Magnetic resonance imaging (MRI) is increasingly being used in cardiology to detect heart disease and guide therapy. It is mooted to be a safer alternative to imaging techniques, such as computed tomography (CT) or coronary angiographic imaging. However, there has recently been an increased interest in the potential long-term health risks of MRI, especially in the light of the controversy resulting from a small number of research studies reporting an increase in DNA damage following exposure, with calls to limit its use and avoid unnecessary examination, according to the precautionary principle. Overall the published data are somewhat limited and inconsistent; the ability of MRI to produce DNA lesions has yet to be robustly demonstrated and future experiments should be carefully designed to optimize sensitivity and benchmarked to validate and assess reproducibility. The majority of the current studies have focussed on the initial induction of DNA damage, and this has led to comparisons between the reported induction of γH2AX and implied double-strand break (DSB) yields produced following MRI with induction by imaging techniques using ionizing radiation. However, γH2AX is not only a marker of classical double-ended DSB, but also a marker of stalled replication forks and in certain circumstances stalled DNA transcription. Additionally, ionizing radiation is efficient at producing complex DNA damage, unique to ionizing radiation, with an associated reduction in repairability. Even if the fields associated with MRI are capable of producing DNA damage, the lesions produced will in general be simple, similar to those produced by endogenous processes. It is therefore inappropriate to try and infer cancer risk by simply comparing the yields of γH2AX foci or DNA lesions potentially produced by MRI to those produced by a given exposure of ionizing radiation, which will generally be more biologically effective and have a greater probability of leading to long-term health effects. As a result, it is important to concentrate on more relevant downstream end points (e.g. chromosome aberration production), along with potential mechanisms by which MRI may lead to DNA lesions. This could potentially involve a perturbation in homeostasis of oxidative stress, modifying the background rate of endogenous DNA damage induction. In summary, what the field needs at the moment is more research and less fear mongering. It is increasingly being used in cardiology to detect heart disease and guide therapy. It is mooted to be a safer alternative to x-ray and radioisotope imaging techniques, such as computed tomography (CT) or coronary angiographic imaging, where there is a clear relationship between exposure to ionizing radiation and increased cancer risk, as recently demonstrated by epidemiological studies for paediatric CT exposures.10 In addition, RF may cause some heating of tissue, and GMF are capable of stimulating nerves and muscles; however, in practice, the operation limits are set to minimize these effects. With regard to biological and long-term health effects associated with MRI exposure and its various components, a number of comprehensive reviews have been published by national and international committees16 along with a recent review of genotoxicity associated with MRI exposures.4 Although the data are somewhat limited, the current consensus is that no clear link exists between MRI or associated magnetic and pulsed radiofrequency fields and subsequent health risks. The relevant data are discussed below. We conclude that it is essential to avoid over interpretation of the limited data available on potential genetic effects of MRI and making uninformed comparisons of these effects with the kinds of DNA damage produced by ionizing radiation.MRI results in exposure of the patient to a mixture of static magnetic fields (SMF), time-varying gradient magnetic fields (GMF) typically in the range 10–100 kHz, and pulsed radiofrequency fields (RF) in the MHz range. While for occupational exposures of staff working with or in close proximity to the scanner, SMF is the most relevant. The known risks of using MRI mainly relate to the interaction of magnetic and radiofrequency fields with ferromagnetic and paramagnetic objects  and that movement of a person through a strong SMF may induce effects such as vertigo and nausea.in vitro and in vivo. DNA damage, especially DNA double-strand break (DSB) and chromosome aberrations (CA), has typically been a main focus of research, since DSB and CA have the greatest potential to lead to carcinogenesis or hereditary effects. The effects of exposure to individual fields, SMF, GMF, or RF have been extensively reviewed by various national and international committees16 who typically concluded that there is no clear link between exposure and an increase in genetic damage. However, current MRI exposures are a complex combination of SMF, GMF, and RF. Only a very limited number of investigations of genetic damage using clinical MRI scanners, and the types of sequences routinely used in the clinic, have been published and are briefly summarized in Table 1. Seven of these studies were also recently critically reviewed by Vijayalaxmi et al.4 What is clear is that the experimental data relating to MRI genotoxicity are limited and inconsistent.The potential of adverse effects associated with exposure to magnetic fields has been studied for a number of years both et al.21 showed no enhancement immediately following in vitro exposure of human leukocytes. In contrast, statistically significant enhancements were observed by Lee et al.19 following in vitro exposure times of 45 min or greater, with the observed level increasing with duration of exposure. For in vivo exposures, Yildiz et al.20 observed no significant increase in lymphocytes taken after the initial non-contrast-enhanced MRI. However, they did report a significant increase in the lymphocytes taken following the subsequent contrast-enhanced MRI.Three of the studies investigated the induction of single-strand breaks (SSB) using the alkaline comet assay. The study of Szerencsi in vitro exposure of human cancer cell lines 0, 1, or 24 h post exposure,18in vitro exposure of human lymphocytes 0, 1, or 20 h post exposure,23 or in human lymphocytes taken 5 min after in vivo exposure of patients undergoing contrast-enhanced cardiac magnetic resonance imaging (CMR).22 In contrast, Fiechter et al.8 reported a statistically significant enhancement in DSB in human lymphocytes taken from patients immediately following contrast-enhanced CMR. While Lancellotti et al.7 reported no enhancement in T lymphocytes taken from volunteers 1 and 2 h post exposure to unenhanced CMR, but significant enhancement was reported at day 2 and 1 month post exposure before returning to base line levels after 1 year (a significant increase was observed at 2 h and day 2 for NK cells but not at other time points).Five studies investigated DSB induction using γH2AX assays . Three of the studies showed no enhancement of DSB following either et al.21 showed no increase in the yield of micronuclei (MN) following in vitro exposure of human lymphocytes, a significant enhancement of both MN and CA (using Giemsa staining) was reported by Lee et al.19 for in vitro exposure times of 45 min and greater, with the observed level increasing with duration of exposure. The most frequent types of aberrations observed were chromatid breaks, with both exchange-type and deletion-type CA being observed for the longest exposure time (89 min). A significant increase in MN frequency was also reported by Simi et al.9 in lymphocytes at 0 h post in vitro exposure, with the frequency increasing with increasing duration of exposure. While for the two shorter exposures no significant increase in MN frequency above controls was observed for cells left for 24 h to repair before performing the assay, a significant increase was still observed for the two longer exposure times. Following in vivo exposure, a significant increase was observed for all participants when blood was collected immediately or 24 h post exposure, although no difference was observed at later collection times .While Szerencsi 17Single experiment using the Ames test shows no mutagenic or co-mutagenic effect.Cells have several well-developed DNA-repair pathways and DNA-damage checkpoints to deal with the frequent DNA lesions produced by endogenous or exogenous sources and the vast majority of lesions will be repaired. However, the efficiency and fidelity of repair, and therefore the relevance, will be dependent on the type of lesion produced. Accurate quantification of these lesions is also not trivial, especially at low levels; therefore, great care must be taken to ensure consistency and with appropriately interpreting the results.24 However, these lesions are typically repaired quickly with high fidelity and so are much less likely to be relevant to long-term health effects compared with DSB.26The standard alkaline comet assay detects strand breaks along with alkali-labile sites, although the technique can also be modified to visualize the presence of base damage by using lesion-specific endonucleases that convert base damage into SSB.27 An early response to DSB is the phosphorylation of the histone H2AX, which can be detected using a fluorescent antibody in conjunction with immunofluorescent microscopy to visualize discrete nuclear foci at sites of DSB.29 Radiation-induced foci have been observed following exposure to ionizing radiation with doses as low as 1 mGy in stationary phase human fibroblasts (∼0.02–0.04 DSB per cell), with the yield increasing linearly with dose.30 Studies have also demonstrated that the yield of γH2AX foci can be a useful quantitative biomarker of human low-level ionizing radiation exposure in blood taken from an exposed individual at doses down to 6 mGy.31 This technique has significantly greater sensitivity in detecting DSB than direct methods which require many hundreds of DSB per cell, but it is also associated with several shortcomings. For instance, studies using Pulse Field Gel Electrophoresis (PFGE) demonstrate that the initial DSB produced are quickly repaired with a half time of ∼20 min, with only a few remaining after 1 h.33 However, γH2AX is a marker of the metabolic activities initiated to repair the damage, rather than a direct marker of DSB. Although a few laboratories are able to observe γH2AX foci within minutes post exposure, their detection is difficult at these early times due to their small size, and they are more reliably scored 15–30 min post exposure. Many laboratories report an initial increase in foci number with time, reaching a maximum ∼30–60 min after exposure, with a reduction being observed at later times. Therefore, the initial yield of DSB may be underestimated due to many of the DSB being repaired during this period; additionally the subsequent rate of DSB repair reported at these later times is often slower than the initial rate of DSB repair.35 It has been proposed that γH2AX may, in some cases, continue to mark the site following the initial rejoining of the break and therefore may not always represent a physical break.36 γH2AX also detects replication-induced DSB in S-phase cells which will contribute to the persistence or delayed response of the signal. Even non-exposed S-phase cells typically have a higher number of background foci than other phases due to endogenously induced damage.37 Replication-induced DSB are also very different chemically to double-ended DSB and potentially much more likely to be repaired; it is therefore not clear whether these secondary foci always reflect the presence of DSB.37 γH2AX may also in some circumstances be generated by DNA transcriptional activity.40 It is important to note that the sensitivity, reliability, and absolute yields obtained with this technique can vary significantly between laboratories depending on many factors such as staining protocol, cell type, cell cycle, temperature, and critically on the imaging and scoring criteria used. This is illustrated by the large variation in results obtained for the 20 patients by Fiecheter et al.,8 with the median number of γH2AX per cell varied from 0 to 0.661 pre-exposure and 0 to 1.065 post-exposure; in contrast, the mean number of foci per cell obtained for 45 patients by Brand et al.22 is significantly smaller and much more consistent, varying from ∼0.09 to 0.17 and consistent with background levels of <0.2 foci per lymphocyte reported for ionizing radiation biodosimeter studies.41DNA DSB and particularly complex DSB (see below) are considered to be particularly important with respect to carcinogenesis as they are intrinsically more difficult to repair than other types of DNA damage with their potential to lead to modification, rearrangement, or loss of chromosomal material.43 As a result, this technique does not maintain the level of sensitivity required to be used as a biodosimeter of ionizing radiation exposure beyond the first hours.41 The flow cytometry results of Fiecheter et al.8 and Lancelloti et al.7 both show significant variation between individuals, with some showing