Our structural analysis revealed that there was no correlation between position of GC soma in the GC layer and location of its PF in the ML, confirming that our AAV-mediated labeling was important to test the projection-dependent variability of the Ca2+ responses in GCs. We then found that the Ca2+ responses of D-GCs differed from those of M-GCs. Pharmacological experiments implied that the different Ca2+ responses were mainly attributable to varied distributions of GABAA receptors (GABAARs) at the synaptic and extrasynaptic regions of GC dendrites. In addition to GABAAR distributions, amounts of extrasynaptic NMDA receptors appear to be also varied, because Ca2+ responses were different between D-GCs and M-GCs when glutamate spillover was enhanced. Whereas the Ca2+ responses of S-GCs were mostly equivalent to those of D-GCs and M-GCs, the blockade of GABA uptake resulted in larger Ca2+ responses in S-GCs compared with D-GCs and M-GCs, implying existence of mechanisms leading to more excitability in S-GCs with increased GABA release. Thus, this study reveals MF stimulation-mediated non-uniform Ca2+ responses in the cerebellar GCs associated with the location of their PFs in the ML, and raises a possibility that combination of inherent functional variability of GCs and their specific axonal projection contributes to the information processing through the GCs.Cerebellar granule cells (GCs) relay mossy fiber (MF) inputs to Purkinje cell dendrites via their axons, the parallel fibers (PFs), which are individually located at a given sublayer of the molecular layer (ML). Although a certain degree of heterogeneity among GCs has been recently reported, variability of GC responses to MF inputs has never been associated with their most notable structural variability, location of their projecting PFs in the ML. Here, we utilize an adeno-associated virus (AAV)-mediated labeling technique that enables us to categorize GCs according to the location of their PFs, and compare the CaThe online version contains supplementary material available at 10.1186/s13041-021-00773-y. Many studies have finely classified cortical neurons into larger number of neuronal types than traditionally identified –5. SpeciThe cerebellar cortex traditionally includes four major types of neurons. Among them, granule cells (GCs) are the only excitatory neurons and convey inputs from mossy fibers (MFs) to Purkinje cells, which are the sole output neurons of the cerebellar cortex , 11. MFsThe cerebellum has been characterized by a uniform cytoarchitecture and a conserved network structure. This perspective is gradually changing by the findings of non-uniform microcircuits and heterogeneous neuronal properties in tandem with the technological advances , 20. Cer2+) concentration upon the MF stimulation among the three groups of many GCs by taking advantage of the acquisition speed and spatial resolution of the widefield fluorescence microscopy. We detected significant differences in the Ca2+ responses of GCs depending on the locations of their PFs, and further obtained results leading to a conclusion that the differences in receptor distributions appear to be responsible for the varied Ca2+ responses in the three groups of GCs. Thus, in addition to the morphological or functional differences of PFs themselves, the GCs’ responsiveness to MF inputs also differs according to the location of their PFs in the ML.In this study, we took advantage of a technique using an adeno-associated viral (AAV) vector that enables distinct labeling of GCs, whose PFs are located in the deep, middle, or superficial sublayer of the ML. We then compared the increase in intracellular calcium (CaAR) α6 subunit (GABRα6) promoter. Stereotaxic injection of AAV-GABRα6 at a proper timing during postnatal development is capable of triggering molecular expression in a group of GCs, whose PFs are bundled together at a certain sublayer of the ML [2+ signals from as many GCs as possible by utilizing widefield Ca2+ imaging. Widefield fluorescence imaging generally affords us both the high spatial resolution and the high-speed imaging, which potentially allow us to identify many GC somas and to accurately capture the time lapse profile of the Ca2+ signals in each GC soma. The widefield Ca2+ imaging combined with labeling by AAV-GABRα6 is therefore capable of simultaneous recording from many GCs and of direct comparison of responses in different groups of GCs. Ca2+ indicator dye, Oregon