Abnormal Ca2+ cycling is linked to arrhythmogenesis, which is associated with cardiac disorders and heart failure. Accordingly, we have generated spontaneously beating CMs from induced pluripotent stem cells (iPSC) derived from patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), which is an inherited and severe cardiac disease. Ca2+ cycling studies have revealed substantial abnormalities in these CMs. Ca2+ transient analysis performed manually lacks accepted analysis criteria, and has both low throughput and high variability. To overcome these issues, we have developed a software tool, AnomalyExplorer based on interactive visualization, to assist in the classification of Ca2+ transient patterns detected in CMs. Here, we demonstrate the usability and capability of the software, and we also compare the analysis efficiency to manual analysis. We show that AnomalyExplorer is suitable for detecting normal and abnormal Ca2+ transients; furthermore, this method provides more defined and consistent information regarding the Ca2+ abnormality patterns and cell line specific differences when compared to manual analysis. This tool will facilitate and speed up the analysis of CM Ca2+ transients, making it both more accurate and user-independent. AnomalyExplorer can be exploited in Ca2+ cycling analysis to study basic disease pathology and the effects of different drugs.Comprehensive functioning of Ca However, cardiac diseases and different drugs can cause changes and variability in Ca2+ cycling that can affect the function and phenotype of CMs. Characterizing these disturbances and abnormalities is vital to improving the studies of the disease pathology and disease prevention and treatment. Intracellular Ca2+ cycling of CMs is analyzed in vitro with Ca2+ sensitive dyes that change their fluorescence properties when binding to Ca2+ ions.Calcium  [2+ cycling analyses have revealed substantial defects and abnormalities in CMs that reflect the cardiac phenotype observed in patients. However, analyzing these Ca2+ transient abnormalities, which consist of cycling patterns that vary in shape and frequency, is difficult, and their quantification is very challenging. To the best of our knowledge, Ca2+ cycling abnormality patterns have thus far only been subjectively analyzed based on visual information, which is labor-intensive, slow and often user-dependent. Manual data analysis performed with visual recognition is also a bottleneck for high-throughput screening and can occasionally be unreliable because of the poor quality of the Ca2+ transients. Having general criteria for Ca2+ cycling abnormality analysis could simplify the comparison between different studies and facilitate the analysis of Ca2+ cycling parameters. In addition, an automated Ca2+ cycling classification software would be beneficial for the future analysis of Ca2+ cycling of CMs on a large scale to screen for adverse cardiac effects of new potential compounds.Induced pluripotent stem cell (iPSC) technology, in which pluripotent stem cells are generated by reprogramming differentiated cells back into the pluripotent state, provides a method for studying the pathophysiology of various disorders in human cells. iPSCs caa (CPVT) , dilateda (CPVT) , hypertra (CPVT) ,12 and Ta (CPVT) . In thes2+ cycling abnormality analysis software based on interactive visualization with a direct manipulation user interface (UI). The software, AnomalyExplorer, can user-independently assist in the classification of the Ca2+ transient patterns detected in CMs by using criteria that are similar to those used in manual analyses that are performed with visual recognition. We demonstrate the usability and capability of the software using iPSC-derived CMs that were generated from patients with cardiac ryanodine receptor (RyR2) gene mutations causing CPVT. Opening of RyR2 Ca2+ channels underlies intracellular Ca2+ release from the sarcoplasmic reticulum in CMs. CPVT is an exercise-induced arrhythmogenic cardiac disease with intracellular Ca2+ cycling defects [AnomalyExplorer is demonstrated with the data generated by two different Ca2+ imaging recording software programs with different sampling frequencies. The analysis of data using AnomalyExplorer is compared with the manual analysis results to establish the reliability of the analysis software. This software tool will improve the accuracy, reliability and throughput rate for Ca2+ cycling data analysis.The aim of the study was to develop and test a Ca defects ; therefo defects ,9 are suThe study was approved by the ethical committee of Pirkanmaa Hospital District (R08070). Patient-specific iPSC lines were established as described earlier and written informed consent was obtained from all the participants. The stud2+ cycling measurements, CMs were dissociated by mechanically excising the beating areas of the cell colonies and treating them with collagenase A (Roche Diagnostics) [Differentiation into CMs was performed by co-culturing iPSCs with murine visceral endoderm-like (END-2) cells  as previously described. For Ca2+nostics) .2+ imaging was conducted in spontaneously beating Fura-2 AM  loaded, dissociated CMs that were perfused with extracellular solution as previously described.[2+ measurements were performed on an inverted IX70 microscope  and CMs were visualized with a UApo/340 x20 air objective (Olympus). Images were recorded with an ANDOR iXon 885 CCD camera  that was synchronized with a Polychrome V light source by a real-time DSP control unit and TillVision  or Live Acquisition software . TillVision and LiveAcquisition are further referred as Recording Software 1 and 2, respectively. Fura-2 AM in CMs was excited at 340 nm and 380 nm light and the emission was recorded at 505 nm. For Ca2+ analysis, regions of interest were selected for spontaneously beating cells and background noise was subtracted before further data processing. The Ca2+ transients were acquired as the ratio of the emissions at 340/380 nm wavelengths and presented as Fura-2 ratio units of F340/F380. Throughout the remainder of this manuscript, the Ca2+ recording of a single cell is called the Ca2+ signal.Caescribed. Ca2+ mea2+ signals were divided into two groups and analyzed separately depending on the recording software. The Ca2+ signals were manually analyzed with visual recognition by identifying the signals as normal or abnormal and categorizing the abnormalities into six subgroups according to their abnormality patterns. In manual analysis, a criterion for the double peak group was a Ca2+ peak with paired peaks that did not reach the baseline. Oscillation was detected if Ca2+ fluctuated for three or more peaks without reaching the baseline. For the low peaks group, the presence of a small amplitude Ca2+ peak of at least 10% of the preceding Ca2+ peak amplitude was required. In the middle Ca2+ peaks, 30 to 80% amplitude of the preceding Ca2+ peak amplitude was required. Plateau abnormality was detected if the rise or decay time of the Ca2+ peak was prolonged, and an irregular beating rhythm was required for the irregular phase group. Here, the Ca2+ peak represents a Ca2+ transient consisting of Ca2+ rise and decay. For comparison studies, the Ca2+ data was analyzed with AnomalyExplorer, in which suitable user-defined percentage limits for each abnormality subgroup were selected for both recording software data groups.The data were generated with two separate softwares (Recording Software 1 and 2) with different sampling frequencies that affected the noise level of the recordings. Therefore, the CaAnomalyExplorer is an interactive software tool that can assist in the visual classification of Ca2+ signals. It is based on the information visualization approach with the following two major components: representation of data and interaction with the data. The representation component of AnomalyExplorer is based on the detail+overview design pattern, and an overview and detailed view of an information space are displayed simultaneously. Generally, the overview provides a high-level view of the information space, and the detailed view shows the current focus of interest in full detail.AnomalyExplorer is a conventional 2D plot of the signal value over time. The plot is similar to what a human observer would use to detect anomalies in the shape of a signal. The idea in AnomalyExplorer is to express the human observer’s criteria for abnormality as rules that can be programmed into AnomalyExplorer. The tool then highlights the detected anomalies in a plot, which allows the user to adjust the sensitivity of anomaly detection by user-defined parameters for signal noise and six different abnormality subgroups.The detailed view in AnomalyExplorer is to define whether the signal is normal or not, and to further analyze the quality and distribution of detected anomalies. Thus, the overview in AnomalyExplorer was designed to be a stacked bar chart of detected anomalies where a bar represents the number of anomalies in a signal, and stacking shows how the anomalies are distributed. Only the abnormal signals are present in the bar chart because the signals deemed normal are not of interest. Information in such a bar chart can be acquired rapidly, and the conventional representation allows for further insight-generating interactions.The most important qualitative aspect in AnomalyExplorer and illustrates the workflow. The current UI design has the following elements: a file chooser to open the signals that will undergo analysis, the stacked bar chart of observed anomalies (overview), dynamic controllers for the anomaly detection sensitivity, and the currently open set of detailed signal views. After these main components there is a section showing how the currently open signals are classified as normal and abnormal , without any observed abnormalities. Overall, 17% of the control WT CMs (n = 23) were categorized as abnormal with AnomalyExplorer analysis and 13% with manual analysis. AnomalyExplorer analysis of different Ca2+ transient abnormality subgroups. To visualize the same results, see 2+ transient abnormalities, which can be observed from the detailed analysis results in the AnomalyExplorer overview in The majority of the spontaneously beating control WT and CPVT CMs were categorized as normal in both the manual analysis  and the AnomalyExplorer analysis of different RyR2 mutated CPVT cell lines, analyses revealed that there were mutation-specific differences in the Ca2+ transient abnormalities with both analysis methods  and in 21 to 26% of the signals with mutations in or near the transmembrane domain (Q4201R and V4653F). Oscillation was also common abnormality type in all of the mutated cell lines in AnomalyExplorer analysis, particularly in V4653F and T2538R mutated CMs . Low peaks were rather common in P2328S and Q4201R mutated CMs , and plateau abnormalities in Q4201R and exon 3 deletion mutated CMs  in AnomalyExplorer analysis. Middle peaks and irregular phases were least common abnormality types in all mutated CPVT CMs. When comparing the consistency of the manual and AnomalyExplorer, it was shown that in P2328S and L4115F mutated CMs the analyses were comparable. Over 5% difference between analyses was seen in low peak abnormalities  with difference of 7–15%, irregular phase abnormalities  with difference of 6–10%, plateau abnormalities (with exon 3 deletion and Q4201R CMs) with difference of 10–18% and double peak abnormalities (with T2538R and Q4201R CMs) with difference of 8–14%.When comparing the manual and  methods . Double 2+ cycling is fundamental for the electrical signaling and mechanical contraction of the heart. It is controlled via ion currents, channels, and exchangers. Comprehensive functioning of Ca2+ cycling is essential for the excitation–contraction coupling of CMs.[2+ cycling abnormalities may cause arrhythmic features such as delayed after depolarizations (DADs), where depolarizing oscillations in the membrane potential follow an action potential after completion of repolarization [2+ cycling may also produce Ca2+ alternans, a beat-to-beat alternation in the amplitude of the intracellular Ca2+ transient.[2+ cycling abnormalities manifest arrhythmic features such as DADs or EADs in their action potential [2+ recordings of long QT-specific CMs have revealed that Ca2+ cycling is involved in prolonging the AP duration and in the formation of EADs as demonstrated by comparable changes in the APs and Ca2+ transients.[2+ cycling, thereby highlighting the importance of the analysis of these transients and abnormalities.Cag of CMs. Ca2+ cycrization , or earlrization –21 Dysfuransient. Previousotential ,11,13. Aansients. Therefor2+ transient abnormalities in many CM disease phenotypes. Consistent rules for the analysis of Ca2+ transient abnormalities are currently lacking; thus, the results obtained from different studies cannot be compared. Thus far, the detection of Ca2+ cycling abnormalities has been based on how the researchers categorize data based on the individual’s reasoning, experience and observations. Previously in our study and in other studies, earlier classification methods have involved categorizing a Ca2+ signal as abnormal if at least one abnormality is detected without indicating the absolute amount or duration of Ca2+ abnormalities in each signal.[2+ transient irregularities, such as multiple events [2+ transients, including at least an irregular rhythm and multiple peaks.[2+ abnormality patterns when comparing the already published reports.After the invention of iPSC technology, disease modeling with patient-specific CMs has been constantly increasing and has revealed Cah signal.–6,11,12 h signal.