an increase while others a decrease in response. The delayed enhancement in γH2AX expression reported by Lancellotti et al.7 at day 2 and 1 month post exposure is inconsistent with DSB being produced during imaging as the majority of DSB are reported to be repaired within hours and the limited sensitivity of the flow cytometry assay at extended times following exposure. This could reflect an enhancement in the background level of DSB production (or other events leading to increased γH2AX expression) post exposure. However, the time course of this increase is still greater than the delayed peak of several hours often observed following exposure of exponentially growing cells to genotoxic agents such as UV and alkylating agents.44Flow cytometry can also be used to analyse γH2AX with the advantage that thousands of cells can be analysed very quickly, through measuring the total fluorescence intensity rather than detecting individual foci. In general though the sensitivity of this approach is lower than that achieved by scoring individual foci, in part due to background foci intensity having a wider inter-individual variability.46 and as a marker of relative risk to different qualities of radiation [The effectiveness of different qualities/types of radiation  at inducing a given biological end point for a given absorbed dose can vary significantly.] exposure.48 In addition, significant evidence exists for the role of CA in early stages of cancer, with data showing that the frequency of CA in peripheral lymphocytes may be a predictor of cancer incidence in human populations.52 MN may also be used as measure of CA induction and may result from acentric chromosome/chromatid fragments or whole chromosomes failing to be incorporated into the daughter nuclei. Evidence indicates a link between increased MN and elevated cancer risks.53 In addition to the dicentric chromosome assay, the MN assay has, to a lesser extent, been established as a technique for biodosimety for ionizing radiation. These have required careful optimization of the sample preparation, data analysis protocols, and harmonization of procedures between laboratories to ensure consistency. However, the sensitivity of these techniques limits their usefulness as a way of retrospectively determining dose in exposures of the order of 100 mGy or above (>2 DSB per cell). Therefore, investigating the induction of CA or MN in situations where there are fewer DSB per cell becomes increasingly challenging, requiring more cells to be scored and great care to ensure reliability of the data. Dicentric aberrations and MN produced by ionizing radiation are essentially stable and can be detected for several months following irradiation, with their loss being directly related to the rate of turnover of cells within the blood. It is therefore interesting that although Simi et al.9 reported an increase in MN in lymphocytes in blood collected immediately after in vivo exposure, no difference was observed compared with controls at later collection times . Although lymphocytes within the blood are typically in G0 phase, the reported dominance of chromatid aberrations reported by Lee et al.19 suggests that rearrangements occur mainly during S-phase. To investigate CA/MN induction, the cells were stimulated to divide. It is therefore possible that the DNA lesions resulting in CA/MN were produced during DNA replication, potentially by replication-induced breaks/DSB resulting from stalled replication forks. So rather than MRI directly producing DNA lesions, it may perturb the homeostasis within the cells for a period of time resulting in an enhancement of endogenous damage.When assessing the relevance of the studies investigating the induction of DNA damage such as DSB, it is important to remember that the production of such lesions does not necessarily lead to long-term consequences. In general, the vast majority of DNA damage produced will be transient because of repair processes. As a result, it is the longer term consequences of this damage that are more critical, the unrepaired fraction and the resultant genetic modifications. A more relevant end point to study would be CA production. These have been extensively used (dicentric aberrations in particular) in peripheral blood lymphocytes for assessment of ionizing radiation, both as a biological dosimeter of exposure55 ROS include superoxide, hydrogen peroxide, hydroxyl radicals, and singlet oxygen and they can oxidize DNA, which can lead to several types of DNA damage chemically similar to those induced by ionizing radiation, although importantly their spatial and temporal distribution will be very different. These lesions are constantly being repaired with high fidelity by the cell via highly effective repair processes which help maintain genome integrity, minimizing the onset of ageing, and tumourigenesis. However, it has been proposed that these ubiquitous background events may account for an important fraction of oncogenic events in humans.56 Identification of these lesions and their production by free radical mechanisms have been the subject of numerous reviews.60 Although few, if any, DSB are formed by endogenous processes,62 events such as DNA damage lesions may lead to stalled replications forks during DNA replication  or interfere with DNA transcription, both of which can be detected by the γH2AX assay as mentioned earlier.40DNA damage is a constant and a very frequent event in the daily life of all cells. Every cell within the body has a background level of the order of at least 50 000 endogenous DNA lesions per day as a result of reactive oxygen species (ROS) and other reactive metabolites, with the number expected to increase in situations with increasing oxidative stress.63 Environmental factors can lead to the activation of endogenous ROS or RNS-generating systems resulting in either a temporary or persistent dysregulation in signalling cascades and a change in ROS and/or RNS concentrations.63 This perturbation in homeostasis of this signalling and associated changes in oxidative stress can lead to a modulation of the background rate of endogenous DNA damage induction. For example, it is well known that very low doses of ionizing radiation can perturb intercellular signalling, levels of ROS, and other signalling molecules and can result in genotoxic effects such as induction of γH2AX foci, chromatid aberrations, MN, and apoptosis in unirradiated cells.67 Interestingly, rather than a continuous increase in effect with increasing exposure, the observed response is often consistent with a switch between two stable states essentially independent of triggering dose (if above a low threshold dose). It has been proposed that MR exposure may be able to increase the rate of production of DNA damage by endogenous processes as a result of an increase in oxidative stress.69It is well known that within the body cells do not act in isolation, with intercellular signalling being vital for maintaining the multicellular organization of tissue and the normal functioning of the constituent cells. ROS along with reactive nitrogen species (RNS) are formed as a consequence of both normal cell metabolism and inflammation and as a result of exposure to environmental factors. Free radicals and resulting reactive non-radical species are important for many physiological processes, including immunological defence, intra- and intercellular signalling, their concentration being determined by the balance between the rate of production and clearance by various antioxidants. As a result, there is a delicate balance between the advantageous and detrimental effects of these radicals.Assuming MRI can induce DNA lesions, the kinetics of induction and repair of any such lesions are also unclear from current studies but remain a crucial question that needs to be addressed. Are these lesions formed directly during imaging where some of the lesions may persist to later times, or are they formed through indirect processes that continue after imaging with a constant turnover as lesions are produced and repaired? This information would be helpful in guiding the understanding of the mechanisms, such as changes in homeostasis or persistent damage. A potential approach to address these questions would be to use live cell imaging using fluorescently labelled DNA-repair proteins and cell cycle markers, to identify the kinetics of formation and repair of individual lesions and cell cycle dependence. The relevance of any short-term increase in DNA damage as a result of an enhancement of endogenous processes has to be considered in the context that similar sites of DNA damage will be continuously produced throughout the lifetime of an individual and therefore any lesions, even if induced by MRI exposure, may not significantly add to the burden of risk associated with the background level of endogenous damage.3 However, due to the way ionizing radiation interacts within the body, the resulting DNA damage produced includes damage unique to ionizing radiation, such as clustered DNA damage, and therefore, it is inappropriate to infer long-term health risks based simply on a comparison of yields of DSB or other non-DSB lesions. It is essential to deconvolute the γH2AX data into different classes of lesions and associated lifetimes, to be able to comment on the risks of MRI to humans.The majority of recent experiments in investigating genotoxicity of MRI use the γH2AX assay as a measure of DSB. It is then tempting to compare induction of γH2AX and implied DSB yields produced following MRI with induction by imaging techniques using ionizing radiation.Figure 2). It is the ability of ionizing radiation to produce clustered DNA damage which make it so biologically effective, with these longer lived clustered lesions dominating the long-term response rather than the much more numerous isolated DNA lesions that are chemically similar to those induced endogenously and are rapidly repaired. In mammalian cells, an exposure of 1 Gy of x-rays or γ-rays will produce ∼1300 base lesions, 1000 SSB, and 20–40 DSB . Although the number of lesions produced by ionizing radiation is significantly less than those produced daily by endogenous processes (∼50 000 per cell per day), it is the clustering of multiple lesions at a site of DNA damage72 and the corresponding reduced ability of their repair that is critical, leading to an increase in genetic modification (clustered lesions produced by endogenous damage are rare and do not match the degree of complexity that can be produced by ionizing radiation74). Although the majority of ionizing radiation-induced DSB will be repaired within ∼60 min, a small fraction may persist for >24 h.78 Theoretical analysis of these DSB using Monte Carlo calculations82 indicates that 20–50% of these DSB are in fact complex DSB (with extra single-strand breaks and/or associated base damage within a distance of 10 base pairs).80 In fact, the frequency and complexity of DSB typically increase with ionization density of the radiation and are important factors in accounting for differences in relative biological effectiveness (RBE) with radiation quality.80 This is also reflected in the variation in biological efficiency per DSB produced as a function of radiation quality, with even the DSB produced by sparsely ionizing x-rays estimate to be 4–40 times more effective than simple DSB produced by high concentrations of hydrogen peroxide.83 Additionally, as DSB are formed along the path of the resulting electron tracks following exposure, the DSB produced can be correlated in space and time increasing the chance that they may interact and lead to genetic and chromosomal rearrangements.84These assertions are founded on the knowledge that ionizing radiation interacts within the body and deposits energy along the resulting electron tracks producing highly structured tracks of ionization and excitation events that are stochastic in nature. This produces a wide variety of molecular damage, including base damage, abasic sites, SSB, and DNA–protein cross links. However, because these electrons produce multiple energy deposition sites along the resulting radiation track on the nanometre scale (correlated in time and space), it is capable of producing sites with multiple lesions on the DNA within a few base pairs of each other (88 While isolated lesions (e.g. base damage or SSB) are repaired quickly with high fidelity, for non-DSB clusters the rate of repair is typically impaired by the presence of additional lesions within the cluster.91 The extent of the reduction is dependent on the types and number of lesions along with their separation and orientation.89 Although processing of certain bistranded lesions may in a few cases lead to repair-induced DSB, the most likely consequence of these non-DSB clustered lesions will be an increase in their lifetime, and therefore, an increased chance of resulting in a stalled replication fork and potentially lead to replication-induced DSB in S-phase . It has been estimated that ∼10% of non-DSB clustered lesions will be converted to DSB as a result of processing,32 with the possibility that some of these DSB may be