Green 488 BAPTA-1 (OGB1), was loaded in the GCL of fresh cerebellar sagittal slices that were obtained from mice subjected to the AAV injection , middle (M-GCs) or superficial (S-GCs) sublayer of the ML, we utilized the labeling method using AAV vectors with the minimum region of γ-aminobutyric acid (GABA) type A receptor  of one side of GCL by utilizing red fluorescence images, which are presumably composed of not only dT fluorescence but also autofluorescence. To estimate ratios of GCs containing genuine dT fluorescence among all GCs, fixed slices were obtained from mice subjected to the injection of AAV-GABRα6 expressing GFP at P7, P9, or P13, and were stained with an antibody of Kv4.2 voltage-gated potassium channels, a marker of GC membranes. We then found that approximately 25–35% of GCs were GFP positive  and other GCs in individual slices. As seen in Fig. 0  confirmed no significant difference between the comparisons performed using the standardized ∫ΔF/F0∙dt . We detected no significant difference in the analysis for D-GCs  of randomly sampled pairs of the dT-positive GCs and the other GCs  on the Ca2+ responses of the GCs. Although we sometimes reduced the intensity of electrical MF stimulation to avoid saturating the Ca2+ responses, overall Ca2+ increase (∫ΔF/F0∙dt) was higher in the presence of bicuculline, a GABAAR antagonist, than in control . Consistently, time to peak and decay time constants of the Ca2+ responses are equivalent between each group of GCs and other GCs . For these experiments, AAV-GABRα6-dT and AAV-GABRα6-mTB were injected into lobe IV/V of the cerebellum at two different time points. The injection at P7 and P9, P9 and P13, or P7 and P13 resulted in the labeling of D-GCs and M-GCs, M-GCs and S-GCs, or D-GCs and S-GCs, respectively . Consequently, the positive value obtained by this calculation denotes a larger Ca2+ response in the former group than the latter group of GCs. Thus, this analysis allowed us to make direct pairwise comparisons of the Ca2+ responses among the three groups of GCs. In control, the Ca2+ responses in M-GCs were larger than D-GCs, whereas the Ca2+ responses in S-GCs were equivalent to those in M-GCs and D-GCs  antagonist, CGP35348 (CGP), together with two NMDAR antagonists, 7-Chlorokynurenic acid (7Cl) and AP5. The differences in the Ca2+ responses were negligible in all comparisons . Schematic diagram of excitatory and inhibitory receptor components considered in this study are shown in Fig. 2+ responses were larger in M-GCs than D-GCs in control -SNAP 5114 (SNAP) and SKF 89976A (SKF), inhibitors of GABA transporter type 3 and 1, respectively, that prevent GABA reuptake to allow stronger activation of extrasynaptic GABAARs. The combination of GABA uptake inhibitors made the Ca2+ responses in M-GCs smaller than D-GCs, as seen in the significantly negative value of the difference in the Ca2+ responses , a glutamate transporter inhibitor. Application of DL-TBOA could lead to strong activation of extrasynaptic NMDARs, and therefore was expected to result in larger difference in the CaBRs on MF terminals and Golgi cells, it has been shown that the postsynaptic GABABRs are also present on GCs [BRs on the difference of Ca2+ responses between M-GCs and D-GCs by applying bicuculline, AP5 and 7Cl, which block GABAARs and NMDARs. In this condition, the Ca2+ increase in M-GCs was equivalent to that in D-GCs  balance in the GCL. A recent study reported that acetylcholine regulates the balance of synaptic excitation and inhibition in the GCL and consequently GC excitability [In the control condition of our experiments, D-GCs were less active compared to M-GCs. When the GABAtability . Anatomitability , 50. Contability , some tytability –57, it wtability . This letability , making 2+ imaging in GCs has demonstrated dense GC activation in behaving animals [Based on the massive number of GCs present, with each having only 3–4 synaptic contacts with MFs, the cerebellar network in the GCL has been theoretically considered to be suitable for pattern separation via sparse GC activity , 60. In  animals –63, cont animals , our res animals . In addi animals  and hete animals . CombinaAll procedures involving mice were performed according to the guidelines of the Institutional Animal Care and Use Committee of Korea Institute of Science and Technology. Because of their nursing ability, ICR mice of both sexes were used in this study.13 vector genome copies per mL were