–6 A commh signal., as sign2+ abnormalities with specific analysis software that could automatically classify and calculate the abnormal signals according to preset specifications. For these reasons, AnomalyExplorer, an interactive software tool that can assist in the visual classification of Ca2+ signals was developed to help researchers working with Ca2+ data of CMs. AnomalyExplorer is based on interactive visualization with a direct manipulation UI, in which the analysis and classification approach of the researcher has been transferred into a software tool to improve the accuracy, reliability and throughput rate for Ca2+ cycling analysis. The user can set the user-defined anomaly detection parameters for each subgroup and the background noise level of the recordings. This direct manipulation of the UI allows for the use of AnomalyExplorer with different measurement settings wherein the recording software, sampling frequency or environment of the measurement setup can vary and optimal settings can be found for each occasion. Once the optimal user-defined anomaly detection parameters have been identified for recordings made with specific measurement settings, there is no need to manipulate the UI.The human visual system is an ingenious pattern recognizer, and our cognitive activity is largely based on this capability. DetectinAnomalyExplorer was to generate task-specific visual representations of data with which the user can interact, thereby amplifying users’ cognition. The means to amplify users’ cognition are numerous, and here the most relevant ones were enhancing the pattern recognition, facilitating the comparisons, and supporting the short-term memory by using external representations. [AnomalyExplorer was based on the detail+overview design pattern, and an overview and detailed view of an information space are displayed simultaneously.[AnomalyExplorer was to define whether the signal is normal or not, and to further analyze the quality and distribution of detected anomalies. Therefore overview in AnomalyExplorer was designed to be a stacked bar chart of detected anomalies where a bar represents the number of anomalies in a signal, and stacking shows how the anomalies are distributed. The first question in the design of an interactive visualization tool is if often whether we need to support a workflow from the overview to detailed view or vice versa. Shneiderman [AnomalyExplorer, the free movement between the overview and detailed views must be supported to reduce the cognitive load. In addition, the views need to be tightly coupled. In the detailed view, the appropriate parameters for anomaly detection are sought, and it must be continuously monitored how this affects the overall outcome, until suitable parameters for data analysis are found. In the overview, the inspection may reveal signals that seem to be outliers or that are otherwise suspicious, and the details on the demand need to be seen.The purpose of information visualization is to use perception to amplify cognition. The idea of tations.  The intetations.  The repraneously. Overviewaneously. The mosteiderman  advocate2+ signal takes from 10 to 120 seconds, and the overall manual analysis time in this study was approximately 90 minutes and 140 minutes with Recording Software 1 and 2 signals, respectively. With AnomalyExplorer, the analysis of the Ca2+ signals is much faster, and was approximately only 10 seconds in this study with both Recording Software 1 and 2 signals, because the analysis results are immediately shown after loading the signals into the software regardless of the number of signals. Therefore, with AnomalyExplorer, the saved time compared with manual analysis increases with the number of analyzed signals. Other benefits of AnomalyExplorer include the fact that the classifications are repeatable and user-independent and that the accuracy and repeatability of the analysis can be enhanced. The definition of signal abnormalities in terms of visual properties, is more flexible than numerical or analytical specification. However, it needs to be highlighted that this type of abnormality analysis does not exclude the need for the numerical analysis of different Ca2+ cycling parameters, such as the amplitude, frequency and peak duration. AnomalyExplorer is anyhow a good tool for screening through the Ca2+ recordings for more extensive analysis and it can facilitate the numerical analysis by determining the abnormality analysis criteria that can be exploited when defining how abnormalities should be considered in numerical analysis, such as when calculating the duration of double or oscillating peaks. To further analyze the mechanism behind different Ca2+ cycling abnormalities, combined electrophysiology and other functional methods together with Ca2+ imaging measurements are needed.It can be roughly estimated that manual analysis of one Ca2+ signals are analyzed with AnomalyExplorer, the results are more comparable between different studies than they are with manual analysis because the user-defined anomaly detection parameters are specifically determined. Once the optimal user-defined parameters have been identified for specific measurement settings, there is no need for manipulating these parameters. With AnomalyExplorer exploiting the interactive visualization, it is easy to catch all of the abnormalities in a sample and obtain a more accurate and complete classification. In this study, we have shown that optimal analysis parameters can be found for data recorded with two different recording software programs and that the detection of normal and abnormal signals as well as the abnormality patterns with AnomalyExplorer is quite consistent with the manual analysis. Some variation in the manual and AnomalyExplorer analysis was observed in the categorization of abnormalities. When classifying the signals as normal or abnormal, inconsistency of the analysis could be seen in 2% and 9% of signals with Recording Software 1 and 2, respectively, which was quite reasonable. When categorizing the abnormalities into subgroups and calculating the number of different subgroup specific abnormalities, the abnormalities were detected with 0 to 5% difference between the manual and AnomalyExplorer analyses. The exception was in the amount plateau and low peak abnormalities in Recording Software 2 signals with 8 and 10% differences between manual and AnomalyExplorer analysis. When comparing manual and AnomalyExplorer analyses of different RyR2 mutated CPVT cell lines it was shown that detection of oscillation and middle peaks were equivalent with 0 to 5% differences in addition to double peak detection, which was quite similar between analyses with 0 to 5% differences in 4 out of 6 mutations. Detection of low peaks, irregular phases and plateau abnormalities differed the most between analyses. Overall, the inconsistency between manual and AnomalyExplorer analysis may be due to differences between human and software analyses because the accuracy of the manual analysis varies since it is performed only by visual recognition without clear abnormality detection rules. Higher percentages of low peak abnormalities were detected in manual analysis than in AnomalyExplorer analysis. This could be explained with background noise of the Ca2+ recordings, which is challenging to recognize visually in the manual analysis. Also irregularity of the beating rhythm as well as the prolonged rise and decay time  of Ca2+ transients are challenging to detect visually in manual analysis since criteria for these abnormality types is difficult to define. With AnomalyExplorer the noise and abnormality detection rules are clearly defined and therefore the anomaly detection is more compatible between signals. Inconsistency between manual and AnomalyExplorer analysis can also be seen due to problems in the Ca2+ recording setup, such as with photobleaching of the Ca2+ indicator or its toxicity together with UV light to CMs, which can interfere with the loading of the cells and cause the transients and thus the whole signal to have extra noise. This can also affect the validity of AnomalyExplorer analysis and may be stated as one limitation of this study.When Ca2+ transient abnormalities like we have previously shown with these cell lines [AnomalyExplorer for detecting cell line- and mutation-specific variability in the Ca2+ transient abnormalities. These types of differences reflect the mutation specific pathology of the disease and reveal that the location of the mutation can affect the disease phenotype. This result is in accordance with our previous study [2+ abnormalities of these different CPVT cell lines are more common in some mutations than in others and that these Ca2+ cycling abnormalities can be rescued or reduced with a specific drug as in actual patients. In the future, AnomalyExplorer can speed up and improve the accuracy of the analysis of this type of drug study, where the effect of a specific type of drug on the amount Ca2+ cycling abnormalities is quantified. Further Ca2+ cycling studies combined with electrophysiological studies, are needed to assess how different Ca2+ cycling abnormalities reflect the mechanism of the disease or drug effect because these are not yet known. Presumably, at least some Ca2+ cycling abnormalities reflect the arrhythmogenic features such as DADs and EADs. Because electrophysiological studies are time-consuming and challenging and because only a restricted number of cells can be measured, these methods are not often combined and more optimization is needed.When comparing the analysis of different CPVT cell lines generated from patients carrying different RyR2 mutations the percentage of different abnormality subgroups varied between cell lines. Overall, double peaks and oscillations were most common and middle peaks and irregular phases least common abnormality types in all CPVT mutations. Other than that, no specific trend between abnormality subgroup percentages and different mutations could be seen and it could be concluded that there were mutation-specific differences in the Call lines .This finus study , where wAnomalyExplorer was designed for the data analysis of <50 Hz sampling frequency data with recording duration of tens of seconds, and it performs flawlessly under these requirements. AnomalyExplorer is able to analyze data with higher sampling frequencies, at least to 100 Hz, but it could be improved to suit for analysis of even higher frequency data in the future. With functional recordings it is problematic to set a certain recording duration limit that is sufficient to define the overall behavior of a cell and there is always uncertainty how well the recording reflects the overall behavior of the cell. In our study the recording duration was implemented for our specific use case with varying recording durations of 11 to 23 seconds. The analysis software does not have a limit for the recording length, but it can be estimated that responsiveness and user experience will suffer with several minute recordings. Short duration signals may not represent the long-term behavior of the cell correctly and for the future Ca2+ cycling analysis, the use of longer recordings with standard duration could be more beneficial, if it would be suitable for the study design. Ín these analyses, the duration of each abnormal Ca2+ transient could be compared to the duration of the whole signal to see the percentage of the abnormal Ca2+ transients in the recording. To improve the analysis of long-term behavior of the cells, long recordings could be also broken up into a series of segments and analyzed separately before averaging. Sampling frequency and recording duration of this study can be stated as a limitation of this study and the current prototype, as it was designed and implemented for a specific use case and for the specific digitization systems. Nevertheless, recording durations of tens of seconds and sampling frequencies of <50 Hz have been utilized successfully before, when studying Ca2+ cycling of iPSC derived disease specific CMs [ific CMs ,23.AnomalyExplorer is suitable for analyzing high number of Ca2+ signals, which can be a bottleneck for high-throughput Ca2+ imaging analysis and screenings in manual data analysis. This tool will facilitate and speed up the analysis of CM Ca2+ transients; furthermore it should be more accurate and user-independent. At present, we are not aware of any similar software that is suitable for single Ca2+ signal analysis that is recorded with any type of recording software. In the future, iPSC-derived CMs can be exploited for screening of potential new chemical compounds and this software and analysis method can be exploited for evaluating different drug responses and in Ca2+ cycling analysis to study basic disease pathology. This method will also be beneficial when analyzing Ca2+ cycling of CMs on a large scale for screening the adverse cardiac effects of new potential compounds.As the use of iPSC-derived CMs for disease modeling and drug screening continues to increase, there is a significant demand for faster and more consistent analysis methods. AnomalyExplorer tool was developed to assist in analyzing and classifying Ca2+ signal abnormalities of disease specific human iPSC-derived CMs. Ca2+ cycling has a central role in CM contraction-relaxation, and it also is essential in the electrical signaling of CMs. As a result, detailed characterization of the abnormal Ca2+ transients present in different cardiac diseases would allow for a more detailed evaluation of the physiological function of the diseased CMs. Because Ca2+ transient abnormalities are difficult to specify and analyze by computational methods, in the AnomalyExplorer tool the abnormalities are recognized by the visual features alone, using interactive visualization with a similar approach as in traditional manual analysis. It is based on the multiple coordinated views paradigm and a direct manipulation UI. Using AnomalyExplorer the classifications are repeatable and user-independent. The software is also capable of screening large datasets faster than manual analysis. By utilizing fast kinetic fluorescence imaging of intracellular Ca2+ levels, together with AnomalyExplorer, the Ca2+ transient abnormalities of spontaneously contracting CMs can be detected and classified, which provides more information regarding the Ca2+ cycling phenotype in these cells.In this study, the S1 Fig2+ signals recorded with Recording Software 1 (A) and 212 Ca2+ signals recorded with Recording Software 2 (B) were analyzed.Totally 132 Ca(TIF)Click here for additional data file.S2 Fig2+ signals in manual and AnomalyExplorer analyzed signals. B) Color-coded pie charts indicate the percentage of the different abnormalities in both manually and AnomalyExplorer analyzed signals. One Ca2+ signal can belong to several subgroups and consist of many abnormalities. In Recording Software 1 signals, totally 171 and 157 abnormalities were found in manual and AnomalyExplorer analysis, respectively. In Recording Software 2 signals, totally 301 and 284 abnormalities were found in manual and AnomalyExplorer analysis, respectively.A) Pie charts indicate the percentage of normal (white) and abnormal (black) Ca(TIF)Click here for additional data file.S1 Supporting Information(DOCX)Click here for additional data file.