complex by virtue of additional lesions close by.92 Experiments have shown that these clustered lesions result in an increase in mutation frequencies in bacteria, yeast, and mammalian cells.94In addition to DSB and complex DSB, ionizing radiation will also form non-DSB clustered lesions. These non-DSB clustered lesions formed contain two or more lesions within the order of 10 base pairs and may occur either on the same strand (tandem) or opposite strands (bistranded). Complex non-DSB damages have been shown to make up a significant component of the lesions induced by radiation, being at least 4–8 times greater in number than prompt DSB for x-rays and γ-rays.Little is known about the mechanisms by which MRI can produce DNA damage, if indeed it does. It is know that the fields associated with MRI are non-ionizing and therefore unable to produce free electrons with sufficient energy to produce clustered damage sites over 1–2 turns of the DNA, similar to that associated with ionizing radiation. Therefore, any lesions that are produced as a result of MRI are much more likely to be repaired with high fidelity than those produced by ionizing radiation.15 did not find clear evidence that SMF are carcinogenic, the data are limited, underpowered, or suffer methodological weakness.11 Both IARC and the recent SCENIHR14 report suggest the need for a large, carefully designed epidemiology study.Although MRI imaging is generally considered to be safe compared with imaging technology using ionizing radiation, there is increased concern about the potential long-term health effects of exposure, especially with the push to higher static fields along with stronger and faster switching fields. Although IARCThe ability of MRI to produce DNA damage and potential mechanisms involved remains unclear. Generally no consistent response has been reported between the very limited number of published studies, with limited or no controls, typically based on data from a small number of subjects or often a single experiment and therefore limited statistical power. It is therefore essential that future studies employ strict standardization in experimental design and scoring criteria, along with quality control measures to ensure reproducibility and consistency. All techniques should be carefully benchmarked against negative and positive controls  to confirm the validity and reproducibility of the results, collected over multiple independent experimental measurements and ideally include inter-laboratory comparisons. There must also be a willingness by the scientists and journals to publish negative results for well-designed experiments. While there is a tendency to focus on the initial induction of DNA damage, it is more relevant to study downstream effects, for example CA production for which there is some evidence to this could be a marker of cancer risk. If a reproducible effect can be observed, then these studies will also need to critically assess the importance of contrast agents, along with potential confounding factors and other potential synergistic effects . An additional issue to be addressed would be to determine the critical component of exposure  if possible to compare responses for different scans between laboratories.40 It would also be appropriate to investigate how imaging may perturb intercellular signalling and associated levels of oxidative stress which may lead to modulation in endogenous damage levels, epigenetic changes, and genomic stability in cells and their progeny. In summary, what the field needs at the moment is more research and less fear mongering.It is important that great care is taken to ensure that any potential genetic effects associated with MRI exposure, if it exists, are not over interpreted. There is a danger that in the absence of clear evidence, physicians and patients are misinformed by the presentation of preliminary data, affecting their choice of potentially life-saving imaging tests. The ability for MRI to produce DNA lesions has yet to be robustly demonstrated. If lesions are formed, then the relative probability of these lesions to produce long-term health effects has to be put in the context of the risk associated with the 50 000 lesions produced daily by endogenous processes. As discussed, the fields associated with MRI are non-ionizing, having insufficient energy to directly break chemical bonds and are thus unable to produce free electrons with sufficient energy to produce clustered lesions including complex DSB with an associated reduction in repairability. The complex DSB produced by ionizing radiation have been shown to be more effective than simple DSB at producing a wide range of biological end points, including mutations, with a greater probability of leading to long-term health effects. It is therefore important not to try an infer cancer risk by simply comparing the initial yields of DSB produced by MRI to that produced by a given exposure of ionizing radiation. The γH2AX assay not only marks classical double-ended DSB, but also stalled replication forks and/or collapse in S-phase or interfere with DNA transcription.


Arabidopsis thaliana cells. Large (20 pA/pF) TPC1 currents were elicited by cytosolic addition of the phosphoinositide phosphatidylinositol--bisphosphate P2) with an apparent binding constant of ~15 nM. The channel is voltage-dependent, activating at positive potentials with single exponential kinetics and currents are Na+ selective, with measurable but low permeability to Ca2+. Cytosolic Ca2+ modulated hTPC1 in dual way: low μM cytosolic Ca2+ increased activity by shifting the open probability towards negative voltages and by accelerating the time course of activation. This mechanism was well-described by an allosteric model. Higher levels of cytosolic Ca2+ induced a voltage-dependent decrease of the currents compatible with Ca2+ binding in the permeation pore. Conversely, an increase in luminal Ca2+ decreased hTPC1 activity. Our data point to a process in which Ca2+ permeation in hTPC1 has a positive feedback on channel activity while Na+ acts as a negative regulator. We speculate that the peculiar Ca2+ and Na+ dependence are key for the physiological roles of the channel in organellar homeostasis and signalling.Two-pore channels (TPC) are intracellular endo-lysosomal proteins with only recently emerging roles in organellar signalling and involvement in severe human diseases. Here, we investigated the functional properties of human TPC1 expressed in TPC-free vacuoles from  The endolysosomal system is composed of a series of internal compartments fundamental for cellular homeostasis and is involved in a variety of different physiological processes from signaling to cell growth up to defence mechanisms, to cite only few135689102+ waves after a period of ischemia and its deletion in mice is cardio protectiveThe physiological importance of TPC channels is underlined by recent findings of their involvement in different severe pathologies. TPC2 plays a role in neoangiogenesis processes linked to vascularization of solid tumors2 sensitivity and the Ca2+ permeability of the channels. Using intracellular Ca2+ measurements101617181920212225261014282+, leading to the suggestion that TPC channels represent the long sought for NAADP sensor2+, but were instead activated by PIP2 and highly permeable to Na+, and an indirect role in lysosomal Ca2+ release was suggested1131Despite the physiological relevance, the functional properties of TPC channels are not fully defined. The major problem in the functional characterization of these proteins is represented by their intracellular localization. This renders very difficult the application of electrophysiological techniques because of the sub-micrometric dimension of animal endosomes and lysosomes. Divergent results have been reported regarding the nicotinic acid adenine dinucleotide phosphate (NAADP)/PI3,5)P,5P2 sens+ selectiveet al.1526273236+ but also Ca2+ permeable. Furthermore we found that both cytosolic and luminal Ca2+ are powerful modulators of hTPC1.An alternative approach to study mammalian lysosomal channels is to express them in plant protoplasts where they localize to the large lysosomal compartment, the vacuole, which is easy to isolate and amenable to classical patch clamp techniques93334In mouse, two TPC1 isoforms have been found, the second one shorter than the first with an N-terminal deletion of 69 aminoacidsArabidopsis thaliana tpc1-2 mutant (AtTPC1 null background)2 reversibly increased the current in response to a depolarizing step of +40 mV from a holding potential of −70 mV (2 washout (2-evoked currents were observed in hTPC1-EGFP expressing vacuoles but not in untransformed control vacuoles (2-responses are due to activation of hTPC1 channels. As NAADP has been proposed to be a TPC-channel agonist2 responding vacuoles did not elicit an increase of membrane current (2 but not of NAADP with hTPC1.Human TPC1 (hTPC1) was transiently expressed in mesophyll protoplasts from the f −70 mV . The cur washout . PI3,5)vacuoles , strongl,5vacuole current ; these d2-activated current increased with increasing PIP2 concentration with a similar apparent binding constant at +40 mV and −40 mV, i.e. 15 and 18 nM Pnd 18 nM , suggest2  from positive to negative values was recorded upon a cytosolic sodium concentration change from 10 to 200 mM. The full agreement of the experimental Vrev with the theoretical Nernst voltage for Na+ shown in + permeable channel. In +) was due to the presence of 2 mM of cytosolic Mg2+: removing cytosolic Mg2+ eliminated the voltage dependent current inhibition  from the cytosol of 0.36 ± 0.3 with an affinity constant at 0 mV (KMg) of 20 ± 3 mM P2 . The res reduced . Figure hibition . A simil+ by K+ in the presence of luminal Na+ resulted in a total disappearance of PIP2 activated outward currents . To focus on Ca2+ permeability, tail currents were recorded by stepping from +70 to −100 mV (10 mV decrement), after a 500 ms activating pule to +50 mV (top). Exploring the responses on an expanded scale around the Vrev, small inward tails were detectable. Ion permeability ratio between Ca2+ and Na+ (PCa/PNa), measured by reversal potentials (bottom) was between 5 and 10% (rev (i.e. the higher was the apparent Ca2+ permeability). We attributed this effect to the entrance of Na+ into the lumen and mathematically corrected it  and Ca2+ bound (i = 1) channels, respectively. Details of this four-state model are presented in the The cyt, this effect is compatible with physiological concentration changes of cytosolic Ca2+; a simple mathematical model is able to quantitatively describe the measured shift together with the kinetic effects.In addition to the intrapore site, we discovered that TPC1 harbours a rather high affinity cytosolic Ca2+ binding site on the luminal side of the channel. In this case the binding of Ca2+ induced a decrease of the activity by shifting the open probability to positive voltages, with a ΔVhalf of about +60 mV when [Ca2+]lum was changed from 1 μM to 1 mM. This might be physiologically relevant since the average luminal concentration of Ca2+ has been reported to be around 500 μM and it may vary from 1 mM to less than 1 μM for example upon experimental manipulation of lysosomal pH2+ effect.Finally, we showed functional evidence of a third Ca+-selectivity in the second pore loop and confirmed that human TPC channels are sodium channel with low calcium permeability.Recently the structure of the Arabidopsis plant counterpart of hTPC1, namely AtTPC1, was published6392+ ] and an increase of cytosolic [Ca2+]. In this context Na+ is actually a negative regulator since its release from the luminal to cytosolic side depolarizes the luminal membrane and limits Ca2+ release. We can speculate that this interplay between Ca2+ and Na+ is necessary to generate a precise shape and dynamic of Ca2+ release or, in other words, a defined Ca2+ signature. Our conclusion is therefore that hTPC1 is a Na+ and slightly Ca2+ permeable, outwardly rectifying channel, working as a sensor of luminal and cytosolic Ca2+.Overall, our data show that calcium permeation mediated by hTPC1 channels provides a positive-feedback mechanism amplified by both the simultaneous reduction of luminal [CahTPC1 coding sequence was PCR amplified from HEK293 (human embryonic kidney) cells cDNA with the Thermo Scientific® Phusion high fidelity polymerase using the following primers: 5′-CATGAAGCTTCATGGCTGTGAGTTTGGATGACGA-3′ and 5′-CATGGAATTCCGCGGTAACGGTCTGGGAGCGCTGG-3′ (underlined are HindIII and EcoRI restriction sites used for cloning).The The PCR product was subsequently digested and ligated into the plant expression vector pSAT6-EGFP-N1 in the Multiple Cloning Site between the HindIII and EcoRI sites downstream of the 35SS strong constitutive promoter in frame with an enhanced GFP. The construct was verified by sequencing and identified as the human TPC1 which is the two pore calcium channel protein 1 isoform X1 coded by XM_011538492.1:246.2696 (mRNA transcript variant X4) that encodes for one of the shorter TPC1 isoforms (816 amino acids).Arabidopsis thaliana tpc1-22, 154 NaCl, 5 KCl, 2 MES-KOH, 5 glucose, pH 5.6, ampicillin 