editors, and another is James Bartles.Thank you for submitting your work entitled "Myosin III-mediated Dimerization and Stimulation of Actin Bundling Activity of Espin" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.This manuscript sheds light on the molecular details underlying the way in which Myo3 binds to Espin1 to regulate actin bundle assembly in hair cells. Espin1 contains a set of ankyrin repeats that bind to Myo3. The authors identify two related sequences within Myo3, ARB1 and ARB2, and show that these sequences bind independently to the ankyrin repeat domain (ARD) of Espin1. Structures of the Espin1 ankyrin repeats bound to ARB1 and to ARB2 peptides from Myo3 show that each binds similarly to the ARD, suggesting that a single Myo3 can trigger dimerization of Espin1. The authors also provide structural and biophysical evidence that the Espin1 autoinhibitory sequence, which inhibits Espin1 binding to actin, resembles the two Myo3 ARB sequences and competes for a common site in the ankyrin repeats. Studies in hair cells support a role for the ARB1/2-ARD interaction in colocalization of Myo3 and Espin1, while light microscopy and electron micrographs provide evidence that Espin1 mediates higher order bundling of actin.Review summary:All three reviewers were enthusiastic about this study and its combination of approaches that provide fresh insights into Myo3-Espin1 interactions in hair cells. There were several concerns raised by the reviewers, one of which will require further experiments, with the remainder most likely addressable using available data and through revision of the figures and text.Essential revisions:d and switch from exothermic to endothermic binding reaction. The authors should assay binding of ARB either to an Espin1 fragment truncated just before the AI sequence (and no more) or introduce mutations in AI domain residues that relieve autoinhibition, and then show that Myo3a binds more tightly to this Espin1 fragment.1) The ITC data showing a lower affinity of Myo3a-ARB1 for full-length Espin1 or a fragment extending through the inhibitory domain are consistent with an autoinhibition model. However, these data do not show that it is the putative AI domain, as opposed to some other region of Espin1, that is responsible for the elevated K2) The authors state that ARB1 and ARB2 bind in a similar manner to the ankyrin repeats, yet the cartoon/ribbons figures in panels 2A and 2D appear to show differences that could reflect somewhat different contacts, at least for a subset of the residues. There should be a detailed comparison of these two structures in figures that would enable the reader to do a side-by side comparison of contacts by ARB1 versus ARB2. Differences in the way in which the two bind to the AR may underlie the apparent differences that mutations in ARB1 versus ARB2 have in the in vivo experiments, where mutations of the Tyr residues in ARB2 seem to have a much stronger effect on recruitment . It may 3) The schematic  which sh4) One issue that needs to be discussed further in the context of the proposed mechanism is the relatively low levels of these two proteins in the stereocilium. For chick utricle, the data are listed in the Shin et al. 2013 Nature Neuroscience paper, which is included in the current list of references.5) The authors should explicitly acknowledge the different methodology used in the current manuscript, which uses post-polymerization bundling, and the previous study by Zheng et al. (2014), which used co-polymerization bundling to examine Espin 1 autoinhibition and the regulation of the Espin 1 xAB domain by the MYO3 peptide.6) The use of the term "dimerization" is misleading and should be replaced with another term that more accurately describes the fact that the two adjacent ARB motifs, each of which recruit an Espin, clusters two Espins close to one another. Essential revisions:d and switch from exothermic to endothermic binding reaction. The authors should assay binding of ARB either to an Espin1 fragment truncated just before the AI sequence (and no more) or introduce mutations in AI domain residues that relieve autoinhibition, and then show that Myo3a binds more tightly to this Espin1 fragment.1) The ITC data showing a lower affinity of Myo3a-ARB1 for full-length Espin1 or a fragment extending through the inhibitory domain are consistent with an autoinhibition model. However, these data do not show that it is the putative AI domain, as opposed to some other region of Espin1, that is responsible for the elevated KThanks for the great suggestions. We have performed ITC titration experiments between Myo3a-ARB1/2 and Espin1-1-494 (truncated right before the AI sequence). Consistent with our prediction, the affinities are in a similar range with that between Myo3a-ARB1/2 and Espin1-AR. The bindings of ARB1/2 to Espin1-1-494 also become exothermic. We have integrated the ITC data between Myo3a-ARB1 and Espin1-1-494 into 2) The authors state that ARB1 and ARB2 bind in a similar manner to the ankyrin repeats, yet the cartoon/ribbons figures in panels 2A and 2D appear to show differences that could reflect somewhat different contacts, at least for a subset of the residues. There should be a detailed comparison of these two structures in figures that would enable the reader to do a side-by side comparison of contacts by ARB1 versus ARB2. Differences in the way in which the two bind to the AR may underlie the apparent differences that mutations in ARB1 versus ARB2 have in the in vivo experiments, where mutations of the Tyr residues in ARB2 seem to have a much stronger effect on recruitment . It may Following the reviewers’/editor’s suggestion, we have added a paragraph and a supplementary figure  discussi“Comparing the structures of Espin1-AR in complex with Myo3b-ARB1 and Myo3b-ARB2, the “YY” motif and the “KxL” motif are essentially in the same places . Despite the high similarity, there are still a few minor differences. First of all, the C-terminal α-helix of ARB2 is shorter. The interaction is mediated by a hydrogen bond between Asp1294 and Tyr144, instead of the more extensive interaction observed in Myo3b-ARB1 complex . Moreover, the positively charged residues in the N-terminus of ARB2 cannot be reliably built, probably due to the high salt concentration in the crystallization buffer (1.6 M ammonium sulfate). Nonetheless, clear electron density can be observed near the negatively charged surface . Indeed, substitutions of these positively charged residues with Ala weakened the binding .”3) The schematic  which shThanks for pointing this out. We have modified the schematic representation to include the involvement of xAB and ABM of Myo3a in the revised 4) One issue that needs to be discussed further in the context of the proposed mechanism is the relatively low levels of these two proteins in the stereocilium. For chick utricle, the data are listed in the Shin et al. 2013 Nature Neuroscience paper, which is included in the current list of references.The reviewers/editors have raised a very interesting point. The protein level for Espin in stereocilia is relatively low when comparing to fascin and plastin, two additional actin filament crosslinking proteins. It is unlikely that Espin plays a major structural role in the cross-linking of F-actin in stereocilia. When reading the Nature Neuroscience paper by Shin et al. 2013, it is interesting to note that the protein level for Myo3a/b together is at a similar range as Espin (a few hundred copies per stereocilium). What we propose here  is that Myo3 and Espin  might work together to perform certain regulating role in elongation and widening of actin bundles. We have expanded our Discussion in the revised manuscript by adding the following:“It has been proposed that Espin may play a regulating role in elongation and widening of actin bundle by concentrating at the tip of stereocilia instead of major structural cross-linking roles . Interestingly, the abundance of Myo3 (both Myo3a and Myo3b together) is similar to that of Espin (a few hundred copies per stereocilium) , indicating that Myo3 and Espin1 may work together to regulate higher order actin bundle structure formation and stability in stereocilia.”5) The authors should explicitly acknowledge the different methodology used in the current manuscript, which uses post-polymerization bundling, and the previous study by Zheng et al. (2014), which used co-polymerization bundling to examine Espin 1 autoinhibition and the regulation of the Espin 1 xAB domain by the MYO3 peptide.Thanks for pointing this out. We have modified the manuscript to acknowledge the different methodology in the current manuscript as follows:“By adding ARB1 or ARB2 to Espin1 and F-actin containing solution, some of the actin bundles were cross-linked and formed clusters under FM and TEM . Similar6) The use of the term "dimerization" is misleading and should be replaced with another term that more accurately describes the fact that the two adjacent ARB motifs, each of which recruit an Espin, clusters two Espins close to one another.Thanks for alerting us on this point. We now use the term “cross-linking” which should be more accurate in describing assembly of two Espin1 molecules by the two adjacent ARB motifs of Myo3.