Fusarium toxins  were also found in the feed, but at levels which did not explain the observed effects. The results indicate that ergot alkaloids may have been the cause of tail necrosis, which is supported by literature data showing that rabbits are especially sensitive towards these toxins.This study describes the association between tail necrosis in rabbits and mycotoxins in rabbit feed. Clinical cases of tail necrosis were observed in 14 out of 103 rabbits kept in an outdoor group housing, fed with hay and a commercial pelleted feed. The observed clinical symptoms, alopecia, erosions, crusts and necrosis were restricted to the tail area and exclusively occurred in young rabbits aged 113 ± 20 days. Dermatological examination suggested that ischemia had caused necrosis. Analysis of blood samples showed an elevated level of creatine kinase. No weight loss occurred in affected rabbits. Trauma caused by injuries or technopathic lesions was also excluded. Histopathologically, the lesions were characterized by acute muscle fibre degeneration and chronic active dermatitis with granulation tissue formation. Necropsy of one rabbit revealed hepatocellular degeneration and necrosis as remarkable findings. Feed analysis for ergot alkaloids by enzyme immunoassays yielded a mean and maximum ergot alkaloid content of 410 ± 250 μg/kg and 1,700 μg/kg, respectively. Faeces of affected rabbits contained ergot alkaloids at levels up to 200 μg/kg. The mean and maximum dietary intake of total ergot alkaloids were 17 and 71 μg/kg bodyweight, respectively. The online version of this article (doi:10.1007/s12550-014-0208-0) contains supplementary material, which is available to authorized users. Fusarium toxins  are regarded as the most important mycotoxins with regard to animal health. For example, fumonisins lead to pulmonary edema, thoracic effusions and hepatocellular necrosis , zearalenone, T-2 toxin/HT-2 toxin and ochratoxin A  of Justus Liebig University Giessen, in groups of seven to nine animals per cage on a perforated plastic floor. Male and female animals were housed in separate compartments. They also had the possibility to withdraw in a wooden box, spread with shavings and straw. The stocking rate and the availability of space for each animal were in accordance with European Union directive 63/2010 . The study was carried out according to the German regulations with regard to animal welfare.Animals were fed with hay and a commercial pelleted feed preparation ad libitum and had unrestricted access to water from a nipple drinker. The composition and the analytical constituents of the pelleted feed are given in Table All rabbits in the colony were regularly vaccinated against myxomatosis  and rabbit haemorrhagic disease  according to manufacturers’ specifications.The first rabbits with clinical symptoms of tail lesions were observed on 12 November 2012. The last observed case was on 07 January 2013. The mean age of the rabbits when showing first clinical signs was 113 ± 20 days. Until then, no pathological findings were observed. Feed and management factors were consistent since the start of the original breeding experiment. The daily feed consumption was assessed by weighing rabbits of two different groups and feed daily over a 1-week period as basis to calculate the amount of mycotoxin which was incorporated per kilogram bodyweight. Also, four healthy adult rabbits of corresponding breeds were included in this procedure to compare their dietary intake per kilogram bodyweight with that of the affected growing animals. All affected rabbits were weighted  up to four times at a 1-week interval, starting when the first lesions became apparent.In total, 14 rabbits, which were offspring of four different breeding pairs, were clinically affected, and all of these affected rabbits were included in this study. Two unaffected animals served as controls for the blood parameters.To rule out behavioural abnormalities and injuries caused by group mates, the rabbits were observed in several intervals during the day (for 10 min every 2 h) between 0700 hours (morning) and 1700 hours (afternoon). Cages and housing equipment were checked for possible sources of technopathic lesions.Every affected animal underwent a complete general clinical examination. The physiological range was determined according to Schall .Blood samples were collected from three affected  and two unaffected  animals and analysed for clinical chemistry  and haematology .Three rabbits  underwent a detailed dermatological examination 9 days after clinical lesions were visible.To avoid secondary infections, animals with ulcerative lesions received antibiotic treatment with enrofloxacin  for 5 days; additionally, chlortetracycline was applied topically .For two severely affected animals, tail amputation became necessary because of cranial progression of the lesions. Surgery was performed under ketamine hydrochloride/medetomidine anaesthesia  were also tested by EIAs. All tests were performed similarly as already described by Liesener et al.  for total (generic) ergot alkaloids (GEA-EIA) and ergotamine (ergotamine-EIA). Additionally, four randomly selected samples of the second batch were analysed by an EIA for fumonisins. More r et al. . FurtherPooled faecal samples of four groups of animals  which were fed to the rabbits and samples of straw  which were used as bedding were also analysed for ergot alkaloids. Macroscopically, no signs of mould were visible in these materials. From each dry material, a 300-g composite sample was collected, cut into pieces of 2- to 4-cm length and further ground to a mean particle size of <2 mm in a laboratory mill before analysis.0 × 100) between 25 and 75 % were used to calculate the toxin content. Highly positive extracts, resulting in absorbance values outside the measuring range (<25 %), were further diluted and re-analysed by EIA.All EIAs were performed as standard microtiter plate tests. Sample preparation was performed as already described earlier  rabbits was 42 g per kg body weight, the average feed intake of adult rabbits was 35 g per kg body weight.The general clinical examination of all 14 affected rabbits yielded results within a normal range, body weight development was normal. Neither aberrant behavioural patterns within each group of rabbits nor indications for technopathic lesions were observed.Symptoms were first observed in November 2012 in six female rabbits . All affected animals showed, to varying degrees, moist, sanguineous lesions at their tails, partly exposing the spine  had only mild skin alterations, such as alopecia and scaling  significantly higher creatine kinase values in the three affected rabbits (The blood biochemistry profile (Table Leporacarus gibbus), was detected in one rabbit (82050). Differential diagnoses included ischemic dermatopathies , automutilation, barbering (trauma by cage mates), technopathic lesions and—less likely—dermatophytosis for cases with mild symptoms such as alopecia and scaling.Three rabbits , which were presented for dermatological examination, all showed varying degrees of alopecia, scales, multifocal crusted erosions and ulcerations on the distal part of the tail. Other acra, namely the ears, were not affected. Slight scaling on the back, due to infestation with low numbers of fur mites  was in a good nutritional state. The liver was firm, irregularly mottled and showed multiple, sharply demarcated pale foci (diameter 0.5–1.0 cm), multifocally distributed within the parenchyma. The spleen was swollen due to acute congestion, and there was an acute diffuse alveolar edema in the lungs. Histopathologically, the liver showed multifocal areas of coagulative necrosis Fig. , randomlFusarium toxins by EIA yielded performance data similar as described previously , the concentration of ergotamine was 120–140 ng/g .Total ergot alkaloids (TOT) could be detected in all 44 samples from three batches of the commercial rabbit feed, at a mean concentration of 410 ng/g . Ergotamine (TAM) could also be detected in all samples, in a mean concentration range of 370 ng/g (range 140–910 ng/g). An overview of analytical results is given in Table Low levels of fumonisins could also be found in all four samples in a concentration range of 5.3–6.5 ng/g. All analysed samples, three from each feed batch, also contained DON (260–540 ng/g), T-2 toxin/HT-2 toxin (8.7-17 ng/g) and zearalenone (105–130 ng/g).n = 20)   and from the unaffected group (F3) contained total ergot alkaloids in a concentration range of 51–180 ng/g  Fig. . The meaNo ergot alkaloids could be detected in straw and hay samples (detection limit 130 ng/g).In this case series, 14 out of 103 rabbits, living under equal conditions, spontaneously showed tail lesions. Exclusively, young, growing animals were affected. As a comparable outbreak has never been reported before in scientific literature, we put some effort in identifying the likely causative factors.We assumed that the tail lesions may be the result of peripheral ischemia due to vasoconstriction caused by mycotoxins, most likely by ergot alkaloids. In the histopathological examination, acute muscle fibre degeneration was coexisting with a chronic inflammation with granulation tissue formation. Vasculitis or thrombosis was not detectable.The lesions observed in the deceased rabbit  could have been a possible consequence of a toxin or a toxic effect  (404 ± 140 ng/g) but this can be addressed as a concentration which is commonly found in cereal-based feedstuff. The DON level was considered far too low to cause typical acute toxic effects, which are primarily characterized by feed refusal and reduced weight gain. This assumption is supported by recent data from toxicological studies  than that of healthy adult rabbits (35 g per day and kg body weight), toxic effects caused by ergot alkaloids are expected to be more pronounced in young rabbits. Using the average daily feed intake of 42 g per kg body weight of growing rabbits and the mean total ergot alkaloid content in feed (410 ng/g), the mean ergot alkaloid intake was calculated to be 17 μg per kg body weight. Using the same intake and the maximum ergot alkaloid levels found in one sample , the maximum daily alkaloid intake could be estimated to be 71 μg per kg body weight. However, it has to be considered that coprophagy of soft pellet faeces results in a re-ingestion and a second passage of ergot alkaloids. Therefore, the effective ergot alkaloid intake may have been much higher.Interestingly, relatively high levels of ergot alkaloids were found in faeces from the three affected groups F1, F2 and F4. Groups F1 and F4 contained the most severely affected rabbits, while symptoms in group F2 were somewhat milder. Group F3 which was unaffected had slightly lower total ergot alkaloids in faeces, but was still positive. This is no surprise because all animals in groups F1–F4 received the same feed Figs. , and 6. Our hypothesis is that ergot alkaloids in rabbit faeces could possibly provide a marker for the oral intake level of mycotoxins and that the intake levels observed in this study circumscribe the toxicologically relevant dose.Furthermore, as rabbits are a coprophagic species, ergot alkaloids in faeces undergo a recycling process, which could enhance toxin resorption. In fact, high ergot alkaloid levels were found in the faeces in the animal groups with clinical symptoms, while faeces from clinically symptom-free rabbits mostly contained no detectable alkaloids at all, and only trace amount levels were measured by the ergotamine EIA in two samples.Gangrene of extremities  has been described for many animal species ESM 2(DOCX 14 kb)ESM 3(DOCX 13 kb)Below is the link to the electronic supplementary material.


Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca This is particularly true for the cardiac Tn isoform (cTn) for which we only have a single incomplete crystal structure of the Ca2+ saturated core complex Troponin (Tn) is a large (∼77 kDa), dynamic protein complex that is located on the thin filament of striated muscle and is responsible for controlling the interaction of myosin with actin. Contraction in striated muscle  is initiated by the simple binding of calcium (Ca2+ binding subunit (TnC), the 24 kDa thin filament binding inhibitory subunit (TnI), and the 35 kDa tropomyosin anchoring subunit (TnT). TnC consists of two globular metal binding domains, the C-lobe and N-lobe, which have structural and regulatory roles, respectively. It is the binding of Ca2+ to the regulatory N-lobe that is responsible for initiating contraction. Following Ca2+ binding, the signal is propagated from the N-lobe to the TnI subunit and subsequently to the other members of the thin filament (tropomyosin and actin). This cascade of conformational events modifies the interaction between the actin thin filament and the myosin thick filament, leading to muscle contraction.The troponin complex  is compo2+ in the presence of the other Tn subunits of the complex 2+, two regions of TnI  partial structure in the Ca2+-free state, were also determined 2+-bound Tn core complex for both isoforms showed Tn to have two distinct sub-domains. The first, called the structural ‘IT-arm’ domain , which can be phosphorylated in vivoA crystal structure of a 52 kDa cardiac Tn core complex (cTn), comprising cTnC (residues 1–161), cTnI (residues 31–210), and cTnT (182–288), was solved in the presence of Ca’ domain , contain2+-bound cTn and skTn core crystal structures did provide confirmation of binding of the TnI switch region to the hydrophobic pocket of the N-lobe of TnC, which was also observed by fluorescence resonance energy transfer (FRET) experiments 2+2+ crystal structure, suggesting a highly dynamic region. Despite a decade now passing since these crystal structures were reported, the structure of the cardiac isoform in the absence of Ca2+ has remained elusive. Without the ability to compare structural models of Ca2+-bound and free states of cardiac Tn, questions have continued to exist over whether the movements suggested by the crystal structures even occur in muscle cells. Indeed, there is still no direct evidence collected in solution using full-length Tn subunits that support the large scale movement of TnI away from TnC for the cardiac isoform. Therefore, a more detailed structural understanding of the Ca2+ regulation mechanism for the release of the switch region from cTnC is needed.Nevertheless, the Ca2+-controlled conformational interplay between the key functional regions of TnI with TnC in the cardiac isoform. PRE-NMR extends the capabilities of traditional NMR approaches to probe protein structure and dynamics by providing long-range distance information up to 30 Å for a proton experiencing relaxation enhancement from a nearby paramagnetic moiety, such as a nitroxide spin label 2+ sensitive movement of several regions of cTnI  NMR spectroscopy to further understand the Ca of cTnI . The reg of cTnI . Four spEscherichia coli BL21 (DE3) cells for expression.Cysteine-less constructs of rat cardiac TnC (cTnC) and rat cardiac TnI (cTnI) were expressed in the pET-3d expression vector (Novagen) as previously described Monocysteine constructs of cTnI were expressed in Terrific Broth medium. Cell lysis was achieved with 2–3 passes through a chilled French Press at 10,000 psi. The insoluble cTnI inclusion bodies were separated from soluble proteins in the cell lysate by centrifugation . The inclusion bodies were solubilised via dialysis into a buffer containing 6 M urea, 1 mM DTT, 1 mM EDTA and 20 mM Tris, pH 8.0, and Complete EDTA-free Protease Inhibitors (Roche). After overnight dialysis, the sample was clarified by centrifugation . The supernatant was loaded onto a 1.5 cm×15 cm carboxymethyl (CM) Sepharose column . cTnI was eluted with a linear NaCl gradient from 0 M NaCl to 0.3 M NaCl, as previously described et al (1989) 15NH4Cl, using the IPTG induction protocol of Studier et al (1990) 15N-cTnC was then purified by hydrophobic interaction chromatography using a 1.5 cm×15 cm Phenyl Sepharose 6 Fast Flow column , as previously described Cysteine-less cTnC was expressed in M9 minimal media as described by Sambrook 2, 3 mM MgCl2, 50 mM MOPS pH 7.2).The paramagnetic nitroxide moiety was introduced onto the sulfhydryl group of the cysteine side-chain of each mono-cysteine cTnI construct by the covalent attachment of the spin label 1-oxyl-2,2,5,5-tetra-methylpyrroline-3-methyl-16 methanethiosulfonate (MTSL) . Immediately prior to labeling, the purified cTnI constructs (∼7 mg/mL) were incubated with 20 mM DTT (dithiothreitol) for 2 h at 4°C to ensure complete reduction of the single cysteine sulfhydryl group. DTT was then removed from the sample using a 5 mL HiTrap desalting column  and the reduced cTnI was eluted in labeling buffer . A 7.5 fold molar excess of MTSL was added immediately to the reduced cTnI. After 2 h incubation at 4°C, an additional 2.5 fold excess of MTSL was added to the sample before overnight incubation at 4°C with gentle stirring. Excess MTSL was then removed by exhaustive dialysis against 2 M urea, 1 mM EDTA, 100 mM KCl, 50 mM MOPS pH 7, before dialyzing into reconstitution buffer  spectrometer using a standard rectangular TE cavity at room temperature. First derivative EPR absorption spectra were collected with a microwave power of 5.0 mW, modulation amplitude of 1.0 G and sweep width of 140 G. EPR spectra were analyzed with a LabView package  of EPR spectral analysis programs. Spin labeling yields for each sample were determined from the integrated area under the EPR absorbance spectrum by interpolation from a calibration curve constructed from MTSL standards. Complete modification with MTSL (>95%) was achieved for all cTnI mutant constructs.15N-cTnC/MTSL-cTnI constructs are referred to according to the position of the spin-labeled cTnI residue , complex formation was verified using SDS-PAGE and size exclusion chromatography (SEC) on a Superdex 75 10/300 GL column .The reconstituted binary 2+ state were prepared by exhaustive dialysis into NMR buffer  containing 3 mM CaCl2. To prepare calcium free samples (−Ca2+), Ca2+ was removed by dialysis against 0.1 M EGTA for 2 h at 4°C followed by exhaustive dialysis into decalcified NMR buffer  with 5 mM MgCl2, as previously described NMR binary samples in the +Ca1H-15N-TROSY spectra were collected for each of the four  spin labeled binary 15N-cTnC/MTSL-cTnI samples. After acquiring each paramagnetic spectrum, the nitroxyl radical of the MTSL label was reduced to its hydroxylamine equivalent by the addition of ∼1/250 volume of a 5-fold molar excess of ascorbic acid (from a 0.5 M stock) directly to the NMR sample. One hour at room temperature was sufficient time to allow for the reduction reaction to go to completion as confirmed by EPR. 1H-15N-TROSY spectra were then collected for each reduced diamagnetic sample. The small volume of ascorbic acid did not significantly dilute the sample, thus enabling the direct comparison of peak intensities between paramagnetic and diamagnetic spectra for calculation of the relaxation enhancement. Spectral assignment and analysis was performed with the program Sparky 2+-free state NMR data were collected on a Bruker Avance 600 MHz spectrometer equipped with a cryoprobe. All experiments were performed at 303 K and spectra were processed using Topspin 1.3 . All NMR samples were 550 µL, and concentrations of the binary complex (cTnC-cTnI) ranged from 150 µM to 250 µM. These low concentrations were required to prevent intermolecular PRE effects 2Γ) was calculated from the ratio of peak intensities measured in the paramagnetic state (paraI) and the diamagnetic state (diaI) for each amide peak assigned in the 1H-15N-TROSY spectra of the binary constructs (2R) was estimated from the peak width at half-height (2 = πΔv1/2R) t) of 10 ms.The PRE caused by the MTSL spin label (nstructs . Peak he2Γ) were converted to distances (r) using a modified form of the Solomon-Bloembergen equation Hω is the Larmor frequency of the proton; K is a constant (1.23×10−32 cm6 s−2) which describes the spin properties of the nitroxide spin label cτ is the correlation time of the electron-proton interaction [sτ) compared to the protein rotational correlation time (rτ), the value of cτ is effectively equal to the overall correlation time of the protein cτ value used in cτ causing, at most, <6% change in the PRE calculated distance 2Γ values to distances using The calculated PRE rates  with >95% efficiency, as judged by double integration of the EPR spectrum. To obtain the intersubunit PRE-NMR constraints, cysteine-less cTnC was isotopically labeled with 15N and the four binary cTnC-cTnI complexes were prepared by individually reconstituting each spin-labeled cTnI construct with the 15N-cTnC protein. Binary complex formation was confirmed by SDS-PAGE and analytical size exclusion chromatography , and presence of Ca2+ (+Ca2+). A 1H-15N-TROSY spectrum was first acquired for each sample in the presence of the paramagnetic spin label .Assignment of all four +Ca spectra  was madeparaI/diaI<1). The PRE-broadening of the NMR signal is proportional to 61/r  calculated for each assigned cTnC resonance in the −Ca2+ and +Ca2+ are plotted in 2+ cardiac Tn crystal structure (1J1D) in a continuous color scale. Strong PRE peak broadening effects (para/IdiaI≪1) are shaded red and are indicative of close proximity (<12 Å) of the cTnC residue to the nitroxide spin label on cTnI. cTnC resonances which were unaffected by the spin label (para/IdiaI∼1) are shaded in blue and represent residues >25 Å away from the spin label. cTnC residues for which no peak intensity ratio could be reliably assigned, either due to uncertainty in assignment or peak overlap, are shown in white. This comparative mapping of the PRE broadening effects enables the rapid visualisation of the relative positioning of each of the functional cTnI regions under investigation with respect to cTnC. The mapping also reveals the effect of Ca2+ binding on the relative positioning of the two subunits.For all four binary complex