100 μg/ml). The transformation efficiency judged by Enhanced Green Fluorescent Protein (EGFP) fluorescence in protoplasts was routinely >50%. An efficient vacuole release was achieved by perfusion of transformed protoplasts with vacuole release solution methylamino) propane (BTP), 5 ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 3 MgCl2, pH 7.5, 450 mOsm with D-sorbitol).Plants of 2, pH = 7.5). TPC-expressing vacuoles were identified by EGFP fluorescence.Patch-clamp experiments on Arabidopsis vacuoles were performed ≥48 h after protoplast transformation, as described elsewhere49502, 10 MES, pH 5.5 (with NaOH). The standard bath (cytoplasmic side) solution contained (in mM): 100 NaCl, 10 Hepes, pH 7.5 (NaOH). In the selectivity experiments of 2 2 mM was added in the cytosolic solution. NaCl 10 mM was obtained by substituting 90 mM of NaCl with equimolar concentration of KCl. In the K+-based bath solution of 2+-permeability, the luminal concentration of NaCl (100 mM) was substituted by 50 mM CaCl2. In this condition we observed that the presence of VRS as bath solution increased the quality of the seals. We measured a liquid junction voltage between the two solutions of 9.9 ± 0.6 mV and corrected offline (V = Vapplied − 9.9 mV). For the sake of clarity in 2+ concentration was determined by Plasma Emission Spectrometry (ICP-OES), instrument model Vista PRO Varian , with the following main operating conditions: RF Power: 1100 W; Plasma gas flow rate: 15.0 L min−1; Sample uptake rate: 0.8 mL min−1. Free calcium concentration in the presence of EGTA was calculated by using a dedicated program2 was purchased as dioctanyl ester (diC8) from AG Scientific or Echelon Biosciences Inc (USA). Other chemicals were purchased from Sigma-Aldrich . PIP2 and NAADP were prepared as 0.9 mM and 1 mM stock solutions respectively and stored at −20°.The standard pipette  solution contained (in mM): 100 NaCl, 2 MgClA total number of more than 95 vacuoles expressing hTPC1 P2) were investigated in this work.Positive currents correspond to cations flowing from the cytoplasmic side of the vacuole to the lumen or anions moving in the opposite direction. Unless otherwise indicated, data are reported as mean ± sem. For the voltage dependent inhibition mediated by cytosolic calcium, we used the Woodhull modelHow to cite this article: Lagostena, L. et al. The human two-pore channel 1 is modulated by cytosolic and luminal calcium. Sci. Rep.7, 43900; doi: 10.1038/srep43900 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Acute myeloid leukemia (AML) is a biologically and clinically heterogeneous cancer of the bone marrow that is characterized by the rapid growth of abnormal myeloid cells.de novo AML. We explored the functional impact of the genes identified in the mutational analyses through an integrated Gene Ontology (GO) and pathway analysis.We performed a mutational analysis to identify AML somatic mutations using the whole-exome sequencing data of 36 tumor-normal sample pairs from Korean patients with NEFH (p = 6.27 × 10−13 and q = 1.18 × 10−8) and TMPRSS13 (p = 1.40 × 10−10 and q = 1.32 × 10−6), also demonstrated q values less than 0.1 in 36 Korean AML patients. Five out of the 11 novel genes have previously been reported to be associated with other cancers. Two gene mutations, CEBPA (p = 5.22 × 10−5) and ATXN3 (p = 9.75 × 10−4), showed statistical significance exclusively in the M2 and M3 subtypes of the French-American-British classifications, respectively. A total of 501 genes harbored 478 missense, 22 nonsense, 93 frameshift indels, and/or three stop codon deletions and these gene mutations significantly enriched GO terms for signal transduction , plasma membrane , and scaffold protein binding . The mitogen-activated protein kinase  was the most enriched Kyoto Encyclopedia of Genes and Genomes pathway.A total of 11 genes, including Morphological AML subtypes may in part reflect subtype specific patterns of genomic alterations. Following validation, future studies to evaluate the usefulness of these genes in genetic testing for the early diagnosis and prognostic prediction of AML patients would be worthwhile.The online version of this article (doi:10.1186/s12881-017-0382-y) contains supplementary material, which is available to authorized users. FLT and NPM1 or CCAAT/enhancer binding protein (C/EBP), alpha (CEBPA) could be used to predict NK-AML prognosis, most patients did not have this mutation set [Acute myeloid leukemia (AML) is a highly malignant cancer of the bone marrow that is characterized by the rapid growth of abnormal myeloid cells. In 2012, leukemia accounted for 2.5% and 3.2% of all new cancer cases and deaths worldwide, respectively [tion set . Two systion set . AML is CDH24, PCLKC, GPR123, EBI2, PTPRT, KNDC1, SLC15A1, and GRINL1B) and insertions in the coding regions of the FLT3 and NPM1 genes [IDH1 gene [et al. (2010), approximately 22% of 281 AML patients had DNMT3A mutations that were newly discovered using targeted sequencing [DNMT3A Arg882 mutation showed poor prognosis among AML-M5 individuals [WAC, SMC3, DIS3, DDX41, and DAXX [de novo AML using WGS and whole-exome sequencing (WES), respectively. They found 23 significantly mutated genes and 237 gene mutations that recurred in at least two patients, which were grouped into nine categories according to their biological functions [Next-generation sequencing technologies have extended AML genetic studies to a genome-wide scope at a single-base resolution. Ley et al. 2008) performed the first AML WGS study on one Caucasian woman with the cytogenetically normal AML subtype M1 and reported non-synonymous single nucleotide variants (nsSNVs) in eight genes  and biological pathways.de novo AML who visited the Division of Hematology and Medical Oncology, Seoul National University Hospital, from 1995 to 2013 who had not received a bone marrow transplant prior to sampling. All subjects provided matched tumor-normal sample pairs that passed a DNA quality control (QC) test. The Institutional Review Board for Human Research at Seoul National University approved the study protocol (IRB number 1201-099-396), and all participants signed informed consent forms for WES. A clinician conducted a retrospective medical record review to obtain clinical data including disease status and blood chemistry. An approximately 10-mL bone marrow sample was aspirated from each participant by a clinician in an aseptic environment, and genomic DNA was isolated using the QIAamp DNA Blood Maxi Kit following the manufacturer’s instructions . A 2-ml whole-saliva sample was obtained from the same individuals for the matched normal samples, and the genomic DNA was extracted using the Oragen DNA Self-Collection kit .We included 36 Korean patients with Inc., Santa Clara, CA, USA). The entire exome regions for both the tumor and normal samples from 36 AML patients were sequenced using the HiSeq 2000 platform with a 100 bp paired-end read protocol . Each the tumor and the normal sample were sequenced to an average read depth of 76X. The fastq quality score and read length cutoff determined by the NGS QC-toolkit were set to 20 and 70, respectively [We captured the target DNA sample using the Agilent SureSelect Human All Exon 50 Mb Kit  [p value less than 0.05 in any patient group. We categorized a mutant gene as a subtype-specific gene if the statistical significance of any one subgroup  represented exclusively the significance of the total patient group. Finally, we systematically searched the PubMed database (www.ncbi.nlm.nih.gov/pubmed) to review previous studies on the relevance of the genes with p values less than 0.05 and q values less than 0.1 for AML and/or other cancers.We performed a mutation significance test using MutSigCV v1.4 with a significance threshold of a te (FDR) . To idenhttp://www.genome.jp/kegg/pathway.html) to identify pathways for the genes that were frequently mutated in AML cells [To investigate the biological relevance of the mutations, we performed a GO enrichment analysis with the Database for Annotation, Visualization, and Integrated Discovery (DAVID 6.8 beta) . We cateML cells .n = 43), M2 (n = 11), M3 (n = 12), M4 (n = 3), and M5 (n = 3). However, four patients were not placed into any of the AML subgroups (Table The mean age of the 36 AML patients was approximately 46 years. The mean percentage of blasts in the bone marrow (60.93%) in patients in this study was approximately 12-fold higher than the level in patients undergoing complete remission. Overall, the abnormally high WBC count and low platelet count observed in the study subjects represent typical characteristics of AML patients. A total of 32 patients were grouped into five AML subtypes as follows: M1 . Among them, 11 genes demonstrated q values less than 0.1 in 36 AML patients . Frameshift indels potentially lead to the disease since they alter protein amino acid sequences which were found in olfactory receptor family 2 subfamily T member 35 (OR2T35) and proprotein convertase subtilisin/kexin type 5 (PCSK5). Missense SNVs were found in transmembrane protease, serine 13 gene (TMPRSS13) in six samples, keratin associated protein 4–5 (KRTAP4-5) in one sample, and G protein regulated inducer of neurite outgrowth 1(GPRIN1) in one sample  and E1A binding protein p400 , that showed statistical significance exclusively in the M2 subtype, as well as one gene, ataxin 3  that was exclusive for the M3 subtype. In other words, these mutations were found to be more frequent in either M2 or M3 subgroup cases than in the total cohort of 36 AML patients , 10 among the 16 genes listed in Table CEBPA mutations and AML. Figure p value for each mutation (y-axis) was plotted against their respective chromosomal position (x-axis). Specifically, the three subtype-specific mutant genes with a p value of less than 0.01were labelled in the plot.We identified three significantly mutated genes with a p = 1.77 × 10−3, Fold Enrichment = 1.63). In terms of cellular components, the plasma membrane showed the most significant alteration . Scaffold protein binding was the most significantly altered molecular function . To investigate the pathway-level relationship of the mutated genes, we performed a pathway analysis in the KEGG database  and long-term potentiation  pathways were significantly altered in AML.We identified total of 501 genes harbored 478 missense, 22 nonsense, 93 frameshift indels, and/or three stop codon deletions. We performed GO and KEGG pathway enrichment analyses with the input consisted of the 501 genes. The GO analysis demonstrated that 43 mutated genes could contribute to AML tumorigenesis through the biological processes such as signal transduction (p < 0.01 and q < 0.1). Specifically, NEFH, hepatitis A virus cellular receptor 1 , interferon, alpha-inducible protein 27 , PCSK5, and the arylsulfatase D gene  have been implicated in a variety of cancers. For instance, the variants of NEFH gene, a tumor suppressor, were suggested as prognostic markers for renal cell carcinoma (RCC) and contributed to susceptibility of esophageal squamous cell and hepatocellular carcinomas [TMPRSS13, 11q23), a splice variant of mosaic serine protease large form (MSPL), encodes a family of the type II transmembrane serine proteases which plays critical roles in maintaining homeostasis, infection, and tumorigenesis [HAVCR1 gene is a biomarker for diagnosing renal cell, ovarian, and colorectal carcinoma [IFI27, the most highly up-regulated gene in human whole blood, is related with immune response through activation of T lymphocytes and dendritic cells [PCSK5 gene  has been reported to be systematically down-regulated in intestinal tumors of the knockout mouse model and human [A total of 11 genes were significantly mutated in 36 AML patients  was reported to be associated with cancers such as RCC and colorectal cancer. All three genes specific to the M3 subtype , and HCLS) also showed possible associations with cancer risks, such as B-cell chronic lymphocytic leukemia (B-CLL).The genes mutated exclusively in the patients with the AML subtypes M2 and M3 have been reported more frequently in previous studies to have an association with AML and/or other types of cancers than the genes identified in the total AML patient group. For instance, previous NGS studies for AML evaluated the prognostic impact of the gene patients , 32. ThiGene set enrichment analysis showed that MAPK signaling pathway was significantly enriched in the 36 Korean AMLs. MAPKs play important role in converting extracellular stimuli into cellular responses and are often altered in cancers . The genIn this study, we replicated multiple gene mutations reported by previous European studies in Korean patients. We also discovered novel genes significantly mutated in AMLs and some mutated genes that showed subtype-specific patterns of mutations. The effects of novel genes and subtype-specific somatic mutations in AML warrant further validation in larger cohorts. Following validation, it would be worthwhile in future studies to evaluate the usefulness of these genes in genetic testing for the early diagnosis and prediction of the prognosis of AML patients.