Although Ca2+ signaling in astrocytes has been intensively studied in the past, our understanding of the signaling mechanism and its impact on tissue level is still incomplete. Here we revisit our previously published data on the strong temperature dependence of Ca2+ signals in both cultured primary astrocytes and astrocytes in acute brain slices of mice. We apply multiscale modeling to test the hypothesis that the temperature dependent [Ca2+]i spiking is mainly caused by the increased activity of the sarcoendoplasmic reticulum ATPases (SERCAs) that remove Ca2+ from the cytosol into the endoplasmic reticulum. Quantitative comparison of experimental data with multiscale simulations supports the SERCA activity hypothesis. Further analysis of multiscale modeling and traditional rate equations indicates that the experimental observations are a spatial phenomenon where increasing pump strength leads to a decoupling of Ca2+ release sites and subsequently to vanishing [Ca2+]i spikes.Changes in the cytosolic Ca The human brain is by far the most complex organ; it hosts our consciences, is the source of our inspiration, and controls many of our vegetative functions. The extraordinary complexity of cellular interactions in terms of structural as well as dynamic properties enables the huge flexibility of our behavior and the amazing plasticity required for learning . While tGlia cells are generally known for their supportive function for neurons. The minority of glia cells are oligodendrocytes that myelinate axons . More reBesides their supportive role in metabolism, astrocytes are now also known to modulate neuronal activity through active signaling mechanisms. Astrocytes are sealing up the dyadic cleft facilitating neurotransmitter diffusion and are essential for glutamate uptake and recycling . Since a3) mediated Ca2+ signaling. In general, Ca2+ signaling is a versatile and universal second messenger that translates extracellular signals into intracellular responses like molecular motor activation or modulation of gene expression i) to single [Ca2+]i transients. The most common Ca2+ signals in astrocytes are repetitive cytosolic [Ca2+]i transients often referred to as [Ca2+]i oscillations [2+]i spiking.The collection of the Catool kit  can genellations  that are2+ signaling is frequency coded [2+]i spiking is indeed stochastic and therefore not truly oscillatory [2+]i time courses for hundreds of cells of different cell types and stimulation antagonists, we found a preserved linear relation between the average period (Tav) and the standard deviation (σ) of the inter-[Ca2+]i-spike intervals. Applying methods from the theory of stochastic processes, we have shown that this pathway specific linear σ-Tav relation characterizes the internal feedback strength i activity has been reported for astrocytes in culture i activity in cultured astrocytes compared to astrocytes within acute brain slices is only barely investigated [2+]i activity is typically measured. Here we revisit the experimental data and integrate it with our mechanistic multiscale modeling framework to quantitatively test our hypothesis on the molecular foundation of the temperature dependency.Despite all these insights, the common mechanism of this spontaneous activity is still not fully understood. In particular, the higher abundance of spontaneous i dynamics changes and use here computational modeling to investigate potential reasons for this phenomenon.Motivated by the typically observed and reported significant differences in Caworkflow . The mosworkflow , 48. In constant . While tIn the experimental study  we used 2+ activity at room temperature and separately at more physiological temperatures with Fluo4-AM loaded to the cells before . In cor2+ activity at room temperature (20°C) and then abruptly changed the perfusion buffer to one of 28°C or of 30°C in another setup. For these cultured astrocytes we observed a similar significant decrease of cells exhibiting Ca2+ signals already within the first minute after the temperature shift  of individual Ca2+ transients by the full width half maximum (FWHM) method. Despite the cell specific heterogeneity, spike durations of the Ca2+ transients of astrocytes in acute brain slices decreased significantly when the temperature was raised from 24°C to 37°C. The decrease from 45 ± 17 s to 13 ± 4 s was fully reversible, since lowering the temperature back to 24°C increased the spike duration again to 45 ± 18 s . We additionally determined the spike duration at 31°C which averaged at 22 ± 5 s   .T30°C 15 cells, 26 spikes; at T20°C 30 cells, 56 spikes) .Within the analogous analysis of cultured astrocytes, we found that the spike duration changed from an average of 63 ± 18 s at 20°C to 21 ± 5 s at 30°C. This effect of temperature on spike duration was again reversible as spike duration increased again to 72 ± 20 s at 20°C after lowering the temperature from 30°C  . Within f(T) = aebT− with a = 620 s and b = 0.109/°C indicating a functional relation between spike width and temperature.The similarity of the signal form and particularly of the quantified spike widths of cells obtained from cultures and brain slices around 30°C points to temperature as the main reason for the typically reported differences in the signals. To analyze this behavior further we plotted the temperature dependence of the spike duration by pooling data from the two different experimental systems. The resulting data points can be fitted nicely by the exponential function 2+ influx and intracellular Ca2+ release from intracellular stores. While Ca2+ free buffer has reduced the total number of responsive cells only slightly to 78 ± 34% , blocking SERCA pumps by loading 1 μM thapsigargin for 15 min has abolished Ca2+ signals in more than 90% of cells at 31°C. Overall, these findings indicate a rapid change of the intracellular signaling mechanism. In particular, the astonishing reversibility of the Ca2+ activity and its signal forms when resetting the temperature to its previous value after tens of minutes suggests a molecular activity dependency on temperature.Within the experimental comparison study we also investigated the role of extracellular Ca2+]i signal forms. These studies report an increase of the pump strength with increasing temperature from 5°C to 38°C. Thus, the Ca2+ uptake at 5°C amounts only 5% of the uptake at 38°C. This can have a large effect on CICR that represents the spike generating mechanism because the IP3Rs are not homogeneously distributed within the cell but form channel clusters that are typically separated by 1 μm or more. This separation may lead together with Ca2+ binding buffers and increasing SERCA activity to steeper gradients close to open channel clusters and subsequently to a weaker coupling between release sites and hence to a reduced CICR.As previously speculated, the experimentally reported temperature dependent activity of the SERCA pumps , 50 may 2+]i signals and was used to investigate the experimentally quantified stochastic nature of [Ca2+]i spiking including the functional robustness of the σ-Tav relation  and [Gk]. The first terms on the right hand side characterize diffusion with the individual diffusion coefficients Di. The following terms in (σl) and release through channels (Pc). The space dependence of Pc reflects the location of channel clusters and the time dependent stochastic opening and closing of the channels. This time dependent source term is governed by stochastic simulation of the channel states. γ is the volume ratio Vcyt/VER. The third term results from Ca2+ uptake into the ER from the cytosol by SERCA pumps with a flux strength Pp and the SERCA dissociation constant kd. The remaining terms in  see i spiking is the nonlinear release flux inducing the CICR by the bell shaped dependence on the Ca2+ concentration i spiking occurs for an intermediate pump rate where Ca2+ is removed fast enough from the cytosol to prevent too frequent repetitive stimulation but where the spatial coupling between the release sites is preserved to allow for cell wide coordinated signals.For this purpose we simulated in silico cells with our GCA implementation that allows for controlling all physiological parameters and obtaining mechanistic insights. From the activity patterns of identical in silico cells that only differ in the SERCA activity shown in Pp that was varied in simulations. From the obtained two exponential dependencies of the spike width on the temperature in experiments g(T) = aexp[−bT] and on the SERCA activity in simulations f(Pp) = αexp[−βPp] we can infer the relation between these two quantities by setting g(T) ∝ f(Pp) asAs a more quantitative analysis we analyzed the spike width in the simulation data in analogy to the experimental findings where we found that the experimental spike width followed an exponential dependency on temperature independently if the width was determined from cultured astrocytes or from astrocytes in acute brain slices . RemarkaPp and the temperature T as shown in 2+ signals at different temperatures.This relation predicts a linear relation between the SERCA activity Pp we first observed the general tendency of less coordinated Ca2+ signals for higher temperature in accordance with the experimental data. More strikingly, we found an exponential dependency on Pp of the spike width in the simulation data in analogy to the exponential dependency of the spike width on the temperature in experiments. The exponential relation was not included explicitly within the model but followed naturally from the physiological simulations indicating the importance of the spatial signal organization. With the two obtained exponential relations for the spike width g(T) from experiments and f(Pp) from simulations, we then inferred the linear relation between SERCA activity Pp and temperature T shown by the line in Pp, no specific relation has