samples, a unique pattern of paramagnetic peak broadening effects arising from the inclusion of the spin label on cTnI were observed . Peak in2+ show that the spin label on I57 (cTnI N-region) had a similar effect on cTnC residues in both Ca2+ states . This indicates that the position of the N-region of cTnI . This movement of the two lobes of cTnC relative to one another could result from an increase in dynamics of the central linker, a change in the interdomain angle of cTnC towards a more extended conformation, or a combination of both. Ca2+ associated changes in intermolecular cTnC PRE effects were also observed for the other three spin labeled sites  were observed for cTnC N-lobe residues in the complexes with the spin label in the switch region (I151 and I159) , indicating release of the switch peptide from the N-lobe of cTnC. We did not observe any other localised region of strong PRE broadening upon release of the switch region in the −Ca2+ state  . This we2+ state . It is t2+-induced differences in PRE effects on cTnC residues were observed for the spin label attached to I143 on the inhibitory region of cTnI , and downstream (by the N-region of cTnI through its interaction with C-lobe of cTnC). In the absence of Ca2+, the effect of the I143 spin label was considerably weaker for a large number of residues throughout the N-lobe and central linker of cTnC. In the C-lobe, there was little difference in the PRE effects observed for most residues in the +Ca2+ and −Ca2+ states, except for helix H, for which considerably stronger PRE effects were observed in the +Ca2+ state. Together, these observations suggest that upon removal of Ca2+, the inhibitory region still remains in close proximity to cTnC through its downstream interaction with the C-lobe of cTnC (shown by label at I57), but due to the release of the switch region from the cTnC N-lobe (as shown by labels at I151 and I159), the inhibitory region samples a much larger range of conformations in the Ca2+-free state.The most dispersed and largest Ca of cTnI . The spiC-domain . This su2+ conditions and its release upon removal of Ca2+. Peak intensity ratios were then used to calculate the PRE rates (Γ2) (2+ (1J1D) were in very good agreement (2+ conditions.The mapping of the PRE broadening patterns for label sites I151 and I159 suggested the tight binding of the switch region to the N-lobe under high Caes (Γ2)  for the es (Γ2) . Distances (Γ2) . Comparigreement , confirm2+ and −Ca2+ states for I151 and I159 reveal the Ca2+-dependent direction of movement of the switch region. These distance changes between the +Ca2+ and −Ca2+ states are also mapped onto the cardiac Tn core crystal structure  indicating an increase in the average distance between the spin label and the cTnC residue. Similarly, blue coloring indicates an increase in distance between the spin label and cTnC when Ca2+ is removed.Comparison of the distances measured in the +Care 1J1D,  in Figur2+-bound state, the distances measured in the Ca2+-free state are substantially longer for N-lobe helices A, B, and D. Slightly longer distances were also observed for cTnC residues located in the defunct Ca2+ site I between helices A and B (residues 28–40), and the central linker region (residues 85–93) of cTnC, indicating that upon release of Ca2+, the switch region moves away from these regions of cTnC. Conversely, distances for the C-domain helices F and G  are longer in the −Ca2+ state, indicating that upon release from the N-lobe in the Ca2+-free state, the switch region moves towards these helices in the C-lobe of cTnC.Compared to the Ca2+ and −Ca2+ states are listed in 2+ are further given in 2+ state since the switch region is tightly bound to the N-domain of cTnC. However in the Ca2+-free state, our data suggests that the switch region is not bound to cTnC, thus the interpretation of the distances measured in the −Ca2+ state should be treated with caution.All distances measured from the switch region (I151 and I159) to cTnC residues that were able to be assigned in both the +Ca2+ to troponin within the binary cTnC-cTnI complex. Using the site-specific placement of nitroxide spin labels in cTn, we have mapped the quaternary interactions and described the change in average distance between key regions of the cTnI inhibitory subunit and the cTnC Ca2+ binding subunit. Our PRE-NMR studies clearly show that the switch peptide is tightly bound within the N-lobe of cTnC in the presence of Ca2+, whereas the inhibitory region exhibits conformational freedom, but remains in the vicinity of the central linker region of cTnC. In the absence of Ca2+, the switch peptide is completely released from the N-lobe of cTnC. Upon release, it moves ∼10 Å towards the C-lobe of cTnC. However, we see no evidence of an alternative binding site on cTnC for the switch peptide in the Ca2+-free state. Our PRE measurements also show that the interaction of the N-region of cTnI with the structural C-lobe of cTnC is, unsurprisingly, Ca2+-independent.We have used PRE to follow the conformational changes associated with binding of Ca2+ to the N-lobe of cTnC have been proposed with the molecular details for the positioning of most functional regions of cTnI reinforced by the crystal structure of the cardiac Tn core structure made available in 2003 2+-induced changes, is lacking, since there is no crystal structure of the cardiac complex in the low Ca2+ state. Determination of such a structure is likely to remain a significant challenge due to its dynamic nature.The geometrical positioning of key functional regions of the TnI subunit with respect to TnC and the subsequent movement of TnI accompanying activation, for both skeletal and cardiac isoforms, has been the subject of many biochemical studies over the past two decades 2+ state 2+-bound and Ca2+-free states. To perform PRE-NMR in the cTnC-cTnI binary complex required the spin label to be placed on 14N-cTnI and reconstituted with 15N-cTnC. This was necessary in order to simplify our 1H-15N-TROSY spectra, since it has previously been demonstrated that skTnI resonance peaks are poorly dispersed, as is typical for partially disordered proteins 1H-15N-TROSY spectra, the PRE broadening effect caused by the cTnI spin labels enabled us to indirectly monitor the functionally important regions of cTnI in the binary complex using full-length cTnC and full-length cTnI. Using this approach in the 42 kDa binary complex, we have identified the regions of cTnI that are most sensitive to Ca2+ binding. Sufficient inter-subunit measurements were obtained to describe the magnitude of movement, or lack of movement, for each of the cTnI regions upon removal of Ca2+.While other NMR studies have also been performed on the complete binary cTnC-cTnI complex in the +Ca2+, the cTnI switch region interacts with a small, well-defined small region of cTnC, consistent with tight binding of this region to the N-lobe of cTnC. This was in agreement with the binding mode of the switch region in the crystal structure of cardiac Tn Overall, our PRE experiments showed no large scale structural rearrangement of the two cTn subunits with respect to each other; only the selective and modest movement of the switch and the inhibitory region were observed. The PRE broadening patterns for the two labels on the switch region  showed tThe PRE broadening effects for the spin label on the inhibitory region were observed to map to a large, but well-defined area of cTnC centred on the central linker. The inhibitory region is likely held in close proximity to the central linker of cTnC as a result of being tethered at either end by the strong interactions between the N-region of cTnI with the C-lobe of cTnC, and the switch region with N-lobe of cTnC. The broad region over which the strong PREs were observed also suggests that this region is highly mobile in solution. Lindhout et al 2+, in particular the positioning of the cTnI switch region upon release from cTnC. The magnitude of movement we observed for the switch region upon release from the N-domain in the Ca2+-free state for both label sites was only modest. The average distance change measured between the switch peptide and cTnC was only up to ∼10 Å  SDS-PAGE (12.5% acrylamide) was used to assess the purity of the preparation of binary samples for NMR. Samples of purified cTnI (Lane 1: I) and cTnC (Lane 2: C) were reconstituted as a binary cTnC-cTnI complex (Lane 3: I+C). (B) NMR samples of binary cTnC-cTnI were monomeric and homogenous, as judged by analytical size exclusion chromatography (SEC) on a Superdex 75 10/300 GL  column. Samples of the binary cTnC-cTnI complex (black line) were found to consist of a single species of MW of ∼42 kDa which was significantly greater than that of isolated cTnC of ∼18 kDa (grey). NMR buffer  was used for all analytical SEC runs.(DOCX)Click here for additional data file.Figure S21H-15N-TROSY spectra of binary troponin complexes.Two dimensional  MTSL spin label attached to cTnI residues: I57-MTSL in the (A) +Ca2+ and (B) −Ca2+conditions; I143 in the (C) +Ca2+ and (D) −Ca2+conditions; I151-MTSL in the (E) +Ca2+ and (F) −Ca2+conditions; and, I159 in the (G) +Ca2+ and (H) −Ca2+conditions. The paramagnetic spectrum (red) is superimposed on diamagnetic (blue) 1H-15N-TROSY spectrum after reduction of the spin labels for each sample.(DOCX)Click here for additional data file.Figure S3PRE rates (Γ2) in cTnC residues caused by the switch region probe site cTnI151 (circles) and cTnII159 (squares). PRE rates measured in the absence of Ca2+ (green) are compared to those measured in the presence of Ca2+ (black). The helices of cTnC are indicated with light grey shading .(DOCX)Click here for additional data file.Table S1PRE-NMR distances between cTnI switch (cTnI151) and cTnC. Distances measured from the cTnI151 spin label to cTnC residues, for residues which were measurable in both the +Ca2+ and −Ca2+ states. The highlighted distances were used to calculate the averages values presented in (DOCX)Click here for additional data file.Table S2PRE-NMR distances between cTnI switch (cTnI159) and cTnC. Distances measured from the cTnI159 spin label to cTnC residues, for residues which were measurable in both the +Ca2+ and −Ca2+ states. The highlighted distances were used to calculate the averages values presented in (DOCX)Click here for additional data file.