Yet, little is known about the impact of acute elevated glucose on the temporal properties of AP-induced Ca2+ transients and ionic underlying mechanisms that lead to muscle dysfunction. Here, we used high-speed confocal Ca2+ imaging to investigate the temporal properties of AP-induced Ca2+ transients, an intermediate step of ECC, using an acute in cellulo model of uncontrolled hyperglycemia . Control and elevated glucose-exposed muscle fibers cultured for five days displayed four distinct patterns of AP-induced Ca2+ transients ; most control muscle fibers show phasic AP-induced Ca2+ transients, while most fibers exposed to elevated D-glucose displayed biphasic Ca2+ transients upon single field stimulation. We hypothesize that these changes in the temporal profile of the AP-induced Ca2+ transients are due to changes in the intrinsic excitable properties of the muscle fibers. We propose that these changes accompany early stages of diabetic myopathy.A common comorbidity of diabetes is skeletal muscle dysfunction, which leads to compromised physical function. Previous studies of diabetes in skeletal muscle have shown alterations in excitation-contraction coupling (ECC)—the sequential link between action potentials (AP), intracellular Ca Diabetes mellitus (DM), a complex metabolic syndrome, is due to the inability of the pancreas to produce and/or secrete insulin, referred as insulin deficiency or improper insulin signal transduction by tissues like hepatic, fat, and skeletal muscle, known as insulin resistance. In either insulin deficiency or resistance, the cells are unable to adequately metabolize the glucose, leading to hyperglycemia, the hallmark of the disease. Late complications of diabetes affect both the quality and quantity of life, resulting in major health costs . The disA common comorbidity of both T1D and T2D is sarcopenia and dynapenia—the loss of muscle mass and strength, respectively, and is termed diabetic myopathy , 7. The 2+ homeostasis and signaling in different models of long-term diabetes mellitus [2+ transients. Direct acute effects of hyperglycemia could have implications for the skeletal muscle myopathy seen in diabetes, especially in patients with poor glycemic control. In particular, studies of experimental diabetes in skeletal muscle have shown alterations in the excitation-contraction coupling (ECC)—a coordinated chain of cellular events that link membrane AP, intracellular Ca2+ release, and contractile machinery [2+ signals produced by single brief electric stimulation [2+ transients and the underlying ionic mechanisms that lead to muscle dysfunction. Here, we used ultra-high-speed confocal Ca2+ imaging to investigate the temporal properties of AP-induced Ca2+ transients, an intermediate step of ECC, using a cellular model of acute hyperglycemia. Our results reveal that elevated glucose-exposed fibers predominantly display abnormal AP-induced Ca2+ transients.While previous studies have investigated the link between changes in skeletal muscle function and muscle mass , 17–23, mellitus –27, few achinery , 29. We mulation . Yet, liflexor digitorum brevis (FDB) muscles of 4- to 5-week-old C57BL/6J mice as previously described [2 exposure followed by cervical dislocation using protocols approved by the University of Maryland Institutional Animal Care and Use Committee. FDB skeletal muscle fibers were isolated, dissociated, and cultured in a humidified incubator at 37°C (5% CO2) as previously described [μg·ml−1 gentamicin . This medium was used as a control isotonic condition (288 mOsm/kg). Two hours after plating, cultures were treated with cytosine β-D-arabinofuranoside  to reduce proliferating cells and to minimize fiber dedifferentiation [Studies were performed on skeletal muscle fibers enzymatically isolated from the escribed –33. Miceescribed –33. FDB ntiation , 33. ForμM fluo-4 AM  in L-15 medium . The ionic composition of L-15 in mM is 137 NaCl, 5.7 KCl, 1.26 CaCl2, and 1.8 MgCl2, pH 7.4) supplemented with 0.25% w/v bovine serum albumin  for 1 h. at room temperature. The dishes were rinsed once with L-15 medium for 5 min to remove residual fluo-4 AM. Individual muscle fibers were imaged with a 60×/1.3 NA water-immersion objective lens. Excitation for fluo-4 was provided by the 488 nm line of a 100 mW diode laser, and emitted light was collected at >505 nm. Action potential- (AP-) induced Ca2+ transients were triggered using a brief electrical field stimulus. External field stimulation and inclusion and exclusion criteria of the characteristics of muscle fibers used in this study were performed as previously described [F0). The average intensity of fluorescence within selected regions of interest  mode  were background corrected by subtracting an average value recorded outside the cell. The average F0 value in each ROI before electrical stimulation was used to scale Ca2+ signals in the same ROI as ΔF/F0.Fluo-4 measurements were carried out on a high-speed confocal system  as previously described , 35. Musescribed . Suprama2+ transient is exclusively determined not only by the sampling rate but also by the kinetic properties of the dyes. Here, we used fluo-4, a high-affinity dye, instead of low-affinity dyes merely because in our imaging system this dye provides brighter responses than the low-affinity indicators. Based on our calibration data, fluo-4 was at most 40% saturated with Ca2+. The length of the fibers used was 400–600 μm and the width was 25–80 μm. No attempts were made to distinguish muscle fiber types or to estimate the actual cytosolic Ca2+ concentration. Ca2+ imaging experiments were carried out at room temperature, 21–23°C.It is important to note that the temporal resolution of the Ca2+ transient, 5-day-old cultured fibers were exposed to either gadolinium , apamin , or Jingzhaotoxin-III , blockers of mechanosensitivity, SK channels, and Nav1.5 channels, respectively. Ion channel blockade treatment was carried out using semilocal perfusion. The working concentration of the blockers used here was based on maximal blocking effects described in previous reports [2+ transient were first identified, then the time course of the Ca2+ profile was assessed before and 10 minutes after blocker application.To assess the contribution of different ion channels to the development of the biphasic action potential Ca reports –39. Fibe2+ signals and statistical analysis were conducted using Origin Pro 8  and SPSS for Windows ver. 24.0 . Summary data were reported as mean ± SD. Normal distribution of data was assessed using the Kolmogorov-Smirnov test. Unpaired two-sample Student's t-test was used to test for differences between the means of the interspike interval of AP-induced Ca2+ transients from two different samples. For experiments that involved the measurements of AP-induced Ca2+ transients before or after the addition of ion channel blockers, the interspike interval was compared before and after the addition of blocker in the same muscle fiber. The statistical significance of interspike interval was analyzed using a paired Student's t-test on raw data. Cross-classifications and crosstabs were used to examine the relationship between two categorical variables. To test for significant differences, we compared the proportion in each variable, with condition (control and elevated glucose) as the independent variable and AP-induced Ca2+ transient pattern  as the dependent variable, using Pearson chi-square (X2). Differences were considered significant when p value < 0.05.Images of arbitrarily selected muscle fibers were collected and evaluated blindly, using the same settings and enhancing parameters so that all images could be directly compared. Line-scan images were analyzed using LSM examiner . AP-induced Ca2+ transients that correlate with the number and frequency of field stimulation used. In response to a single 1 ms field stimulus, they respond with one single action potential, a single Ca2+ transient, and a single twitch) [https://doi.org/10.1155/2017/1509048). In a previous study, we analyzed Ca2+ handling and action potential- (AP-) evoked Ca2+ transients in 5-day-old cultured control and high-glucose-exposed FDB fibers using a ratiometric Ca2+ dye and low temporal resolution [2+ transients, we next monitored fluo-4 transients during stimulation of FDB fibers using an ultra-high-speed (100 μs/line) confocal microscope in line-scan mode. x-t confocal line-scan images , biphasic (18%), phasic-delayed (12%), and phasic-slow decay (3%)  , 33, evesolution . To gainn images  and corransients  of four profiles  show the(3%) see . The risesponses . In cont2+ transients in fibers challenged with either D-glucose or L-glucose  43.08 p = 1.12E − 7, implying that elevated glucose-exposed fibers exhibit a different distribution of patterns of AP induced-Ca2+ transients when compared to control counterparts. Note that the biphasic pattern was more commonly observed in D-glucose challenged fibers, while in L-glucose exposed fibers the phasic pattern was predominant, as seen in control fibers . The effects of D-glucose on the distribution of the AP-evoked Ca2+ transients were distinct to those observed in muscle fibers exposed with the same concentration of metabolically inactive L-glucose . We assessed the properties of AP-induced Ca-glucose  using thifferent . In D-glifferent  the distifferent  the distifferent . We testl fibers . Also, t-glucose , suggest2+ signals, fibers were subjected to suprathreshold field stimulation of alternate polarity. The Ca2+ transients in fibers with phasic responses occur with a similar short latency (<2 ms) in response to external stimulation of either polarity  and could, in principle, result from alterations in AP propagation which translate into AP-induced Ca2+ propagation deficits . We wanted to evaluate whether the delay was caused by the absence of a direct response to stimulation at the site of imaging combined with a slow propagation of an AP from the fiber end away from the recording site and towards the recording site. If this were the case, then inverting the polarity of the stimulus applied to a phasic delayed fiber would trigger an AP-induced Ca2+ transient with a considerably shorter delay at the site of recording since propagation would no longer be required. 2+ transients in a fiber with delayed responses using pulses of opposite polarity. As in the phasic fiber, suprathreshold pulses elicited synchronous AP-induced Ca2+ transients across the fiber width. The comparison of the time course of AP-induced Ca2+ transients measured at the same fiber end but with field stimuli of opposite polarity shows similar delayed time courses and only a modest shift (<3 ms) in their latency upon polarity change. This finding suggests that the Ca2+ transients are not delayed because of the time for propagation of the action potential or another signal from one end to the other end of the fiber. In that case, the signal recorded at the end of the fiber where the action potential is initiated would not exhibit the delay. However, this was not observed. Since the delay was similar for both polarities of stimulation , we conclude that the delay could be due to either a delay in the activation of the action potential  or a delay in the activation of the Ca2+ transient after the action potential propagation along the fiber when the action potential is activated at the opposite end of the fiber where the recording is taking place. These possibilities are considered further in the discussion.In fibers with phasic-delayed CaNext, we investigated whether modifications of the excitable properties of the muscle fiber could account for the occurrence of the biphasic phenotype. We used blockers of ion channels known to modulate the membrane potential and AP properties of the skeletal muscle.