been assumed but is only caused by the spatial signal mechanism.It is worth emphasizing that we varied in our multiscale simulations the pump strength motivated by the experimentally reported temperature dependent SERCA activity but did not include any specific relation. From the unbiased parameter scan on Strikingly, the two independently obtained relations for the SERCA pump activity both exhibit a linear dependency on the temperature questioning the applicability of the Arrhenius law in this case.Eact in the order of the thermal energy RT or a more complex reaction system than what the Arrhenius law is considering. While the activation energy of SERCA is larger than the thermal energy as indicated by the use of ATP for the pump reaction, the underlying effective reaction scheme is more complex. The pump reaction consists of several  binding steps of Ca2+ and ATP to the SERCA protein. Since each of the binding and unbinding processes will be affected by temperature changes, the resulting reaction may exhibit already different characteristics from the collision of free particles assumed in Arrhenius law. More importantly, we determine here the cell wide averaged SERCA activity. The modulated SERCA activity is changing the spatial coupling between the release sites which leads to a modified hierarchical signal organisation by changing diffusion properties within the diffusion mediated CICR mechanism. This renders the reaction scheme even more complex and may be the main reason for the deviation from Arrhenius law.The finding either indicates an activation energy Pp in the simulations were smaller than those we would expect from the inference. Furthermore, the slope of the relation from inference is twice as large as the linear fit to the experimental data. This quantitative discrepancy is potentially caused by slightly different spatial characteristic like Ca2+ buffer content and physiologic settings of the cells. Nevertheless, the integrative approach shows how molecular properties can be characterized from global cell behavior by mechanistic modeling.When comparing the different scales for the pump strength-temperature dependence in 3R activity was significant modulated. This is a further evidence that it is the cell level that shapes the [Ca2+]i signals and not local properties. In line with these findings, we found that the rate equation based model 3R activirameters .2+ spikes. The nonlinear dependence of Tav on the pump strength might indicate a possible control mechanism. A recent study [2+ signaling pathway, cells can control this behavior by the expression level of SERCA. From our findings here, we would expect a negative feedback of SERCA expression on fast oscillations and a positive feedback on slow oscillations with high concentration peaks and plateaus.Interestingly, we found in the multiscale GCA simulations a regime of optimal signaling in terms of regularity of Cant study  has repo2+ signaling is probably not an astrocyte-specific phenomenon. In cardiac muscle the frequency of Ca2+ spikes decreases dramatically at 37°C compared with 22°C. In other cell types like in rabbit renal tubules, hepatocytes, and parenchymal and endothelial cells acute hypothermia affects the intracellular Ca2+ homeostasis and goes along with a rise in cytosolic Ca2+ levels [2+ signaling, this mechanism might be used in many temperature sensitive cellular processes, which could make SERCA a key temperature sensor.The impact of temperature on Ca+ levels –68. Due


The biologic modifier, omalizumab, helps to reduce lesions of chronic urticaria within 1–2 weeks.Chronic urticaria is defined as episodic or daily hives lasting for at least 6 weeks and impairs quality of life. Two main subtypes include chronic idiopathic (spontaneous) urticaria and inducible  urticaria, but some patients have urticarial vasculitis. “Autoimmune chronic urticaria” implies the presence of histamine releasing or mast cell activating autoantibodies to IgE or FcϵRI, the high affinity receptor on mast cells and basophils. In patients not readily controlled with labeled dosages of second generation HThe online version of this article (doi:10.1186/1939-4551-7-31) contains supplementary material, which is available to authorized users. The lifetime prevalence of chronic urticaria, defined as episodic or daily hives lasting for 6 weeks, occurs in approximately 1.8% of the adult population with a period prevalence (past 12 months) of 0.6 to 0.8% . Chronicdamage of the vessel wall, nuclear debris, extravasation of red blood cells) despite the phenotype being an urticarial lesion that doesn’t leave residual pigment or ecchymosis  sombe tried .1 (and H2 receptor) receptor antagonist and is effective in some patients without either intolerable or any perceived drowsiness. In a study of 50 patients, doxepin 10 mg three times daily was compared with diphenhydramine 25 mg three times daily [The tricyclic antidepressant, doxepin, has been studied in 2 double blind, controlled trials , 34. Whies daily . “Total es daily . And in 4 cause wheal/erythema reactions [Because intradermal injections of very small dosages of LTDeactions , 36, theeactions  and monteactions , 39 haveeactions . In retreactions . In a doeactions . The liteactions .The immunosuppressive drugs may be therapeutic as monotherapy for patients with uncontrolled chronic urticaria. There may be a clear-cut response to immunosuppressive drugs in the initial 1–4 weeks of therapy. Some patients respond after 3–5 months of treatment. Benefit-risk considerations should be assessed, and patients must be monitored for clinical harm and laboratory abnormalities. Cyclosporine –45, tacrColchicine –59, dapsIn that elevated concentrations of D-dimer reflect activation of the external pathway of the coagulation system and evidence of fibrinolysis, patients with treatment-resistant chronic urticaria received a low molecular weight heparin, nadroparin  daily and oral tranexamic acid as an inhibitor of fibrinolysis . There wApproved for Leishmaniasis because of its anti-trypanosomatid parasite activity and recognized as a drug with antineoplastic effects, the protein kinase B inhibitor, miltefosine, was reported to reduce the urticaria activity score in antihistamine resistant patients (UAS7) over a 4 week period compared with placebo . The int1 receptor antagonist. While increasing the dosage up to 4 fold of a second generation agent, levocetirizine [1 receptor antagonists (wherein hydroxyzine 25 mg is comparable to cetirizine 10 mg) [Chronic urticaria impairs quality of life and in about ½ of patients doesn’t respond readily to labeled dosages of a single Htirizine  or deslotirizine  has beene 10 mg) . The oppe 10 mg) . The num


The downstream effects of altered Ca2+ handling in the distinct subcellular organelles for proper cellular function are beginning to be elucidated. Here, we summarize the evidence that vulnerable neurons may be exposed to homeostatic Ca2+ stress which may determine their selective vulnerability, and suggest how abnormal Ca2+ handling in the distinct intracellular compartments may compromise neuronal health in the context of aging, environmental, and genetic stress. Gaining a better understanding of the varied effects of Ca2+ dyshomeostasis may allow novel combinatorial therapeutic strategies to slow PD progression.Parkinson’s disease (PD) is a major health problem affecting millions of people worldwide. Recent studies provide compelling evidence that altered Ca L-DOPA , a DA precursor, indicates that DA neurons are crucial to the disease. However, the neuropathological hallmarks of PD, which are the presence of proteinaceous intracellular deposits called Lewy bodies or Lewy neurites in surviving neurons, are more distributed and not exclusive to DA neurons. Non-DA neurons which show pathology in PD include cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) and basal forebrain (BF), noradrenergic neurons in the locus ceruleus (LC), and serotonergic neurons in the raphe nuclei  is an incurable late-onset neurodegenerative disorder which is strongly associated with aging, as evidenced by the exponential increase in incidence above the age of 65 . Due to clei RN; . Neurodeclei RN; . Thus, eDistinct mechanisms have been proposed to account for the preferential loss of DA neurons in PD. One hypothesis proposed that DA itself may be the culprit, as oxidation of cytosolic DA and its metabolites can lead to the production of cytotoxic free radicals and oxidative stress . HoweverAnother hypothesis has linked PD to mitochondrial dysfunction . MitochoThere is extensive evidence for mitochondrial involvement in both sporadic and genetic PD. Toxins such as MPTP, rotenone, and paraquat, which inhibit complex I, can cause a Parkinsonian phenotype . In addiper se cannot explain the selective vulnerability of neurons which die in PD.However, the observed decrease in complex I deficiency in homogenates from nigral tissue from PD patients is too big to be restricted to SNc DA neurons, and only a proportion of PD patients show complex I inhibition in the SNc . In addiFigure 1).A hypothesis, put forward by Surmeier’s group, suggests that specific and shared physiological features are responsible for the risk of a subset of neurons to degenerate in PD , and com+ and K+ gradients) across their plasma membrane to integrate incoming chemical signals, and pass them on to other neurons. Voltage-dependent Ca2+ channels in most neurons are only opened by strong depolarization during an action potential. These channels close relatively slowly during membrane repolarization, such that the total Ca2+ influx during a spike is very sensitive to spike duration. To minimize global increases in Ca2+, neurons which need to spike at high frequencies tend to restrict Ca2+ entry by keeping spikes very brief, and tend to express Ca2+ buffering proteins to help manage intracellular Ca2+ levels , mitochondria, Golgi, and lysosomes ] to move Ca2+ from the cytoplasm into the ER lumen. Ca2+ sequestered in the ER can be released at sites where it can be pumped back across the plasma membrane, or can be used locally to modulate cellular function  receptors which reside at direct ER-mitochondrial contacts termed MAMs (mitochondria-associated ER membranes) allow for direct flux of Ca2+ from ER into mitochondria . Indeed, stimulation of Ca2+ release from the ER by ryanodine, accompanied by an increase in cytosolic Ca2+ levels, was found to protect DA neurons from spontaneous or induced neurodegeneration , with obvious downstream effects both on plasma membrane functioning/signaling and lysosomal degradative capacity.Apart from its signaling function, Caresponse . Too mucdiseases . Similaror CFTR; , 2004. P2+ store. The first is the Golgi apparatus, which shares some functions and biochemical markers with the ER. The Golgi complex is a highly dynamic intracellular organelle which processes and sorts membrane proteins derived from the ER to the cell surface, secretory vesicles or lysosomes, and which also receives retrograde transport input. Thus, damage to neuronal Golgi structure can have important functional consequences for protein and vesicular trafficking , and at least in some cells contains ryanodine receptors  and nicotinic acid adenine dinucleotide phosphate (NAADP) which have been shown to activate ryanodine receptors , and TPCs (two-pore channels), which are members of the TRP channel superfamily as well . TPC channels located on endosomes and lysosomes have been reported to be targets for NAADP, the most potent Ca2+ mobilizing messenger  have been identified, and many of them affect brain function. Conversely, many neurodegenerative diseases also exhibit lysosomal dysfunction . Lysosomensation . Whilst 2+ levels on autophagy, a process employed by cells to get rid of protein aggregates and defunct organelles, and deficits of which are also clearly implicated in PD  than many other neurons in the brain, some of which do not display significant loss over 60–70 years  or autosomal-dominant  inheritance account for approximately 10% of PD cases .Similarly, genetic forms of PD would be expected to converge on pathways affected by altered intracellular CaPD cases . Of thoshandling . For exahandling . Depletit stress . Other soduction . Similarergetics . Both paergetics . Whilst Figure 1; 2+ handling and mitochondrial oxidant stress is less clear. Autosomal-dominant mutations in LRRK2 have been shown to cause deficits in Ca2+ homeostasis, leading to mitochondrial depolarization and enhanced mitophagy, which can be prevented by L-type Ca2+ channel inhibitors  which may in turn increase extramitochondrial-derived oxidant stress to further threaten the viability of affected neurons.Apart from directly affecting mitochondrial Cang cells . Such ch+ levels , may lea2+ channel antagonists may be viable therapeutic targets in the early stages of PD. There are oral antagonists [dihydropyridines (DHP)] available, with good blood–brain barrier permeability and a long record of safe use in humans. Adult SNc DA neurons can compensate for L-type Ca2+ channel antagonism and continue pacemaking (2+ channel antagonists (2+ channels, or because other factors may become more prominent during disease manifestation. Such factors may in part derive from alterations in intracellular Ca2+ stores, with the resultant varied downstream effects on cellular proteostasis.The above-mentioned findings indicate that L-type Cacemaking , and micagonists , suggestagonists . Howeveragonists , maybe b2+ dysregulation in the pathogenesis of PD. It is becoming increasingly clear that abnormal Ca2+ handling may have pleiotropic effects on a variety of intracellular events resulting in mitochondrial oxidant stress, deficits in ER proteostasis, endolysosomal/autophagic trafficking and alterations in Golgi function which require further investigation. Thus, whilst L-type Ca2+ channel antagonists may attack the source of the problem, improving the deteriorated cellular functions of mitochondria, ER, lysosomes, or Golgi may be an efficient complementary strategy to attack the varied downstream effects of the increased burden of handling intracellular Ca2+ in vulnerable neurons. Maybe a feasible future therapeutic strategy should not involve a “hit-hard” principle employed for example to treat cancer patients, but rather a “hit-softly, continue hitting, and hit at multiple places at a time” principle aimed at correcting a combination of cellular deficits derived from improper Ca2+ handling employing combination-type therapies.Much work remains to be done before gaining a clearer understanding of the role of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SOCE is activated following the depletion of the endogenous Ca2+ stores, which are mainly located within the endoplasmic reticulum (ER), to replete the intracellular Ca2+ reservoir and engage specific Ca2+-dependent processes, such as proliferation, migration, cytoskeletal remodeling, and gene expression. Their paralogs, Stim2, Orai2 and Orai3, support SOCE in heterologous expression systems, but their physiological role is still obscure. Ca2+ inflow in neurons has long been exclusively ascribed to voltage-operated and receptor-operated channels. Nevertheless, recent work has unveiled that Stim1–2 and Orai1-2, but not Orai3, proteins are also expressed and mediate SOCE in neurons. Herein, we survey current knowledge about the neuronal distribution of Stim and Orai proteins in rodent and human brains; we further discuss that Orai2 is the main pore-forming subunit of CRAC channels in central neurons, in which it may be activated by either Stim1 or Stim2 depending on species, brain region and physiological stimuli. We examine the functions regulated by SOCE in neurons, where this pathway is activated under resting conditions to refill the ER, control spinogenesis and regulate gene transcription. Besides, we highlighted the possibility that SOCE also controls neuronal excitation and regulate synaptic plasticity. Finally, we evaluate the involvement of Stim and Orai proteins in severe neurodegenerative and neurological disorders, such as Alzheimer’s disease and epilepsy.Stim1 and Orai1 are ubiquitous proteins that have long been known to mediate Ca Capacitative calcium entry (CCE) or store-operated Ca2+ entry (SOCE) represents a peculiar mode of Ca2+ entry, which is activated following depletion of the ER Ca2+ pool in non-excitable cells (2+-selective Ca2+ release-activated Ca2+ (CRAC) channels . Moreover, a defective SOCE was associated to severe neurodegenerative disorders, such as Huntington’s disease , in the formation and maturation of dendritic spines and in gene expression. We also analyze the evidence in favor of Stim and Orai activation during synaptic stimulation and of their contribution to synaptic plasticity. Finally, we discuss their involvement in AD and other brain disorders, which hints at neuronal SOCE as a novel therapeutic target for neurodegenerative diseases.Neurons possess a highly developed Caunctions . As virtunctions , neuronsrocesses . Ca2+ in NMDARs; . The maic spines . ER-depele cells . This pachannels . The Ca2lization , 2014b; decoders . Stromalrane PM; , whereasrane PM; . SOCE harane PM; , which g neurons  and dent neurons . These sease HD; , Alzheimease HD; , and spoease HD; . It is, ease HD; . Herein,Drosophila melanogaster to humans. Stim1 is a type I transmembrane (TM) protein of 685 amino acids embedded either in ER membrane or on the PM where it is targeted after N-glycosylation of Asn131 and Asn171 . The ER-luminal portion contains a canonical EF-hand domain (cEF), which serves as ER Ca2+-sensor, and a sterile alpha-motif (SAM) domain required for protein–protein interaction. A hidden, non-canonical EF-hand domain (hEF), unable to bind Ca2+, is also present between cEF and SAM . The cytosolic domain comprises three coiled-coil (CC) regions (CC1-CC2-CC3), which overlap with an ezrin-radixin-moesin (ERM) motif, a serine/proline-rich (S/P) sequence and a polybasic lysine rich (K-rich) domain. In addition, the ERM domain presents crucial Orai-activating regions, which have been termed Orai1-activating small fragment (OASF), CRAC-activating domain (CAD), or Stim1–Orai1 activating region (SOAR), and include CC2 and CC3 . When ER Ca2+ concentration falls below a threshold level due to InsP3R or RyRs activation, Ca2+ dissociates from cEF, thereby causing the unfolding of the adjacent EF-SAM domains and Stim1 multimerization . Stim1 oligomers rapidly redistribute to peripheral ER sites, termed puncta, in close proximity to PM, bind to and activate Orai1 . Orai1 is composed of 301 amino acids, both NH2 and COOH termini reside in the cytoplasm, and each of them has been implicated as a critical accessory region in Orai1 activation via direct interactions with Stim1. Ca2+ influx is indeed gated by the physical interaction between an NH2-terminal domain proximal to the first TM alpha-helix of Orai1 and a COOH-terminal CC domain of the channel protein with CC2 and CC3 on Stim1 . The crystal structure of Drosophila Orai1 revealed a hexameric structure, while the optimal Stim:Orai1 subunit stoichiometry seems to be 2:1  > +60 mV, permeability to Ca2+, but not to other monovalent cations, fast and slow Ca2+-dependent inactivation, and a single-channel conductance in the order of femtosiemens  and three Orai proteins . Stim isoforms are expressed in almost all mammalian tissues and are highly conserved from d Asn171 . Stim1 pte Orai1 . Orai1, on Stim1 . The chao be 2:1 . Stim1 aic cells , i.e., nytoplasm , 2011. Tomplexes . In addiic oxide  and aracic oxide  productiic oxide , cell cyic oxide , mitochonergetics, exocytonergetics, and pronergetics.2-terminus, while Stim2 requires a longer ER-localization peptide , and both Orai2 and