FLT3, NPM1, CEBPA, MLL, WT1, IDH1/2, RUNX1, ASXL1, TET2, DNMT3A), EVI1 overexpression screening, and 6.0 single-nucleotide polymorphism array (SNP-A) analysis in diagnostic samples of the 278 AML patients enrolled in the ALFA-0701 trial. In cytogenetically normal (CN) AML (n = 146), 38% of the patients had at least 1 SNP-A lesion and 89% of the patients had at least 1 molecular alteration. In multivariate analysis, the independent predictors of higher cumulative incidence of relapse were unfavorable karyotype (P = 0.013) and randomization in the control arm (P = 0.007) in the whole cohort, and MLL partial tandem duplications (P = 0.014) and DNMT3A mutations (P = 0.010) in CN-AML. The independent predictors of shorter overall survival (OS) were unfavorable karyotype (P < 0.001) and SNP-A lesion(s) (P = 0.001) in the whole cohort, and SNP-A lesion(s) (P = 0.006), DNMT3A mutations (P = 0.042) and randomization in the control arm (P = 0.043) in CN-AML. Interestingly, CN-AML patients benefited preferentially more from GO treatment as compared to AML patients with abnormal cytogenetics . Although the interaction test was not statistically significant, the OS benefit associated with GO treatment appeared also more pronounced in FLT3 internal tandem duplication positive than in negative patients.We recently showed that the addition of fractionated doses of gemtuzumab ozogamicin (GO) to standard chemotherapy improves clinical outcome of acute myeloid leukemia (AML) patients. In the present study, we performed mutational analysis of 11 genes ( FLT3 internal tandem duplication (FLT3–ITD), NPM1 and CEBPA mutations. Mutations of these three genes allowed a refined prognostic classification of CN-AML and improved risk stratification in this subset of AML patients . The only independent predictors of OS we identified in the whole cohort were unfavorable cytogenetics and SNP-A lesions. Unfavorable cytogenetics was also the only independent prognostic factor for relapse risk in the whole cohort. The potential of genetic profiling to refine prognosis in CN-AML has been highlighted by many studies , 19, 20. reports –20. The  our own . Howeverenotypes , 23–26. FLT3–ITD has been recognized as a major predictor of outcome in AML, especially in the CN-AML subset [FLT3–ITD did not significantly impact on CIR and OS in univariate analysis, suggesting that treatment with GO may improve outcome of FLT3–ITD positive patients. Actually, we found that the OS benefit associated with GO treatment was more pronounced in FLT3–ITD positive than in negative patients. Interestingly, these findings are consistent with results from Jawad et al. who found that leukemic stem and progenitor cells carrying FLT3–ITD were more sensitive to GO and that FLT3–ITD status was an independent predictor of in vitro chemosensitivity to GO [FLT3–ITD mutations were found independently prognostic in older AML patients, but not predictive of response to GO. However, the cumulative dose of GO in this study was much lower than in the ALFA-0701 trial, which may account for these discordant results [We and others –15 previL subset . Neverthty to GO . HoweverFLT3–ITD positive AML. Although patients with FLT3–ITD positive AML are usually considered as candidates for allogeneic stem cell transplantation , the benefit of this high-risk procedure in first CR for those patients is still a matter of debate [FLT3 mutated AML. Several anti-FLT3 compounds are undergoing evaluation in different phases of clinical trials, as monotherapy or in combination with standard chemotherapy. Despite promising in vitro studies, clinical responses to single-agent FLT3 inhibitors in AML patients are generally incomplete and not sustained, and no significant improvement in OS has been demonstrated so far [FLT3–ITD positive AML patients, particularly in those not eligible for allo-SCT, the addition of GO to standard chemotherapy appears as an interesting treatment option, that would be worthy of further investigation in clinical trials.Overall, the current information about GO supports the efficacy of this agent in newly diagnosed AML, with acceptable toxicity , 29. In f debate –32. Durid so far , 34. TheDNMT3A mutations represent adverse prognostic factors, particularly in CN-AML. Both markers add independent prognostic information and, therefore, could contribute to improve risk stratification in patients with CN-AML. Importantly, our results suggest that addition of GO to standard chemotherapy might overcome the poor prognosis of FLT3–ITD. However, further studies based on larger patient cohorts are required to formally identify the molecular determinants of response to GO.In conclusion, our study emphasizes that SNP-A lesions and From January 2008 to November 2010, 278 patients aged 50–70 years with previously untreated primary AML were included in the randomized multicentric Phase 3 ALFA-0701 trial (NCT00927498), investigating the benefit of the addition of low fractionated doses of GO to standard front-line chemotherapy (Supplementary Data) . The stuCytogenetic R-banding analysis was performed on diagnostic bone marrow samples using standard methods. The karyotypes were described according to the International System for Human Cytogenetic Nomenclature recommendations  and clasMononuclear cells from pretreatment bone marrow or peripheral blood were isolated by Ficoll density gradient centrifugation . The blast percentage following enrichment was above 60% in all samples. Genomic DNA was extracted from mononuclear cells using the QIAamp DNA Mini Kit®  following the manufacturer’s instructions. Total RNA was extracted from the same specimen and reverse transcribed using the High-Capacity cDNA Archive Kit  and according to the standardized protocol developed within the Europe Against Cancer (EAC) program .MLL-PTD [FLT3–ITD [FLT3 tyrosine kinase domain (FLT3–TKD) (FLT3D835/I836) [NPM1 (exon 12) [CEBPA [WT1 (exons 7 and 9) [IDH1R132 [IDH2R140 [IDH2R172 [RUNX1 (exons 3–8) [ASXL1 (exon 12) [TET2 (exons 3–11) [DNMT3A  [EVI1 overexpression [TET2, IDH1/2, and DNMT3A mutations was restricted to CN-AML. Further details regarding gene mutation analysis are available in the Supplementary Data.MLL-PTD , FLT3–ITFLT3–ITD , mutatio35/I836) , NPM1 (eexon 12) , CEBPA [) [CEBPA , WT1 (ex7 and 9) , IDH1R13IDH1R132 , IDH2R14IDH2R140 , IDH2R17IDH2R172 , RUNX1 (ons 3–8) , ASXL1   and EVI1pression  were assPatient genomic DNA was processed and hybridized to Genome-Wide Human SNP 6.0 arrays  according to the manufacturer instructions. Raw data were analyzed using the Genotyping Console version 4.1 software (Affymetrix). In order to distinguish somatic from constitutional SNP array lesions, we adopted a stringent and conservative algorithm. Aberrations were excluded as known copy number variants if there was > 50% overlap with variants from the public Database of Genomic Variants (DGV Beta version 10). Interstitial UPD < 20 Mb were considered as constitutional and excluded from the analysis. Subsequently, all CNA and UPD were validated by visual inspection and annotated based on the hg19 human genome assembly.P-values are two-sided, with a nominal type I error of 0.05.Median with interquartile range and percentages were computed as summary statistics. Clinical endpoints considered for prognostic analyses were CR/CRp rate, CIR and OS. CIR and OS were calculated after censoring patients who received allo-SCT in first CR at allo-SCT time. We focused here on the prognostic influence of CC, SNP-A, and molecular alterations. First, univariate analyses for CR/CRp were based on logistic regression models with prognostic influence measured by odds ratio (OR) with 95% confidence interval (95%CI). Univariate predictive analyses for CIR and OS were based on Cox models with prognostic influence measured by hazards ratio (HR) with 95% CI. Estimation of survival curves used the Kaplan-Meier method, compared by the log-rank test , 47. Ste


However, the identity of epithelial Ca2+ channel that activates these transcriptional responses is unclear. In many other non-excitable cells, store-operated Ca2+ entry is a major route for Ca2+ influx and is mediated by STIM1 and Orai1 proteins. This study was performed to determine if store-operated Ca2+ channels were expressed in human bronchial epithelial cells and, if so, whether they coupled Ca2+ entry to gene expression. Cytoplasmic Ca2+ measurements, patch clamp recordings, RNAi knockdown and functional assays were used to identify and then investigate the role of these Ca2+ channels in activating the NFAT and c-fos pathways and EGF expression. STIM1 and Orai1 mRNA transcripts as well as proteins were robustly in epithelial cells and formed functional Ca2+ channels. Ca2+ entry through the channels activated expression of c-fos and EGF as well as an NFAT-dependent reporter gene. Store-operated Ca2+ entry was also important for epithelial cell migration in a scrape wound assay. These findings indicate that store-operated Ca2+ channels play an important role in stimulating airway epithelial cell gene expression and therefore comprise a novel potential therapeutic target for the treatment of chronic asthma and related airway disorders.Ca A common theme in chronic asthma is significant remodelling of the airway wall Airway epithelia lie at the interface between a host and its environment and thereby comprise a first line of defence against air-borne allergens. Although long considered a passive component to the remodelling process, recent work has now established that airway epithelia respond directly to environmental risk factors associated with asthma 2+ in airway epithelial cells 2+ entry is particularly important for airway epithelial cell function. Ca2+ influx is required for EGF secretion 2+ entry 2+ influx is through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane 2+ stores, as occurs following stimulation of G protein-coupled receptors or growth factor receptors that couple to phospholipase C to generate the second messenger inositol 1,4,5-trisphosphate (InsP3). The two key components of the CRAC channel pathway are the ER resident protein STIM1, which senses the amount of Ca2+ within the store 2+ entry through Orai1 activates the Ca2+-dependent transcription factor NFAT 2+ influx was present and required Orai1 expression 2+ influx through this pathway increased interleukin 8 expression. Despite its importance in airway epithelial cell remodelling, the molecular identity of the Ca2+ influx pathway that activates expression of EGF and other signalling molecules is not clear. Here, we show that store-operated CRAC channels are present and functional in human airway epithelial cells. Ca2+ entry through these channels stimulates gene expression including transcription of EGF. We also show that the channels are regulated by cold, a common pre-disposing factor in asthma The house dust mite allergen and physiological triggers including histamine increase cytoplasmic Ca2+ entry in the human bronchial epithelial cell line (16HBE) by stimulating cells with the sarcoplasmic/endoplasmic reticulum Ca2+ATPase inhibitor thapsigargin (2 µM) in Ca2+-free external solution 2+ uptake into the stores, thapsigargin unmasks a Ca2+ leakage