+ and divalent cations [2+ levels and/or membrane depolarization [2+ transient. To test whether the occurrence of the biphasic action potential-induced Ca2+ transient depended on MsC, the time course of fluo-4 Ca2+ elicited by field stimulation and a priori identified as a biphasic signal was measured in control external solution, followed by the addition of gadolinium ; although the amplitude of the Ca2+ signal was reduced after the Gd3+ addition, this effect was not further evaluated. After washout of Gd3+, the amplitude of the Ca2+ transient remained reduced (data not shown). The above results suggest that MsC do not contribute to the occurrence of the biphasic action potential-induced Ca2+ transients in 5-day-old cultured muscle fibers.The mechanosensitive ion channels (MsC) in the skeletal muscle are activated by membrane stretch and strong membrane depolarization and are permeable to Na cations . Increasrization . We hypoCa2.3, is normally expressed at low level; however, its expression is markedly increased in denervated and myotonic dystrophy muscle [Ca2.3 channel activity in the T-tubules of denervated skeletal muscle causes a local increase in potassium ion concentration that leads to hyperexcitability [Ca2.3 could be involved in the development of the biphasic action potential-induced Ca2+ transient. To test whether the occurrence of the biphasic Ca2+ transient involved KCa2.3 channels, the time course of fluo-4 Ca2+ elicited by field stimulation was measured in a control external solution, followed by the addition of apamin (1 μM) , although a reduction in the amplitude of the Ca2+ signal was also observed  . KCa2.3 y apamin . Ten minobserved . These rv1.5 channels is low in 1- to 2-day-old cultured muscle fibers. However, Nav1.5 expression increases in fibers cultured for over 3 days [v1.5 increased expression could explain the occurrence of abnormal excitability and Ca2+ signals. To test whether increased Nav1.5 function is involved in the altered action potential-induced Ca2+ signals seen in 5-day-old cultured fibers, we exposed the fibers to JZTX-III, a Nav1.5 channel blocker [μM; 10 min) to the external solution did not affect the time course of the biphasic response . Contrary to Gd3+ or apamin, the addition of JZTX-III to the recording solution did not reduce the amplitude of the AP-induced Ca2+ signals  on the temporal properties of AP-evoked Ca2+ signals. The present study shows that muscle fibers cultured in control medium (5 mM D-glucose) for 5 days display 4 distinct temporal waveforms of AP-induced Ca2+ transients: phasic, biphasic, phasic delayed, and phasic-slow decay, in order of predominance. Our study also shows that fibers challenged with elevated extracellular D-glucose  also exhibit these 4 distinct patterns. However, under these conditions, the biphasic pattern is the predominant waveform, suggesting that elevated glucose promotes the biphasic responses. To our knowledge, this study is the first report of these abnormal AP-induced Ca2+ signals in relation to elevated glucose in fibers of normal morphology.Numerous studies have investigated how changes in skeletal muscle excitability, Caglycemia , 47, 48;glycemia , contracglycemia , and Ca2ignaling  of the s2+ transients? We have previously shown that the electrode array used in our study allows for the application of field pulses resulting in the depolarization of the end of the fiber close to the cathode, and hyperpolarization of the opposite end of the fiber (near the anode) [phasic fibers, pulses of alternating polarity elicited propagated AP-induced Ca2+ transients at the end of the fiber facing the cathode, near the recording site, and its longitudinal propagation along the fiber [2+ transient peak following single AP stimulation occurs in ~1-2 ms in phasic muscle fibers. The delayed responses (time to Ca2+ transient peak > 3 ms) and the second phase of biphasic responses were variable from fiber to fiber. We do not currently know the nature of this variation. We found that fibers with delayed AP-induced Ca2+ transient pulses of opposite polarity resulted in subtle latency changes of the Ca2+ transient  field stimulus capable of generating such delayed (>3 ms) or biphasic AP-induced Cae anode) . In phashe fiber   delay will be similar at both ends of the fiber for a given polarity stimulation or at the same end of the fiber with alternating polarity stimulation  hyperosmotic stress in the cell periphery of cultured muscle fibers [2+ signals are present in muscle fibers challenged with hyperosmotic stress induced by elevated glucose for more prolonged periods (>24 h.) remains to be determined.Cafunction , 58. A pe fibers . Whether2+ signals seen in long-term cultured fibers and experimental hyperglycemia occur in patients with diabetes? Most adults with diabetes have at least one coexisting condition, either acute or chronic [2+ signals and excitable properties are critical for skeletal muscle function [2+ signals observed in five-day-old cultured fibers occur in vivo, these could contribute to the development of muscle weakness, fatigue, and diabetic myopathy. Further work exploring the underlying mechanisms and relationship between diabetes and skeletal muscle disuse  would be of pathophysiological interest.Do these defects on AP-induced Ca chronic . Muscle  chronic  and long chronic  and are  chronic , 63. Becfunction , 57, we 2+ signals that we observed may be influenced or caused by fiber disuse and/or denervation which may occur in long-term cultured muscle fibers [In this study, we used an in cellulo model of hyperglycemia using 25 mM glucose for 1-2 days. This paradigm is an extreme model of hyperglycemia, and it is restricted to a short spectrum of metabolic abnormalities and hormonal changes seen in diabetes . The abnormalities in AP-induced Cae fibers  and in se fibers . Both mue fibers , 45. Bece fibers   exposure to elevated D-glucose enhances AP-evoked Ca2+ transients in cultured muscle fibers. Comparison of the time course of AP-induced Ca2+ transients in muscle fibers before  and after 1 h. challenge with 25 mM D-glucose, cyan trace (A), or after 1 h. challenge with 25 mM L-glucose, red trace (B). Summary of rest and at peak indo-1 ratio measurements for fibers exposed to 25 mM elevated D- or L-glucose (C). No significant (N.S) changes in resting indo-1 ratio were found in fibers challenged with either 25 mM elevated D- or -L-glucose. Exposure for 1 h. with D-glucose induced a significant increase in peak indo-1 ratio. ∗Indicates p < 0.05 compared with control, two-sample unpaired Student's t-test, n = 14-15 fibers, 3 mice.Supplemental Figure 2. Ara-C treatments minimize in vitro de-differentiation. Exemplar transmitted light images of muscle fibers cultured in serum-containing medium (A) and serum-containing medium and treated with cytosine β-D-arabinofuranoside  to minimize de-differentiation. Images of the same fibers were taken at day 1 (upper rows) and day 5 (lower rows) after fiber plating. Scale bar 100 µm. Note that in fibers treated with ara-C, the morphology was retained after 5 day in culture, whereas cell proliferation, intercellular fusion, and fiber sprouting were evident in the serum-containing medium


ID1 expression and its prognostic value in acute myeloid leukemia (AML). Herein, we further reported ID2 expression and its clinical significance in AML. Real-time quantitative PCR was performed to detect ID2 transcript level in bone marrow mononuclear cells of 145 de novo AML patients. ID2 expression was significantly up-regulated in AML patients compared with controls. ID2 overexpression occurred with the highest frequency in poor karyotype , lower in intermediate karyotype , and the lowest in favorable karyotype . Moreover, high ID2 expression correlated with lower complete remission (CR) rate, shorter overall survival, and acted as an independent prognostic biomarker in whole-cohort AML and non-M3-AML patients. Importantly, the prognostic value of ID2 expression in AML was validated by The Cancer Genome Atlas (TCGA) data. In the follow-up of patients, ID2 expression at CR phase was decreased than at the time of diagnosis, and was increased again at the time of relapse. These findings demonstrated that bone marrow ID2 overexpression was a frequent event in AML patients, and predicts poor chemotherapy response and prognosis.Dysregulation of ID proteins is a frequent event in various human cancers and has a direct role in cancer initiation, maintenance, progression and drug resistance. Our previous study has revealed  As one of the most common type of hematological malignancies, acute myeloid leukemia (AML) is clinically, cytogenetically, and molecularly heterogeneous disease . DespiteID gene family is composed of four members , and each member plays a different role in carcinogenesis  might serve as a potential biomarker for distinguishing AML form controls  analysis, ID2 expression in AML, the whole-cohort AML patients were divided into two groups (ID2high and ID2low) by the cut-off value  based on ROC analysis. The comparison of clinical manifestations and laboratory features between ID2high and ID2low groups of AML patients was presented in Table ID2high patients had significantly older age (P=0.001) and a tendency of higher white blood cells (P=0.068). Significant differences were also observed in the distributions of French-American-British (FAB) and World Health Organization (WHO) classifications . High ID2 expression occurred with the lowest frequency in M3 or t subtype , which was lower than M0/M1/M2 subtypes  (P=0.010) and M0/M1/M2/M4/M5/M6 subtypes  (P=0.001). Moreover, among the karyotype classifications, ID2high patients occurred with the highest frequency in poor karyotype , lower in intermediate karyotype , and the lowest in favorable karyotype  (P=0.004).In order to analyze clinical relevance of ID2 expression with the tested gene mutations, besides that high ID2 expression tended to be associated with IDH1/2 and FLT3-ITD mutations .As was shown in Table ID2high patients had significantly lower CR rate than ID2low patients in whole-cohort AML  vs 31% (8/18), P=0.068].Follow-up data for complete remission (CR) rate after induction chemotherapy was obtained in 126 patients. ID2 expression had significantly shorter OS than those with low ID2 expression among whole-cohort AML  was obtained in 124 patients. Cases with high ID2 expression in AML. The univariate analysis included the variables showed in Table P<0.100 in univariate analysis. After adjusting for other covariates, ID2 expression acted as an independent prognostic biomarker in whole-cohort AML and non-M3-AML  to determine the prognostic value of ID2 expression in AML, we searched and analyzed an independent assessment in AML patients from The Cancer Genome Atlas (TCGA) databases. By the median level of ID2 expression set as the cut-off value, patients with higher ID2 expression showed a significantly shorter OS among both whole-cohort AML  cells (NB4) and were rapidly induced upon all-trans retinoic acid (ATRA) treatment  and negative controls (ddH2O) were included in each assay. Relative ID2 expression levels were calculated using 2−ΔΔCT method.RQ-PCR was performed to detect reported . PCR reareported  was usedID2 mRNA expression (RNA Seq V2 RSEM) in a cohort of 200 AML patients [http://www.cbioportal.org) [patients  from TCGtal.org) , 37.U test and Pearson Chi-square/Fisher exact test was employed to compare the difference of continuous and categorical variables between two groups. ROC and AUC were conducted to assess the value of ID2 expression in distinguishing whole-cohort AML, non-M3-AML and CN-AML patients from controls. Kaplan-Meier and Cox regression analyses were performed to analyze the impact of ID2 expression on OS. For all analyses, a two-tailed P value less than 0.05 was determined as statistically significant.Statistical analyses were performed through SPSS 20.0 software package. Mann-Whitney’s