Orai3 lack the ∼18 amino acid NH2-terminal hydrophilic stretch of Orai1, which together with the nearby Orai1-specific proline-rich domain mediate reactivation after fast Ca2+-dependent inactivation . Furthermore, Orai3 can be activated by 2-aminoethoxydiphenyl borate (2-APB), which in turn inhibits both Orai1 and Orai2  or over-expressing Orai1 and/or yellow fluorescent protein (YFP) or enhanced green fluorescent protein (EGFP)-tagged Stim proteins. These experiments have been conducted on both mouse and rat neurons. Co-expressed Orai1 and Stim1 (both in its YFP and GFP tagged forms) are evenly distributed in the soma, primary dendrites and post-synaptic dendritic spines of mouse cortical neurons, thereby confirming the localization of the endogenous proteins . The phaclusters , as obseclusters . Moreoveendrites . Surprisc spines . Althougc spines . Likewisc spines , in whicc spines . This moc spines . Overall2+ store depletion with thapsigargin reversibly enhances the association of endogenous Stim1 and Stim2 with the PM in cortical neurons; however, when the cells are co-transfected with either Stim1 and Orai1 or Stim2 and Orai1, this treatment increases the number of Stim1–Orai1 puncta more than nine-fold, while it does not significantly stimulate Stim2 redistribution into sub-membranal clusters .The scenario is different in rat cortex and hippocampus, which clearly show higher levels of Stim2 as compared to Stim1. Caclusters . Similarclusters . These dl milieu . Therefo+ levels . Likewis+ stores . Thus, S2+ content, and gene expression in mouse. Post-synaptic dendritic spines are the primary recipient of excitatory inputs in most central neurons and may be broadly classified into three groups depending on their morphology: mushroom spines, thin spines, and stubby spines  activation and stress fiber formation in microvascular endothelial cells  Canonical 3 (TRPC3), as shown in mouse cerebellar Purkinje neurons . It is, therefore, clear that more work is required to fully understand the structural and functional relationships between SP, Stim1 and Orai1. While the role of Stim1 and Orai1 in the control of spine architecture is still uncertain, Stim2-mediated SOCE maintains mushroom spine structure in mouse hippocampus both in vitro and in vivo  to support long-term stabilization of mushroom spines even in the absence of synaptic activity  is enhanced, thereby favoring resting Ca2+ entry and stimulating SOCE-dependent downstream targets. As described in the paragraph “Evidence about Stim- and Orai-mediated Ca2+ entry in brain neurons,” this mechanism is triggered by Stim2  in order to refill the ER Ca2+ store in cortical neurons , thereby abrogating InsP3- and mGluR1-dependent intracellular Ca2+ release and impairing cerebellar motor behavior  phosphorylation. Moreover, membrane depolarization  refills ER Ca2+ load, thereby dismantling Stim1 puncta, deactivating SOCE and, ultimately, restoring Sp4 abundance , nuclear factor of activated T cells (NFATs) and nuclear factor-κb (NF-κB), are usually activated by membrane depolarization, rather than hyperpolarization . We cannot rule out the possibility that other yet unknown transcription factors are selectively activated by the constitutive influx of Ca2+ via store-operated channels in brain neurons. This would permit them to activate or de-activate the expression of two distinct sets of genes depending on the extent of membrane excitation .Basal SOCE does not only modulate spinogenesis and ER Cale cells . Sp4 is le cells . Stim1 ibundance . This stbundance . Furtherbundance . We shourization . The pre2+ levels and tune gene expression. However, SOCE could also play a role during neuronal excitation. Even a single synaptic stimulus fully depletes the ER Ca2+ pool in dendritic hippocampal spines , which are required for LTP expression and maintenance, respectively  and Stim2 (in mouse cortex and hippocampus) activate Orai2 to mediate SOCE in silent neurons to regulate spine morphogenesis, preserve ER Cal spines  and has,l spines . Earlierl slices . Unfortua2+ pool . If SOCEs in LTP . One mignduction . This hyectively . Moreove2+ currents in cortex, hippocampus, cerebellum and neuroendocrine system  trigger SOCE-dependent pathways involved in LTP induction and expression  and/or (2) limit voltage-dependent Ca2+ inflow, thereby preventing cytotoxic Ca2+ accumulation. This hypothesis makes physiological sense as Orais are low-conductance, Ca2+-selective channels tightly coupled to their decoders (2+]i (2+ entry during synaptic activity by decreasing CaV1.2 and CaV1.3 activity with (CaV1.3) or without (CaV1.2) Orai1 contribution. This subtle regulation of Ca2+ influx could prevent detrimental Ca2+ entry into firing neurons and, therefore, it would be interesting to examine the interaction between Stim1 and VOCCs not only in healthy neurons, but also in the presence of neurodegenerative disorders.Stim1 does not only associate with Orai1 and Orai2 (and TRPC3) in brain neurons. CaV1.2 (α1C) mediates L-type voltage-operated Cae system . Recent e system . Store dcurrents . In the currents . Similarr5 cells . Notably+ influx . More re+ influx . Similarng cells . It is s T cells , in whic T cells . The reldecoders , while Vrs -causing mutations in presenilins (PSEN1 and PSEN2 genes) has revealed an increase in ER Ca2+ concentration that leads to a compensatory increase in InsP3R and RyR expression and SOCE down-regulation  or TRPM5 . We have the opportunity to witness the beginning of a new era in the study of neuronal Ca2+ dynamics. This is why only scarce preliminary information is currently available regarding the localization and pathophysiological roles served by the diverse Stim and Orai isoforms in central neurons.It has long been thought that excitable cells, including neurons, do not require SOCE to replenish their endogenous Cabehavior . NeverthFirst, there is a tissue- and species-dependent pattern of expression. In the mouse, which provides a multitude of transgenic models suited for the investigation of cognitive process in health and disease, Stim1 reaches the highest expression levels in the cerebellum, while Stim2 is far more abundant in the hippocampus. This is consistent with preliminary findings implicating Stim1 in the control of motor coordination  and StimTable 4). This feature is surprising when considering that Stim2, but not Stim1, activates Ca2+ inflow in response to mild-store depletion in other cell types. It turns out that Stim2 should activate basal SOCE in cerebellum as well. However, Stim1 is far more abundant in this region than in other brain areas. Moreover, the ER becomes rapidly depleted of Ca2+ in the absence of Ca2+ influx in mouse cerebellar granule cells  and cortex/hippocampus (supposed to be higher due to the constitutive activation of Stim2). The situation is clearer in rat neurons, in which Stim1 and Stim2 accomplish two distinct roles: Stim2 gates resting Ca2+ entry, whereas Stim1 elicits Ca2+ inflow in response to larger ER depletion (Table 4).Second, both Stim1 (cerebellum) and Stim2 (cortex and hippocampus) trigger SOCE in mouse central neurons even in the absence of synaptic activity (le cells : this su2+ in response to ER emptying (Table 4). This issue gains more relevance when considering that no clear-cut role has hitherto been attributed to this isoform in any cell type  of the underlying channel, which generates sub-pA currents in the whole-cell mode of patch-clamp and often fall below the resolving power of current amplifiers  could confirm the notion that Orai2 and Orai1 mediate SOCE, respectively, in mouse and rat by exploiting their electrophysiological differences (Table 1).Fourth, despite the fact that Stim1, Stim2, Orai1, and Orai2 were shown to mediate SOCE in brain neurons, no study has attempted to measure Iplifiers . This tac)-KO mice (2+ homeostasis and excitability.We foresee that future work will unveil new yet undiscovered aspects of the pathophysiological role fulfilled by Stim and Orai proteins in central neurons. For instance, SOCE amplitude is dramatically enhanced in cerebellar granule neurons obtained from cellular prion protein (PRP-KO mice  and in H-KO mice ; howeverThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Initial rapid peak and subsequent reduction of [Ca2+]c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca2+, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]c). The elevation of [cAMP]c was blocked by transduction of the dominant-negative Gs, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca2+]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor.Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca Elevation of ambient glucose leads to a gradual depolarization of 10 to 15 mV, which is followed by an initiation of action potentials. Initial depolarization induced by glucose is brought about by a decrease in K+ permeability of the plasma membrane. It is now known that glucose enters the cells, is metabolized through the glycolytic pathway and in mitochondria, and the resultant increase in ATP/ADP ratio causes closure of the ATP-sensitive K+ channel (KATP channel) c) are monitored in pancreatic β-cells, the addition of a high concentration of glucose reduces [Ca2+]c rather rapidly [2+]c lasts for a few minutes and is followed by an oscillatory elevation of [Ca2+]c [2+]c is thought to be due to sequestration of Ca2+ mainly to endoplasmic reticulum (ER) via the ER Ca2+ pump (SERCA) [2+]c is accompanied by an increase in Ca2+ concentration in ER c –11. The  (SERCA) , 13. In on in ER , 15. The2+, glucose also increases cyclic 3’, 5’ AMP (cAMP) in pancreatic β-cells [c) induced by a high concentration of glucose has been thought to be secondary to elevation of [Ca2+]c c activates calcium-dependent AC such as ACVIII, and increases production of cyclic AMP c. This observation raises a possibility that increase in [cAMP]c is rapid and at least partly independent of elevation of [Ca2+]c.Besides changes in Ca β-cells –18. Elev [Ca2+]c . In fact [Ca2+]c , 21. ACVclic AMP . Howeverclic AMP  showed tO-methylglucose [2+]c and [cAMP]c in β-cells [2+]c and [cAMP]c in β-cells by acting on the glucose-sensing receptor. The present study was conducted to investigate whether or not the glucose-sensing receptor is involved in glucose-evoked intracellular signals. If this is the case, immediate changes in [Ca2+]c and [cAMP]c should be observed. We therefore investigated whether glucose elicits immediate actions on [Ca2+]c and/or [cAMP]c in pancreatic β-cells. The results show that glucose induces immediate changes in [Ca2+]c and [cAMP]c by acting on the glucose-sensing receptor.We have shown recently that subunits of the sweet taste receptor  are exprlglucose , 27. Int β-cells , it is rO-methylglucose were purchased from Wako Pure Chemical Industries, Ltd .Nifedipine, N-methyl-D-glucamine (NMDG) and 3-2 at 37°C.MIN6 cells (passages 16–21)  were groThe animal experiment was approved by the Animal Experiment and Ethics Committee, Gunma University School of Medicine (#25–0112), and was conducted according to the guidelines for animal care issued by the Committee. B6; 129-Tas1r3 <tm1Csz>/J mice were purchased from the Jackson Laboratory . They were kept in an experimental animal facility controlled at 23°C room temperature with a 12-hr light and dark cycle, and with free access to standard chow and water. The animal experiment was conducted according to the guidelines for animal care issued by Animal Experiment and Ethics Committee, Gunma University School of Medicine.2+ free HKR buffer  c was performed by using a fluorescent Ca2+ indicator Fluo-8 . MIN6 cells and isolated β-cells were loaded with 2 μM Fluo-8/ AM dissolved in HBSS for 20 min at room temperature. To monitor [Ca2+]c using Cameleon-nano15, MIN6 cells were transfected with PM-Cameleon-nano15 c in MIN6 cells using an ultrasensitive Ca2+ indicator, yellow Cameleon-nano15 [2+]c in these cells [2+]c was observed. A typical response of [Ca2+]c is shown in 2+]c was observed within seconds and [Ca2+]c peaked within 10 sec. [Ca2+]c then rapidly decreased to a plateau level, which was lower than the basal level. [Ca2+]c remained decreased for 1 to 5 min and then [Ca2+]c rose abruptly. The initial rapid peak of [Ca2+]c was observed in approximately 60% of the cells examined while the following plateau phase and the second large elevation of [Ca2+]c were observed in almost all of the cells tested. In cells without the rapid peak of [Ca2+]c, a small hump of [Ca2+]c was instead observed immediately after the addition of glucose  of the subsequent oscillatory elevation of [Ca2+]c was smaller than that in cells with an initial rapid peak of [Ca2+]c. Thus AUC was significantly smaller than that in cells with a rapid peak of [Ca2+]c . More importantly, when cells expressing PM-Cameleon-nano15 were simultaneously loaded with fura-2, the initial rapid elevation of [Ca2+]c monitored by PM-Cameleon-nano15 was abolished (2+]c. Triphasic changes in [Ca2+]c were observed when cells were stimulated with 8.3 mM and 16.7 mM glucose (2+]c. Both the first rapid peak and AUC of the second oscillatory elevation of [Ca2+]c were increased in dose-dependent manners (2+]c was markedly inhibited by the addition of a non-specific inhibitor of phospholipase C (PLC) U73122 [2+]c was also abolished by U73122  U73122  2+. To thYM254890 . SimilarYM254890 . It shou glucose .2+]c by using conventional Ca2+ indicators and found that the initial rapid elevation of Ca2+ was detected by using fluo-8 in a fraction of the cells. As shown in 2+]c induced by 25 mM glucose was observed in approximately 40% of the fluo-8-loaded cells. In these cells, triphasic changes in [Ca2+]c were qualitatively similar to those observed by using PM-Cameleon-nano15. When extracellular Ca2+ was removed, glucose-mediated initial peak of [Ca2+]c was observed but the large elevation of [Ca2+]c observed several minutes later was markedly inhibited . This rapid elevation of [cAMP]c was observed in the presence of mannoheptulose  since the rapid elevation of [Ca2+]c was observed in the absence of extracellular Ca2+. Since the rapid elevation of [Ca2+]c was inhibited by an inhibitor of PLC and an inhibitor of Gq, it may be due to a release of calcium from ER caused by inositol trisphosphate. This rapid elevation of [Ca2+]c was observed even if glucose metabolism was blocked by mannoheptulose, indicating that it is not dependent on glucose metabolism. Instead, the rapid elevation was blocked by lactisole, an inhibitor of T1R3 [2+]c was never observed in T1R3-null β-cells. The rapid peak of [Ca2+]c evoked by glucose is therefore caused by activation of the glucose-sensing receptor. In agreement with this notion, activation of the receptor by an administration of nonmetabolizable glucose analogue 3-O-methylglucose reproduced the rapid elevation of [Ca2+]c. Collectively, the rapid elevation of [Ca2+]c is a signal due to activation of the glucose-sensing receptor. It should be mentioned that the rapid elevation of [Ca2+]c induced by glucose is different from the elevation of [Ca2+]c induced by typical calcium-mobilizing agonists, for example, carbachol [2+]c, [Ca2+]c response induced by glucose is subtle and is not detected by fura-2 whereas that induced by carbachol is easily detectable by fura-2 [2+]c was only transient and [Ca2+]c decayes quickly. In addition, glucose-induced rapid elevation of [Ca2+]c is followed by a sustained decrease in [Ca2+]c while that induced by carbachol is not. Presumably, glucose and carbachol activate PLC differently and elicit slightly different [Ca2+]c signals.The present study was conducted to detect receptor-mediated rapid signals evoked by glucose in pancreatic β-cells. Using an ultrasensitive Can-nano15  targetedn-nano15 , we coulhe cells . This el of T1R3 . Furtherarbachol  in many y fura-2 . Also, g2+]c response to glucose: roughly half of the cells presented a rapid peak of [Ca2+]c induced by glucose whereas rest of them did not. Difference in the expression levels of T1R3 would explain the different patterns of [Ca2+]c response in two populations. However, when we measured the expression of T1R3 in pancreatic islets c, which is due largely to sequestration of Ca2+ into ER through activation of SERCA [2+ into ER is not certain [2+]c induced by glucose . Reduction of [Ca2+]c following a rapid elevation of [Ca2+]c may result from a unique property of the glucose-sensing receptor. In addition, activation of T1R3 leads to reduction of [Ca2+]c at least in some occasions. In human GLP-1-secreting cells, stimulation of the glucose-sensing receptor by acesulfame-K persistently reduced [Ca2+]c [2+]c is due to activation of the plasma membrane Ca2+ pump (PMCA) [2+ pump leading to sustained reduction of [Ca2+]c. In β-cells, activation of the receptor by glucose may cause activation of SERCA and reduces [Ca2+]c. Further study is obviously needed to identify the mechanism of activation of SERCA by the glucose-sensing receptor. In this regard, it is possible that increase in subplasma membrain Ca2+ activates SERCA and promotes uptake of Ca2+ to ER. Alternately, since elevation of cAMP stimulates sequestration of Ca2+ to ER [s would cause sequestration of Ca2+ to ER. Further studies are needed to identify the mechanism. It should be mentioned that the reduction of [Ca2+]c induced by glucose is also dependent on glucose metabolism to some extent. This is because SERCA is an ATP-requiring enzyme and glucose elevates intracellular ATP within a minute c . The redp (PMCA) . Althoug2+ to ER , it is pa minute . Elevatec by paying particular attention to a rapid action of glucose. The results obtained in MIN6 cells and mouse β-cells show that glucose induces an immediate elevation of [cAMP]c. Glucose-induced elevation of [cAMP]c preceded a large oscillatory elevation of [Ca2+]c and was observed even in the absence of extracellular calcium. These results suggest that glucose-mediated elevation of [cAMP]c is at least partly independent of elevation of [Ca2+]c. We have previously shown that the glucose-sensing receptor causes elevation of [cAMP]c by a Gs-dependent mechanism [O-methylglucose increases [cAMP]c (c is secondary to the elevation of [Ca2+]c [c in β-cells. It is likely that at least rapid elevation of [cAMP]c is caused by receptor-mediated activation of Gs.In the present study, we monitored the glucose effect on [cAMP]echanism . In addi [cAMP]c . Our res [Ca2+]c . The rea2+]c [2+]c would activate calcium-dependent PLC leading to generation of DAG c . In β-cen of DAG . Alternaubstrate . In any ubstrate . Collect2+ and cAMP, in pancreatic β-cells. These actions are independent of the glucose metabolism and are mediated by the glucose-sensing receptor. Importantly, inhibition of the receptor or deletion of the T1R3 gene attenuates glucose-induced insulin secretion [The present study demonstrates for the first time that glucose evokes rapid intracellular signals, Caecretion , 45. Speecretion . The gluecretion . Our resecretion  that theecretion , their cecretion . In factecretion , 48. Morecretion , 45 and ecretion , it is cIn summary, we identified the intracellular signals evoked by glucose via the activation of the glucose-sensing receptor. These rapid signals may be important for the priming of the metabolic pathway , 27 and