pathway that gradually leads to Ca2+ store depletion. Once Ca2+ release to thapsigargin had terminated, we readmitted Ca2+ to the external solution. A rapid rise in cytoplasmic Ca2+ occurred, indicating the presence of store-operated influx  in the extracellular loop between transmembrane domains I and II, which are close to the glutamate residue (E106) that confers Ca2+ selectivity 2+ entry in 16HBE cells and immune cells by La3+ would therefore reinforce the view that the Ca2+ entry pathway activated by thapsigargin in 16HBE cells is indeed the CRAC channel. We therefore compared the concentration dependence of block of Ca2+ influx by La3+ between 16HBE and RBL-1 cells, the latter being a model CRAC channel system. La3+ inhibited store-operated Ca2+ influx in RBL-1 cells in a dose-dependent manner  and 10 mM EGTA activated a Ca2+ current with an identical current-voltage relationship, but the current activated more quickly . The mean size of the current (measured at −80 mV) was ∼-0.8 pA/pF  was sufficient to induce robust c-fos transcription 40 minutes later (2+-dependent NFAT (nuclear factor of activated T cells) family of transcription factors. RT-PCR studies demonstrated the presence of NFAT1 and NFAT4 in 16HBE cells  in order to dissect out more clearly the impact of the modest increase in Ca2+ entry rate from the effects of a prolonged cytoplasmic Ca2+ rise that occurs when responses are measured after several minutes stimulation.The accelerated rate of Cacription . In thisCRAC channels have been shown to contribute to migration of vascular myocytes in a scratch wound migration assay 2+ influx stimulates both rapid and long-lasting responses in epithelial cell function, very little is known about the identity of the Ca2+ entry pathways present. In this study, we show that store-operated CRAC channels are functionally expressed in airway epithelia where they stimulate EGF gene expression and are required for cell migration. We also find that CRAC channel activity increases following exposure to cold air, an environmental factor that contributes to airway inflammation.Although Ca2+ influx was present in the epithelia and several lines of evidence suggest the underlying channels are largely indistinguishable from the well-characterised CRAC channels in mast cells, T lymphocytes and related cell lines 2+ current that was activated by store depletion, was non-voltage activated, showed strong inward rectification, was Ca2+-selective, exhibited Ca2+-dependent fast inactivation that had a similar voltage-dependence to that seen in mast cells, and was inhibited by the CRAC channel blocker Synta66 2+ flux measurements identified permeability to Ca2+, Sr2+ and Ba2+, as is the case for CRAC channels 3+ inhibited store-operated Ca2+ influx in airway epithelia and RBL-1 mast cells with similar IC50 and Hill coefficients. Fourthly, structurally distinct small molecule inhibitors of CRAC channels in immune cells also blocked Ca2+ influx in airway epithelia. Finally, siRNA directed against the two key CRAC channels components, STIM1 and Orai1, reduced protein expression and Ca2+ influx to similar extents.Orai1 was strongly expressed in the airway epithelial cells, both at mRNA transcript and protein levels. Orai3 mRNA was also clearly resolvable, although to a lesser extent than Orai1. Both STIM1 and STIM2 proteins were also expressed and at similar levels. Store-operated Ca2+ influx through the channels activated both c-fos and NFAT transcription factors as well as NFAT-driven gene expression. Ca2+ entry through CRAC channels also increased transcription of EGF, which plays an important role in epithelial cell repair and regeneration + channel blockers have also been found to impair EGF-driven epithelial repair 2+ influx through CRAC channels that arises when the membrane potential is depolarised after K+ channel inhibition.We also found that activation of CRAC channels had important consequences on epithelial cell function. Ca2+ entry did not involve increased protein expression. Heating cells to >35°C leads to clustering of STIM1 proteins at endoplasmic reticulum-plasma membrane junctions and results in transient Ca2+ entry through Orai1 upon cooling 2+ entry in epithelial cells, suggesting reduced temperature might impair an inhibitory pathway. Regardless of the mechanism, our findings reveal that a pre-disposing factor linked to asthma susceptibility increases Ca2+ influx and thereby accelerates transcription of c-fos, a regulator of chemokine and cytokine production. CRAC channels might therefore operate in parallel with cold-sensing TRPM8 channels that have been found to trigger mucin hypersecretion in human bronchial epithelia Exposure to cold air is a pre-disposing factor to the development of asthma and other airway diseases. CRAC channel activity increased ∼1.5 fold after transient pre-exposure to cold 2+ entry into airway epithelia that drives expression of genes involved in the remodelling mechanism. Targeting CRAC channels therefore might provide a new therapeutic approach for managing chronic airway inflammatory disorders including asthma and chronic obstructive pulmonary disease.Collectively, our results identify CRAC channels as an important route for Ca2) in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 2 mm l-glutamine, and penicillin/streptomycin.The human bronchial airway epithelial cell line 16HBE was a kind gift from Dr Lin-Pei Ho . Cells were cultured , using the manufacturer's instructions. Transfection efficiency was ∼60%, judged from the fraction of GFP positive cells obtained 48 h post transfection.2, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4 with NaOH, as described 2+-free solution contained 145 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, 0.1 mM EGTA, pH 7.4 with NaOH. Cytoplasmic Ca2+ imaging experiments were carried out using a TILL Photonics system with an IMAGO CCD camera. Cells were alternately excited at 356 and 380 nm, and images were acquired every 2 seconds. Images were analyzed off line using IGOR Pro for Windows. Ca2+ signals are represented at the 356/380 nm ratio.Cells were loaded with Fura-2/AM (1 mM) for 40 minutes at room temperature in the dark and then washed three times with a standard external solution composed of 145 mM NaCl, 2.8 mM KCl, 2 mM CaCl2 1, Ethylene glycol-bis(b-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid (EGTA) 10, HEPES 10, Mg-ATP 2, pH 7.2 with CsOH. Bath solution contained (in mM): NaCl 135, KCl 2.8, CsCl 10, CaCl2 10, MgCl2 2, HEPES 10, D-glucose 10, pH 7.4 with NaOH. The CRAC current was measured by applying voltage ramps (−100 to +100 mV in 50 msec) at 0.5 Hz from a holding potential of 0 mV. For fast inactivation, step pulses (250 msec duration) were applied from 0 mV to −100 mV every 2 seconds. Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitised at 100 ms. Fast inactivation was determined by dividing the steady state current during the hyperpolarising pulse (measured after 240 ms) by the initial current (measured after 1 ms). Capacitative currents were compensated before each ramp by using the automatic compensation of the EPC 9 -2 amplifier. Leak currents were subtracted by averaging 2-3 ramp currents obtained just before ICRAC had started to develop, and then subtracting this from all subsequent currents.Whole cell patch clamp recordings were carried out as previously described 21. Membranes were blocked with 5% non-fat dry milk in TBS plus 0.1% Tween 20 (TBST) buffer for 1 hour at room temperature. Membranes were washed with TBST three times and then incubated with primary antibody overnight at 4°C. Anti-STIM1 and –STIM2 antibodies were obtained from Cell Signalling Technology and used at 1∶1000 dilutions. Orai1 and ERK2 antibodies were from Santa Cruz Biotechnology and were used at a dilution of 1∶500 and 1∶5000, respectively. The membranes were then washed with TBST again and incubated with a 1∶2500 dilution of goat anti-rabbit secondary antibody IgG from Santa Cruz Biotechnology for 1 h at room temperature. After washing with TBST, the bands were developed for visualization using ECL-plus Western blotting detection system . Gels were quantified using the UN-SCAN-IT software package (Silk Scientific).Total cell lysates (50 µg) were separated by SDS-PAGE on a 10% gel and electrophoretically transferred to nitrocellulose membrane, as described2+-free external solution (containing 0.1 mM EGTA) without thapsigargin. After a further 40 minutes (at room temperature.), total RNA was extracted by using an RNeasy Mini Kit (Qiagen), as described. RNA was quantified spectrophotometrically by absorbance at 260 nm. Total RNA (1 µg) was reverse-transcribed using the iScriptTM cDNA Synthesis Kit (Bio-Rad), according to the manufacturer's instructions. Following cDNA synthesis, PCR amplification was then performed using BIOX-ACTTM. ShortDNAPolymerase (Bioline) with primers specific for the detection of c-fos , EGF , NFAT1 , NFAT4 , ORAI1 , ORAI2 , ORAI3 , STIM1  and β-actin  were synthesized by Invitrogen. The PCR products were electrophoresed through an agarose gel and visualized by ethidium bromide staining.16HBE cells were stimulated with 2 µM thapsigargin for 10 minutes (for c-fos measurements) or 1 hour (for EGF measurements) at room temperature in standard external solution. Thereafter, cells were washed with Ca22. Gene expression was defined as fluorescence 3xSD> cell autofluorescence, measured in non-transfected cells. Cells were stimulated in culture medium and maintained in the incubator for ∼24 hours prior to detection of EGFP.24–36 hours following transfection with an EGFP-based reporter plasmid that contained an NFAT promoter , cells were stimulated with thapsigargin (100 nM) and the number of cells expressing EGFP measured subsequently (∼24 hours later), as described2+ imaging experiments. For RT-PCR experiments, the cells were exposed to 2 µM thapsigargin in Ca2+-free external solution for 7 minutes and then 2 mM Ca2+ was readmitted for 1 minute. Thereafter, cells were washed with Ca2+-free external solution without thapsigargin. After a further 40 minutes (at room temperature), total RNA was extracted as described above.16HBE cells plated on coverslips were removed from the incubator (37°C) and kept at 15°C for 2 hours. They were again returned to the incubator for either 30 min or overnight before loading with Fura 2-AM for Ca16HBE cells were allowed to form a monolayer for 48 h. A 100-µl pipette tip was then used to scrape across the culture dish, which was then washed three times with standard culture medium and then returned to the incubator. Bright-field images were captured 5 minutes, 4 hours and 16 hours later, using a Nikon microscope at x40. The images were analyzed using ImageJ and the total number of cells inside the wound were compared with experiments in which Synta66 (10 µM) had been added immediately after wound formation. The perimeter of the wound was marked on the outward facing base of the dish for identification purposes.Results are presented as mean±sem. Data were compared using Student's t test or by analysis of variance (ANOVA) for multiple groups. Differences were considered statistically significant at values of p<0.05.