Odontella mobiliensis, Skeletonema grethae, Phaeodactylum tricornutum, Thalassiosira pseudonana) and one green algae  for their extracellular polymeric substances (EPS) release under model ENP treatments: 25 nm titanium dioxide (TiO2), 10–20 nm silicon dioxide (SiO2), and 15–30 nm cerium dioxide (CeO2). We found SiO2 ENPs can significantly stimulate EPS release from these algae (200–800%), while TiO2 ENP exposure induced the lowest release. Furthermore, the increase of intracellular Ca2+ concentration can be triggered by ENPs, suggesting that the EPS release process is mediated through Ca2+ signal pathways. With better understanding of the cellular mechanism mediated ENP-induced EPS release, potential preventative and safety measures can be developed to mitigate negative impact on the marine ecosystem.Engineered nanoparticles (ENPs), products from modern nanotechnologies, can potentially impact the marine environment to pose serious threats to marine ecosystems. However, the cellular responses of marine phytoplankton to ENPs are still not well established. Here, we investigate four different diatom species (The online version of this article (10.1186/s11671-017-2397-x) contains supplementary material, which is available to authorized users. Engineered nanoparticles (ENPs), which range in size between 1 and 100 nm (in at least one dimension), are used in the fabrication of numerous consumer goods, including printer inks and paints, detergents, bactericides, coatings, cosmetics, sunscreen lotions, tires, computer construction, and drug delivery. Given the promising application of ENPs, funding for the National Nanotechnology Initiative (NNI) in the USA alone approached $1.4 billion in 2017 . EstabliMost marine microbes, whether auto- or heterotrophic, are generally capable of producing exopolymeric substances (EPS), which have diverse functional roles and physical properties in the marine ecosystem acting as growth inhibitors, growth promoters, toxins, metal scavengers, or as substrates for the heterotrophic cycle –26. EPS 2+ is a common second messenger involved in a multitude of intracellular signaling pathways. It has been demonstrated that Ca2+ is required for chemotaxis, motility, and adhesion in the diatom Amphora coffeaeformis [2+ levels are known to lead to the activation of protein kinase C, which is involved in many intracellular signaling pathways [2+-mediated secretion process controls the release of EPS from diatoms [2+ signaling, exocytosis, and correlating Ca2+ signaling with exocytosis has been reported in our previous study [2+ pathways, which are essential for cell signaling [2+ concentration changes are important in cell signaling and secretion processes; however, there are no reports of titanium dioxide (TiO2), silicon dioxide (SiO2), or cerium dioxide (CeO2) to alter intracellular Ca2+ level in phytoplankton.Caaeformis . Enhancepathways . Since t diatoms , and theus study . Past stignaling , 32–34. Odontella mobiliensis, Skeletonema grethae, Phaeodactylum tricornutum, Thalassiosira pseudonana) and one green algae  under ENP treatments. By understanding underlying mechanisms of ENP-induced EPS-release in phytoplankton, implementation of preventative and safety measures can mitigate potentially detrimental effects toward marine organisms.In 2013, Quigg et al.  summariz2, SiO2, and CeO2. The particle size distribution ranged from 7 to 66 nm in TiO2, 9 to 66 nm in SiO2, and 12 to 70 nm in CeO2. Some larger sizes could be due to aggregation or agglomeration while the predominant size for TiO2 is 25 nm, SiO2 is 10 to 20 nm, and CeO2 is 15 to 30 nm, which are consistent with manufacturer’s information  was used to characterize size metrics of the following ENPs suspended in pure water: TiO2+ concentration, phytoplankton cells (OD 600 = 0.8) were loaded with Fluo-4AM dye and exposed to 1 mg/ml of 25 nm TiO2, 10–20 nm SiO2, and 15–30 nm CeO2 ENPs respectively. The change in intracellular Ca2+ concentration, as represented by the fluorescence intensity within phytoplankton cells, was monitored for 150 s. Figure 2+ concentration in SiO2 by approximately 50–300%, TiO2 by approximately 40%, and CeO2 by approximately 150–200%, while the control conditions (L1 medium) remained unchanged. The results show ENPs can induced significant intracellular Ca2+ responses in phytoplankton and suggest that phytoplankton respond to distinct ENPs through Ca2+ signaling pathways. Our data indicates only minor changes in intracellular Ca2+ levels when TiO2 is present, potentially attributed to substantial phytoplankton cell death from TiO2-induced toxicity [2 prompted increase in the intracellular Ca2+ concentration [2 surprisingly showed the most obvious intracellular Ca2+ increase for all phytoplankton species, while CeO2 can only trigger an intermediate intracellular Ca2+ concentration increase. Previous research suggested potential of high CeO2 concentrations (> 50 mg/ml) to induce intracellular oxidative stress and elevation of intracellular Ca2+ levels, though effects were small, and supported our finding [2+ concentration changes in specific phytoplankton, ultimately paving a new avenue for future research.To investigate whether ENPs could induce an increase in intracellular Catoxicity , 38. In ntration  alongsidntration . However finding   and Skeletonema grethae (around 1000–1500%). These data indicate that different ENPs can induce specific EPS release from phytoplankton, and intracellular Ca2+ changes also match EPS release results. By assessing the changes in intracellular Ca2+ concentration, it is evident that there is a direct connection in the Ca2+ cellular pathways in which ENPs evoke the EPS secretion from phytoplankton. The observation here is in agreement with our previous studies based on Phaeocystis EPS release [2+ cellular pathways.Enzyme-linked lectin assay (ELLA) was used to assess the amount of EPS release from phytoplankton cells when stimulated with TiOfor TiO2 , 43 and  TiO2 [2 –46. EPS plankton –49. In t release . The res2 for Dunaliella tertiolecta, Thalassiosira pseudonana, and Skeletonema grethae. It appears that these diatoms are primed to recognize SiO2 particles. However, in Phaeodactylum tricornutum, a strong EPS secretion was not found. This difference represents EPS release triggered by ENPs dependents on the phytoplankton species and ENPs concentration , Dunaliella tertiolecta (UTEX999), Skeletonema grethae (CCMP775), Phaeodactylum tricornutum (UTEX646), Thalassiosira pseudonana  were grown in L1 marine medium  on a 14:10 (light: dark) cycle at 100 μmol m−2 s−1 and 24 °C under axenic conditions. Growth phase of the culture was determined by cell counting with a hemocytometer.Batch cultures of 2, SiO2, CeO2 , were sonicated in pure water before usage. ENPs were reconstituted with filtered L1 medium  before being tested. The size of ENPs was independently confirmed using homodyne dynamics laser scattering (DLS). Briefly, seawater samples were refiltered through a 0.22-μm Millipore membrane (pre-washed with 0.1N HCl) and poured directly into five 10 ml scattering cells that were then positioned in the goniometer of a Brookhaven BI-200SM laser spectrometer . The autocorrelation function of the scattering intensity fluctuations detected at a 45° angle was processed on line by a Brookhaven BI 9000ATautocorrelator, and particle size distribution was calculated by the CONTIN method . Results from each sample were collected in triplicate right after sonication. Calibration of the DLS spectrometer was conducted using standard suspensions of monodisperse latex microspheres .All ENPs, TiO2, SiO2, and CeO2  or L1 medium (control) for 48 h. The supernatant containing secreted EPS was collected and briefly centrifuged at 4000 rpm to remove the residual ENPs. This protocol was adapted from our previous publication [The phytoplankton cells were cultured in a 96-well plate with L1 medium for 24 h. Cells were treated with ENP stocks: 5 mg/ml and 1 mg/ml, 0.5 mg/ml, 0.1 mg/ml, 10 μg/ml, 1 μg/ml of the TiOlication . The con2SO4  in order to terminate the reaction. The optical density was measured at 450 nm by PerkinElmer VICTOR3 . This protocol was adapted from our previous publication [The supernatant containing secreted polysaccharide was collected and briefly centrifuged at 1700 rcf (Megafuge 1.0R) to remove the residual ENPs. The supernatant was then incubated in a 96 well  plate overnight at 4 °C. Afterwards the 96-well plate was washed with PBST (PBS + 0.05% Tween-20) and PBS and then blocked with 1% BSA. The 96-well plate was washed again with PBST and PBS and incubated with lectin  , conjugated to horseradish peroxidase  , at 37 °C for 1 h. The substrate, 3,39,5,59-tetramethylbenzidine , was added to each well at room temperature followed by Hlication , 52.The pellet containing phytoplankton was collected and obtained the ZR-96 Quick-gDNA kit . In brief, 4× lysis buffer was used to break phytoplankton cells and flow through the DNA binding column, eluted by elution buffer in the end. DNA concentrations were measured by NanoDrop ND-1000 . Protocol was adapted from manufactured kit protocol.2, SiO2, and CeO2 respectively. All calcium signaling experiments were carried out on a Nikon microscope . Protocol and conditions were adapted from previous publications [The phytoplankton cells were then loaded with a Fluo-4AM dye (1 mM)  for 60 min . After tications , 34.To measure the surface charges of ENPs, the zeta potential (ζ) of ENPs was measured with a Zetasizer Nano ZS, Malvern, in the presence of artificial seawater at 25 °C. After the data were collected from each sample, the recorded values were averaged.The data is reported as means ± SD. Each experiment was performed independently at least three times. Histograms were made by GraphPad Prism 6.0. .