Choosing the right mentor is crucial for effective mentorship. Yet, many medical students have difficulties finding a suitable mentor. Thus we developed mentoring speed dating (MSD) as a promising matching tool to connect students and faculty mentors successfully. The purpose of this study was to explore mentees’ and mentors’ experience with MSD and investigate the impact of MSD on the perceived mentorship quality and continuance of the mentoring relationship.t-tests and multivariate analysis of variance were used to analyze the impact of MSD on student’s mentorship satisfaction.The authors completed a mixed methods study at the University Medical Center Hamburg-Eppendorf, Germany, between June 2011 and March 2014. They conducted four focus groups with mentees and mentors who participated in a mentoring speed dating event and analyzed transcripts using conventional content analysis with inductive categorizing. In addition, three mentoring cohorts  were surveyed on mentorship satisfaction and the 1-year continuance of their mentorship was monitored. Fifteen mentees and fifteen mentors participated in the focus groups. The authors identified several themes such as short and long term benefits of MSD and fulfillment of expectations. Benefits included finding out about the personal connection, matching expectations, providing an efficient overview of candidates. The survey was completed by 93 students . Independent There were significant differences in responses to the items “Commitment of mentor” (p = .019) and “Constructive feedback” (p = .038) among the students who attended MSD and the students without MSD. After one year far more mentoring relationships existed among those mentees who participated in MSD in comparison to the “no MSD group”.MSD is a valuable matching tool with beneficial effects on the mentorship quality. It enhances essential factors in the mentoring relationship such as commitment and satisfaction. Due to the positive effects of mentoring on the professional development of medical students and young physicians, formal mentoring programs have gained popularity within academic medicine –4. MentoSeveral benefits of mentoring have been reported for mentees (students), mentors (faculty) and institutions  , 3, 7, 8Mismatches in terms of different values, attitudes or work styles are quite common in mentee-mentor relationships  and ofteIn December 2011 we implemented an annual “mentoring speed dating” event to facilitate mentoring relationships between students and faculty mentors, who participated in a formal mentoring program that fosters early introduction to research and promotes academic careers . FurtherMSD, as an innovative matching tool, was adapted from the concept of “speed dating” , 21. TheThe purpose of this study was to gain an in-depth understanding of MSD as an innovative matching tool to connect mentees (students) and faculty mentors. In particular, we were interested in both mentees’ and mentors’ view on and experience with MSD regarding the process of matching and the outcome in terms of satisfaction with the mentoring relationship. Furthermore we aimed to investigate effects of MSD by comparing the mentorship quality and the 1-year continuance of the mentoring of three mentoring cohorts, who were matched either via MSD or chose their mentors via online profiles.The study was conducted at the University Medical Center Hamburg-Eppendorf (UKE), Germany between December 2010 and January 2014. We used a mixed methods approach including method- and data triangulation. Both qualitative (focus-groups) and quantitative data  were collected.Second- and third-year medical students at the UKE who participated in the mentoring program for excellent students  since De“What have been the benefits of the MSD event in your view?”, “How did you feel regarding the surrounding and atmosphere during MSD?”, “What was the time frame like in your experience during MSD?”, “To what extent is MSD able to facilitate a long-term mentoring relationship?”.We conducted two focus groups that were based on semi-structured interviews with open-ended questions. Participants were asked to reflect their MSD experience and used the following prompts to guide the discussion during focus groups: We contacted all students  and mentors  who participated in the previously described MSD by email and invited them to participate in separate focus groups. In total 15 students  and 15 mentors  participated in the focus groups. Informed consent was received before the focus groups started. According to recommendations, focus-groups were heterogeneous regarding gender. Each session lasted 60–90 minutes  and was All mentoring cohorts  were asked to complete a set of items with regard to characteristics of their mentoring relationship after six months of participation in the mentoring program for excellent students. To explore the effect of MSD, the three cohorts were assigned to two groups: Cohort 2010 was assigned to the group without MSD, whereas the cohorts 2011 and 2012 were assigned to the MSD-group. Following our applied definition of mentoring and desirable characteristics of mentors that are recommended by Berk et al., two of the authors (JG and ES) developed the items to reflect a comprehensive assessment of the mentoring relationship between students and faculty mentors , 6. ItemFurthermore the duration of the mentoring relationship was considered as an outcome measure. To assess the 1-year continuance of the mentoring relationship we sent an email to all mentees after 12 months of mentorship asking whether they would like to continue the mentoring relationship. In case we did not receive a response after two reminder emails we contacted the respective mentor to clarify the current state of their mentoring relationship.Each focus group was analyzed by using conventional content analyses with inductive categorization. Data analysis started with reading the transcripts repeatedly to gain a sense of the whole. Subsequently three of the authors  reviewed the data word by word to identify key concepts and generate labels of codes independent from each other. Next, CM, GK and JG sorted codes into categories, which were reviewed by all authors. During this process we developed final definitions for each category and code. Consensus was reached through considering the matrix, which was completed by one of the researchers  during the focus groups . We chost-tests were carried out for the six items illustrating the mentoring relationship from the mentee’s point of view. To explore the possible effect of gender, a second independent t-test with gender as group variable was computed. In addition, a two-factorial multivariate analysis of variance (MANOVA) with MSD and gender as the main factors and MSD and gender as an interaction term was conducted to evaluate the responses of the two groups (no MSD and MSD) to the six items illustrating the mentoring relationship. Gender was also included in the analyses because the mean was different for males and females among some items. We used another chi-squared test to analyze for differences of the 1-year continuance among the group matched via MSD and the no MSD group. For the statistical analyses PASW SPSS 18.0 was used.Descriptive statistics were generated to provide an overview. Pearson’s chi-squared tests were used to evaluate for differences in gender between the mentoring cohorts. To compare the answers of students in the two groups (no MSD vs. MS), two independent The study was carried out in accordance with the Declaration of Helsinki. The study protocol was approved by the Dean of the University Medical Center Hamburg-Eppendorf, Hamburg, Germany since the research protocol was not deemed to be biomedical or epidemiological research. One week before the MSD event and the focus-groups participants received a fact sheet of the study and a consent form by email. Participation in the study was voluntary. There was no disadvantage to those who chose not to participate. At the beginning of the MSD event and the focus-groups the study was fully explained by one of the researchers (JG). Only mentees and mentors who gave written informed consent freely were able to participate in the study. Data were anonymized by one author (JG), who had access to identifying participant information. None of the other authors had access to identifying participant information. The Dean of the University Medical Center Hamburg-Eppendorf, Hamburg, reviewed the study and consent procedure. Participants were able to opt-out of the study without experiencing disadvantages until data were anonymized. Quantitative data underlying the study findings are freely available.We conducted four focus groups in total . 15 students  and 15 mentors  participated in separate focus groups. We identified several themes focused on benefits of MSD, the environment and the time-frame. We found slight differences in the responses by group (mentors vs. mentees). Thus codes and illustrative quotes are presented separately for mentors and mentees.We identified numerous benefits of MSD. Most commonly, mentors and mentees mentioned that MSD helps to account for the ‘chemistry’ of the relationship and personal component. One mentor stated:With MSD… you easily find out whether the chemistry does fit or not.Likewise one mentee said:Chemistry is the most important. It is not only the area of research and what the mentor is doing in his lab all day, but how he is as a person. Everybody is different and you cannot get this [information] from the online profile. And it is the most important to notice if you get along with each other.All mentees and mentors emphasized that MSD is very useful to get a first impression. Participants stated that finding out about the unique personal connection is of great importance for future mentoring relationships:The main aspect is that both participants get a personal impression…that is extremely important in my opinion.Furthermore MSD helps to match the expectations of both parties involved in the mentoring relationship. Both, mentees and mentors pointed out the importance to compare each other’s expectation with regard to academic interest and the role of the mentor, respectively mentee. One mentor stated:I think the main benefit is that both the mentor and the mentee are able to clarify their expectations with regard to the mentoring relationship. Within