Somatic exocytosis of serotonin from dense core vesicles could be triggered by a train of 10 action potentials at 20 Hz in Retzius neurons of the leech. However, the same number of action potentials produced at 1 Hz failed to evoke any exocytosis. The 20-Hz train evoked exocytosis through a sequence of intracellular Ca2+ transients, with each transient having a different origin, timing and intracellular distribution. Upon electrical stimulation, transmembrane Ca2+ entry through L-type channels activated Ca2+-induced Ca2+ release. A resulting fast Ca2+ transient evoked an early exocytosis of serotonin from sparse vesicles resting close to the plasma membrane. This Ca2+ transient also triggered the transport of distant clusters of vesicles toward the plasma membrane. Upon exocytosis, the released serotonin activated autoreceptors coupled to phospholipase C, which in turn produced an intracellular Ca2+ increase in the submembrane shell. This localized Ca2+ increase evoked new exocytosis as the vesicles in the clusters arrived gradually at the plasma membrane. In this way, the extracellular serotonin elevated the intracellular Ca2+ and this Ca2+ evoked more exocytosis. The resulting positive feedback loop maintained exocytosis for the following hundreds of seconds until the last vesicles in the clusters fused. Since somatic exocytosis displays similar kinetics in neurons releasing different types of transmitters, the data presented here contributes to understand the cellular basis of paracrine neurotransmission.The soma of many neurons releases large amounts of transmitter molecules through an exocytosis process that continues for hundreds of seconds after the end of the triggering stimulus. Transmitters released in this way modulate the activity of neurons, glia and blood vessels over vast volumes of the nervous system. Here we studied how somatic exocytosis is maintained for such long periods in the absence of electrical stimulation and transmembrane Ca Experiments were performed at 18°C after 1–8 days in culture.Imaging experiments were performed in cultured Retzius neurons of leeches Electrical stimulation consisted of trains of 10 action potentials produced by 10-ms current pulses delivered at 1 or 20 Hz  produced by the progressive exo/endocytosis of dense core vesicles in the clusters . An advantage of these Fluo dyes is that both can be excited with a single wavelength and therefore allow a fast (100 ms/image) imaging rate and /or long acquisition periods when compared with ratiometric dyes, thus reducing bleaching and neuron damage. This was particularly critical for our experiments when imaging simultaneously Ca2+ and exocytosis, since two images were acquired for each time point over several minutes. To calibrate the Ca2+ concentrations we also tried Fura-2 imaging. However, the high sampling speed necessary to detect the fast Ca2+ transient required to double the imaging rates and neurons in these conditions were damaged rapidly by the Fura-2 excitation and emission wavelengths. For this reason we sacrificed the concentration measurements to correlate the precise kinetics of the Ca2+ transients with those of exocytosis. Dyes had to be injected by iontophoresis since Retzius neurons do not retain esterase-coupled dyes. For this, the dyes were dissolved in water at a concentration of 5 mM and were injected into the soma by a 4 nA hyperpolarizing current for 4-6 min using borosilicate microelectrodes. The electrode tip was back-filled with the dye solution and the microelectrode was then filled with 0.1 M KCl. Imaging was made 10–15 min after the dye had been loaded.Relative changes in the intracellular Ca2+ spikes in response to individual action potentials with both Fluo dyes, the low affinity (2.5 μM) Fluo-5F could not detect the small Ca2+ transient described in the results sections. For this reason most of the experiments reported here were made by using the higher affinity (Kd = 345 nM) Fluo-4, with which we consistently detected the small Ca2+ transient. A major concern of the use of high affinity dyes for our purposes is the possibility that dye saturation distorts the kinetics of the Ca2+ dynamics and exocytosis. However, that the kinetics of the fast Ca2+ transient obtained with either Fluo dye were identical suggested that Fluo-4 was not saturated under our experimental conditions. This was further tested by the addition of ionomycin (5 μM) to neurons injected with high affinity dye after recording the transient evoked by 20 Hz stimulation. Ionomycin produced fluorescence increases to levels high above the peak fluorescence of the Ca2+ transients. In addition, neurons that became damaged during the experiments had similar fluorescence increases. These evidences indicate that the somatic Ca2+ increases in response to electrical stimulation were not produced under dye-saturation conditions.Although we detected Ca2+ kinetics due to buffering by Fluo-4 did not affect significantly our results, since such distortions occur within miliseconds . Fluorescence measurements of FM1-43 or Fluo-4 were performed with excitation and emission wavelengths at 488 and 535 nm respectively. A cooled CCD camera  acquired image sequences of 640 × 480 pixels. To study fast Fluo-4 fluorescence transients in response to the stimulation trains, images were acquired every 100 ms for 60 s. To follow FM styryl dye changes or the long-lasting Fluo-4 transients, separate images were acquired every 2 s for 15 min. The image sequences were stored digitally by using TILLvisION software.2+ signals, confocal imaging of FM4-64 and Fluo-4 fluorescence was carried out using an Olympus Fluoview FV1000 upright confocal scanning microscope using 473 nm for Fluo-4 excitation and 560 nm for FM4-64 excitation. For the simultaneous imaging fluorescence was acquired with a 60X water immersion objective (1.1 NA). Fluorescence was detected in parallel by a spectral-based detector capturing 503–543 nm for the Fluo-4 emission and 600–700 nm for the FM4-64 emission. Time-lapse sequences were made by acquiring an image every 2 s for 20 min. Images were stored digitally by using Fluoview 3.1 software (Olympus).For simultaneous recordings of exocytosis and Ca2 regions of interest (ROIs) that contained a fluorescent spot. These areas were chosen because they correspond to the average area of a vesicle cluster  was subtracted from the intensity of that ROI at each time [F(t)]. The difference was divided by F0, to generate ΔF/F0. For simplicity, throughout the text and figures this normalization is referred to as dF/F. Surface plots were made by using an RGB 8 bit color scale calibrated in the dynamic interval. Plotting and curve fitting were performed using Igor Pro 6.2 software (Wavemetrics). Data are presented as mean ± s.e.m. Statistical comparisons were performed using unpaired two tailed Student's t-test.We used Image-J software  for most of the analysis. The time series of images were aligned with the StackReg plug-in. The mean fluorescence intensity was measured along time or space sequences from 1.8 to 2 μm2+ channels we used nimodipine ; to eliminate Ca2+-induced Ca2+ release we used a combination of ryanodine  and thapsigargin . 5-HT receptors were blocked with methysergide  and to block PLC neurons were preincubated for 30 min with U-73122 . All drugs were applied to the bathing fluid from 500X stock solutions 15 min before images were taken, except for U-73122, which was added 30 min before.To block L-type CaThe general procedures were as in Trueta et al. . In brieElectrical stimulation of individual neurons with a 20-Hz train Figure , left la2+ contributes to this large-scale somatic exocytosis, the kinetics of exocytosis and intracellular Ca2+ were measured simultaneously by imaging the fluorescence of FM4-64 and Fluo-4 dyes. A major concern of these measurements is that high affinity Ca2+ sensitive dyes may affect the intracellular free Ca2+ concentration and therefore exocytosis. However, as shown in Figure 2+ sensitive dyes to measure Ca2+ dynamics and exocytosis simultaneously. Moreover, as will be seen below, there was also good correlation between the kinetics of exocytosis and intracellular Ca2+ in pharmacological experiments.To explore how Ca2+ . The second Ca2+ transient was small and purely submembrane . This small Ca2+ transient appeared between the peak of the large Ca2+ transient and the large-scale exocytosis . The large transient was also restricted to the submembrane shell  with a time constant of 2.0 ± 0.2 s  in every neuron tested, where it also peaked by 600 ms , again with the resolution limited by our 100 ms/image sampling rate. The temporal summation of the subsequent Ca2+ spikes produced that the maximal Fluo-4 fluorescence value  was reached after the last impulse of the train. The maximum fluorescence amplitude was only 33% of that of the fast Ca2+ transient upon 20-Hz stimulation. After the last spike of the 1 Hz train, fluorescence decayed exponentially with a time constant of 1.7 ± 0.3 s. This decay time was similar (p = 0.32) to that of the fast Ca2+ transient upon 20-Hz stimulation. At the soma center the maximal amplitude of the transient evoked by 1-Hz stimulation was 72% of that in the periphery.The Ca2+ transient evoked by 20-Hz stimulation was 67% larger than that obtained at 1-Hz  what triggers exocytosis. Moreover, the amplitude of the transient evoked by 20-Hz stimulation was 38.5% larger than the sum of the peak amplitudes of the 10 Ca2+ spikes evoked by a 1-Hz train. Since transmembrane Ca2+ entry upon subsequent voltage steps does not facilitate in the soma of these neurons  substituted Ca2+ in the external solution (not shown) to block transmembrane Ca2+ entry , or after 20-Hz stimulation when Ca2+ had been replaced by magnesium in the external solution to prevent transmembrane Ca2+ entry .The pharmacological dissection of the components of the fast Ca2+ source contributing to the fast transient was Ca2+-induced Ca2+ release. Incubation of six neurons with 100 μM ryanodine and 1.6 μM thapsigargin reduced the amplitude of the fast Ca2+ transient by 41 ± 5.1% . The gray sigmoidal function in Figure 2+ transient. On the other hand data show that different Ca2+ sources cooperate to determine the number of vesicle clusters arriving at the plasma membrane.As predicted before, the third Ca Figures . Ca2+-ine Figure  in our pe Figure  reduced 2+ transient determines the amount of vesicle clusters undergoing exocytosis came from how far the fast Ca2+ transient penetrates the cytoplasm. This is shown by plotting the average amplitude of the fast Ca2+ transients in different experimental conditions over the radial intracellular distance from the plasma membrane . Figure 2+ transients. However, they were not produced by a transmembrane Ca2+ flow, since our voltage clamp records during exocytosis lacked any transmembrane currents , an unspecific blocker of metabotropic 5-HT inverterbrate receptors. Methysergide abolished both submembrane Ca2+ transients without affecting the fast Ca2+ transient in the six neurons stimulated at 20 Hz   and intracellular Ca2+ release  this manipulation abolished both submembrane Ca2+ transients and exocytosis upon 20-Hz stimulation. However, the fast Ca2+ transient remained intact . The cyclic shape of the plots shown in Figure 2+ entry and exocytosis. The feedback loop operates during the on-state, when the released 5-HT produces the submembrane Ca2+ transients and these transients evoke more exocytosis. The intracellular Ca2+ concentration declines when exocytosis fades out, and the feedback loop ends upon exocytosis from the last vesicles in the cluster  to produce a robust response that may persist over long periods in the absence of any further stimulation  have made the following declarations about their contributions: Carolina Leon-Pinzon and Francisco F. De-Miguel conceived and designed the experiments. Carolina Leon-Pinzon, Montserrat G. Cercós, Paula Noguez and FFM performed the experiments. Carolina Leon-Pinzon, Montserrat G. Cercós, Citlali Trueta and Francisco F. De-Miguel analyzed the data. Francisco F. De-Miguel and Citlali Trueta contributed reagents/materials/analysis tools. Francisco F. De-Miguel and Carolina Leon-Pinzon wrote the paper.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.