Growth factor receptor bound protein 7 (Grb7) is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine), however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors. For over 20 years, Src homology 2 (SH2) domains have been explored as targets for the development of potential therapeutics  domain, pleckstrin homology (PH) domain, a between the PH and SH2 (BPS) domain, and lastly a C-terminal SH2 domain -amide) was developed via phage display and was found to specifically decrease binding between Grb7 and tyrosine phosphorylated HER family members in breast cancer cell extracts, but have no effect on the interaction between Grb2 and HER3  experiments confirming that G7-18NATE specifically binds to Grb7-SH2 preferentially over the Grb2-SH2 domain  but with the equivalent specificity . It is important to note that the nomenclature to be used to describe SH2 domain secondary structure is as derived by Eck et al. as illustrated in Figure The structure of the Grb7-SH2 domain in complex with G7-18NATE revealed the amino acid residues forming the binding site for the peptide ligand -amide) was synthesized using standard Fmoc-chemistry and purchased from Purar Chemicals . The synthesis of G7-18NATE-PB ((cyclo-(CH2CO-WFEGYDNTFPC-RRMKWKKK(Biotin))-amide)) has been previously described , with levels reaching between 3289 RU and 6324 RU. The GST fusion proteins were passed over the active chip surface at 0.7–0.9 μM to achieve final immobilization levels of 838-1283 RU. GST alone was immobilized on the reference flow cell (462-616 RU) as a negative control to allow for double referencing. Lyophilized G7-18NATE was resuspended in running buffer containing 150 mM NaPO4, 150 mM NaCl and 1 mM DTT (pH 7.4) and injected over the chip surface for 60 s at 30 μL.min−1 in triplicates. Due to machine error some injections were removed before analysis due to presence of spikes that rendered the sensorgrams unable to be interpreted. The interpretable data were analyzed using Scrubber2.0  and GraphPad Prism.SPR experiments were performed on a Biacore T100 using CM5 series S sensor chips. The GST tagged proteins were immobilized onto the sensor chip surface by amine coupling an anti-GST antibody to the surface of the chip. For this, firstly the chip was activated by 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 M N-hydroxysuccinimide. The polycloncal antibody (Abcam) was passed over the chip surface at 30 μg.mLThe microarray was conducted by the Protein Array and Analysis Core at the MD Anderson Cancer Center  using standard protocols. Briefly, lyophilized G7-18NATE-PB (100 μg) was resuspended in PBST and incubated with the nitrocellulose slide pre-spotted with GST-fusion SH2 domains (full list provided as B1 (B for bicyclic)  amino acid to a serine . Using SPR, we directly compared the binding of the cyclic peptide G7-18NATE to the Grb7-SH2 wild-type (WT) and the mutated Grb7-SH2 (R462S). The Grb7-SH2 proteins were prepared as GST fusions to enable efficient immobilization on the sensor chip surface via anti-GST antibodies. The G7-18NATE peptide was flowed over the sensor chip surface and the measured response at equilibrium used to construct binding curves that were fitted by a single-site binding model.R2 of 0.99 for both interactions. The binding curves indicated that the R462 of the BC loop did indeed contribute to the binding affinity of the G7-18NATE/Grb7-SH2 interaction. While G7-18NATE bound to WT Grb7-SH2 with a KD of 2.43 μM, the R462S mutant had a 3.3-fold reduction in binding affinity for G7-18NATE, with a KD of 7.97 μM , however the calculated equilibrium dissociation constant was higher than the highest peptide concentration measured  could confer binding of the Grb2-SH2 to G7-18NATE. This would indicate the degree to which Arg462 in Grb7 is responsible for binding specificity. We therefore generated the Grb2-SH2 S90R (BC2) mutant protein using site-directed mutagenesis and tested its binding to G7-18NATE using the same SPR experimental procedure described above. As shown in Figure In the course of our analysis of G7-18NATE and its derivatives, specificity was assessed by comparing the binding of the peptides to the SH2 domains of Grb2, Grb7, and Grb10, and in some instances Grb14-SH2 . Here, a biotinylated G7-18NATE derivative (G7-18NATE-PB) was tested for binding against 79 SH2 domains  and considered that this interaction could contribute to the specificity for Grb7 of all the G7-18NATE derived peptide ligands. Using mutagenesis we henceforth determined that Arg462 contributes to the binding affinity between Grb7-SH2 and G7-18NATE. The converse mutation in the Grb2-SH2 domain did not confer binding to G7-18NATE, suggesting other amino acids also define Grb7 specificity. We also probed a panel of 79 SH2 domains with a biotinylated version of G7-18NATE and identified that the peptide is highly specific for the Grb7-SH2 domain. The only other SH2 domain that showed detectable binding by G7-18NATE was that of SH2D2A. Interestingly, while SH2D2A does not possess an arginine at the BC2 position it has a leucine at the βD6 position.CX1FX2GYDNX3X4X5 motif, whereby tryptophan was selected in approximately 50% of clones at the corresponding E3 position. Thus, G7-18NATE derivatives that are modified at the E3 position are predicted to maintain the hydrogen bond interaction with the Grb7 Arg462 (BC2). It should be noted that the formation of this single hydrogen bond, that may only occur transiently , is consistent with the relatively small increase in affinity that is conferred. A 3.3-fold change in KD corresponds to approximately only 1 kcal/mol change in ΔG, which is less than expected for a well formed hydrogen bond.The initial observation that Arg462 may contribute to the binding interaction came from the Grb7-SH2/G7-B1 X-ray crystal structure (PDB ID: 5EEQ). In this structure, the Arg462 side-chain extends and engages the E3 backbone carbonyl in G7-18NATE, as well as the E3 sidechain carboxylic acid. The former of these interactions is likely to be more influential to binding. In previously described crystal structures of cyclic peptides bound to the Grb7-SH2 domain, the E3 sidechain is positioned in various rotamer conformations whereas Arg462 is frequently observed in the proximity of the E3 backbone carbonyl  to determine whether or not Arg462 (BC2) contributes to the interaction with these molecules, and defines Grb7 selectivity for natural substrates.The G7-18NATE peptide and its derivatives are constrained in a β-turn conformation with extensive intra-molecular hydrogen bonds. A hydrogen bond between the F2 backbone carbonyl and the Y5 backbone amine positions the E3 backbone carbonyl for engagement with the Arg462 (BC2) sidechain. This suggests that the Arg462/substrate interaction may only occur in ligands that are naturally restricted in a β-turn conformation. To date, no X-ray crystal structures have been solved of Grb7 in complex with an We have also described how the introduction of the arginine mutation to the Grb2-SH2 at the BC2 position was insufficient to confer binding by the G7-18NATE peptide . This suggests that additional amino acids underlie the specificity of G7-18NATE for Grb7-SH2 over Grb2-SH2. This is supported by our microarray results where the only SH2 domain other than Grb7 to show detectable binding to G7-18NATE was that of SH2D2A, an SH2 domain that contains a leucine at the βD6 position. This finding is consistent with previous reports that report the contribution of leucine at the βD6 position to the specificity of Grb7. When Leu481 is mutated to glutamine (the corresponding amino acid in Grb14), binding to the HER2 receptor is lost, and conversely in Grb14, the glutamine to leucine mutation enables HER2 binding (Janes et al., Thus, in this study we have demonstrated that Grb7 Arg462 contributes to G7-18NATE binding, but that specificity is mediated by multiple factors including the leucine at the βD6 position. We have also established that the G7-18NATE peptide is exquisitely specific for Grb7-SH2 over other SH2 domains. Together this provides insight for the development of even more potent ligands that target the Grb7-SH2 domain, and demonstrates that SH2 domains, despite their common structural features, are readily able to differentiate between ligands and thus have high potential as targets for therapeutics development.GW was responsible for the detailed experimental design, protein overexpression and SPR experiments, figure and manuscript preparation. WL undertook the cloning of SH2 domain mutants and was also involved in protein expression, purification and SPR experiments. MG provided SPR expertise and training. JW designed the conceptual framework for the experiments, oversaw the interpretation of the results and finalized the manuscript with the other authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.