a short time I may present my way of supporting as a mentor and clarify if the expectations match.One mentee compared MSD to the mentor profiles and said:In comparison with the online profiles [speed mentoring] is more important for the decision. The mentor profiles were good to get an overview about what the mentors are doing [area of research and specialization] and then watch their research websites to find out which area of research I am interested in and afterwards meet them in person.In addition, mentors mentioned that MSD enables them to avoid ambiguity with regard to the detailed area of research and daily routines. One mentor stated:A very important thing is that there are mentors who are focused on experimental biomedical research and some with an emphasis on clinical research. In case I conduct clinical research I could send a mentee who would like to work with mouse models for example to a mentor experienced in this research approach. With speed mentoring you can avoid ambiguity.MSD also benefits from an overview of several candidates. Participants frequently outlined the efficiency and comparability inherent to this matching strategy:Within a short period of time you had the chance to meet several scientists. You got to know what exactly they are doing and what you are interested in. They usually have different projects and speed mentoring is helpful to get an overview. It was extremely timesaving and efficient.Furthermore MSD serves as helpful decision aid. Participants mentioned the advantage of the bidirectional process in which both parties choose their preferred candidates. Mentees experienced the mentors’ choice of their person as a pleasant feedback in particular when considering the high academic ranks of mentors  and their numerous duties. One mentee said:I think it is good, that the mentors have to make an ‘active’ choice, too. Thereby you get the feeling, that the mentor wants you as well, and you don`t feel like you impose on him. This feedback is really helpful.Mentees mentioned two additional categories which were not described by the mentors. One mentee said that self-reflection was increased through structured questions of the mentors during MSD. Furthermore some mentees reported that the mentors’ expectations regarding mentorship and the students’ performance became clear.We identified two key aspects with regard to the environment of MSD: Atmosphere and time-frame. Several mentees perceived the atmosphere as tensed and exciting. A few mentees said it felt similar to a job interview. Additionally the hierarchy gap played a role. Likewise mentors mentioned the ‘interview character’ and obvious hierarchy. Mentors reported an open-minded atmosphere when reflecting on their MSD experiences. Both mentees and mentors emphasized that they adapted quickly to the 5 minute sessions and the mentees’ initial nervousness decreased .When we asked the participants in how far MSD might facilitate long-term relationships mentors identified two major categories . Mentees mentioned fulfillment of expectations only. Participants emphasized that the ‘real mentorship’ evolves after MSD as mentors and mentees get to know each other. Still most participants considered MSD as an important basis to build on a serious, long-term mentoring relationship. One mentor stated:I think, if mentees get their selected mentor, they are even more motivated. They feel more comfortable regarding the first meeting with their mentor because they have an idea what to expect. And very likely they are glad that they succeeded to get their preferred mentor.Both mentees and mentors pointed out that the mentoring relationship and process met their expectation. One mentor reported:The matching was ideal. All three of my mentees met my expectation in terms of high motivation for scientific work and mentoring for one and half year by now.Likewise one mentee said:Speed mentoring provides a sound basis for good, long-term [mentoring] relationships later on. The mentoring relationship with my mentor progressed and works very well.In addition mentors hold the opinion that MSD leads to high commitment and results in better team work among the mentees. Finally, mentors gave examples for the success of the mentoring relationships originated in MSD in terms of substantial career moves of their mentees:It became clear, that these [mentees] participate in a graduate school … or applied successfully for a grant from ‘Studienstiftung des deutschen Volkes’ [largest German institution to sponsor young talents] or ‘Cusanus Werk’ [scholarship body of the Catholic Church].F = .273 p = .762) or gender  = .277, p = .871) nor did the two groups MSD vs. non MSD show statistically significant differences in age (t(91) = .340 p = .735) or gender  = .000, p = .988). The response rates of the survey were 78.4% (n = 29) for mentoring cohort 2011, 94.6% (n = 35) for mentoring cohort 2012 and 85.2% (n = 29) for mentoring cohort 2013. Neither the three mentoring cohorts differed significantly in age  = -2.66, p = .009), “My mentor provided constructive and useful feedback” (t(86) = -2.22, p = .029) and “Altogether I am satisfied with my mentor” (t(91) = -2.19, p = .031). The result of an independent t-test using gender as the group variable revealed that male students scored higher than female students in all six items. A statistically significant difference was found for one item only: “Altogether I am satisfied with my mentor” (t(91) = -2.10, p = .039). At a Bonferroni corrected p = .008 no significant difference could be found any longer.The independent F = 1.701, p = .132) and gender  = 1.622, p = .152) nor the interaction MSD*gender  = 1.604. p = .157). On item-level, the items “My mentor is committed to mentoring” (F(1) = 5.768. p = .019) and “My mentor provided constructive and useful feedback” (F(1) = 4.454. p = .038) showed a statistically significant effect of the matching method  = 5.061. p = .027), “My mentor demonstrated content expertise in my area of need” (F(1) = 5.993. p = .016), “My mentor provided constructive and useful feedback” (F(1) = 4.970. p = .028), and “Altogether I am satisfied with my mentor” (F(1) = 7.476. p = .008). Therefore mean responses are displayed separately for gender  as well as matching method  in Gender as a factor showed a statistically significant effect among the items “I have confidence in my mentor’s professional integrity” . In contrast, male students who participated in MSD rated only two items higher in comparison to the “no MSD group”. The interaction of matching method and gender showed no significant effect on item-level.2 = 15.912, p < .000). More than one third of the mentees who chose their mentor via online profiles of the mentors quitted mentoring after one year. In comparison only one mentee of each mentoring cohort matched via MSD resigned the mentoring relationship with his or her mentor after one year (We analyzed the 1-year continuance of the mentoring relationship as a criterion for the success or failure of mentorship. The percentage of mentees who quitted mentoring after one year differed significantly by matching method (MSD vs. no MSD; χone year . Three mPrevious research has shown that successful mentoring is based on matching the mentee and mentor according to personal and attitudinal similarities , 28–30. Formal mentoring programs in academic medicine usually employ three major approaches to match mentors and mentees: randomly assignment of mentees to mentors by an administrator, selectively matching based on certain criteria such as clinical specialty, area of research, personal attributes and/or other aspects conducted by an administrator, or mentees are allowed to choose their mentors on the basis of previously mentioned criteria , 18. RecA recent qualitative study across two academic health centers described that failed mentoring relationships were characterized by several aspects including poor communication, lack of commitment to the mentoring relationship and lack of personal similarities . Our quaIn our study participants reported that MSD largely benefits from the bidirectional process in which both parties chose their preferred candidates. Thus the MSD process allows mentees and mentors to have more influence on the matching process. Two US studies that explored formal mentoring programs in organizational settings found that perceived input into the match was associated with greater mentorship quality, greater career mentoring  and greaThe continuance of mentorship serves as an apparent indicator for the success of mentorship. The differential mentoring program offered at the UKE is designed to accompany students until they finish medical school, still both parties are able to quit the mentoring relationship at the end of each semester . CompariA recent qualitative study across two medical schools identified lack of commitment and communication as characteristics of failed mentoring relationships . ConsideA recent systematic review of the mentoring literature described the complexity of the personal connection for mentoring relationships. The authors reported that mentoring relationships were potentially enhanced by similar interests and ideals . Our quaMentoring in academic medicine is challenged by clinical, research and administrative demands , 10. ThuOur study has several limitations. First focus groups are not suitable for generalization. Even though group interaction distributes substance to the topic, individual opinions can occur. Second a selection bias is possible: we only interviewed participants of one medical school. Third the students were high-performing and invited exclusively to the Mentoring Program for Excellent Students . Thus thThe present data suggest that MSD is a valuable matching tool for a medium group size. It helps to overcome common mentoring barriers such as lack of fit between mentor and mentees  and vuln