produced as described previously [AAV vectors with estimated titers of approximately 10eviously . AAV coneviously . The cDN2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose. To load the cerebellar slices with OGB1, a Ca2+ indicator dye, slices were placed in the recording chamber, a glass pipette connected to pneumatic microinjector  was filled with OGB1 acetoxymethyl ester , lowered ~ 40 um into the GCL of the cerebellar slices, and then a bolus of OGB1-AM was ejected into the cerebellar slice at 10 psi for 5 min to locally label the GCL. Before imaging the slices, slices were incubated in the recording chamber for 30 min to allow continuous perfusion by extracellular ACSF to wash away the excess OGB1-AM, leaving only the internalized OGB1.Chemicals used were obtained from Sigma or Wako Pure Chemical Industries, unless otherwise specified. Slices were prepared as previously described . SagittaLive cell imaging of OGB1 was performed using a microscope (Nikon Eclipse FN1) equipped with a high N.A. water immersion objective CFI75 LWD 16X W, an iXon Ultra 897 camera (Andor) and NIS elements imaging software (Nikon). Cropped images  of the GCLs were taken at 220 Hz, before, during, and after the MF burst stimulation . A concentric bipolar electrode (FHC Inc.) was placed on the white matter to apply the MF stimulation. The timing of the MF stimulation was controlled by the trigger-out feature of NIS elements software.2+ responses in the GCs, several inhibitors were added to the extracellular ACSF before and during the imaging. Inhibitors used were bicuculline , AP5 , CNQX , 7Cl , CGP , DL-TBOA , SNAP , and SKF . All inhibitors were obtained from Tocris Bioscience.To tease apart the individual contribution of ionotropic and metabotropic receptors at the MF-GC synapse on the observed Cabaseline) change over time, a part of smoothed time course before and after MF stimulation-dependent Ca2+ increase was fitted with an exponential decay curve /Fbaseline) × 100. The integration of fluorescence increases upon MF stimulation (∫ΔF/F0∙dt) was calculated to quantify the total Ca2+ increase over time. The resultant values of ∫ΔF/F0∙dt represent the Ca2+ responses in individual GCs. However, each slice would have different distribution of the Ca2+ responses due to different labeling conditions, stimulation efficacy, or addition of inhibitors. To standardize the slice to slice variability, a z-score of the Ca2+ responses (standardized ∫ΔF/F0∙dt) was calculated by the equation (∫ΔF/F0∙dt − μ) / σ, in which μ and σ are a sample mean and a sample standard deviation of ∫ΔF/F0∙dt of all GCs detected in a single slice, respectively. The z-score of each GC was thus basically regarded as its Ca2+ response, and the “Ca2+ responses” in figures indicate the standardized ∫ΔF/F0∙dt (z-score), unless otherwise specified. To estimate the effect size of difference in Ca2+ responses between different GC groups, averaged differences (a) and the averages of total Ca2+ increase (b) are also calculated by using the unstandardized ∫ΔF/F0∙dt, and their ratio (a/b × 100) was used as the estimation  and decay time constant obtained from a single exponential fit. We made a macro in OriginPro software (OriginLab) for the process from smoothing to obtaining the values of ∫ΔF/F0∙dt to automate the analysis.Individual GCs were selected from the dT or mTB images by the spot detection function of NIS Elements. The GCs with the 20 highest dT or mTB intensity were considered dT- or mTB-positive GCs. For the experiments using a single AAV-GABRα6-dT injection, we first identified 100 GCs detectable by their dT intensity, which presumably include both dT fluorescence and autofluorescence, and the GCs with the 20 . The subtracted z-scores thus show the difference in Ca2+ responses between the two groups of GCs. For Additional file 0∙dt and unstandardized peak ΔF/F0 were used to calculate differences in the Ca2+ responses. To minimize the influence of noise and to omit the results obtained from the unhealthy slices, fluorescent intensity was also measured from the whole GCL, and results were discarded if the peak ΔF/F0 of the whole GCL was less than 0.7.To compare the Ca2+ responses while minimizing the contaminating signals from out-of-focus GCs, we manually combed the images of OGB1 and dT, and selected pairs of neighboring dT-positive GCs and other GCs, both of which were in focus, among GCs identified by the spot detection function of NIS Elements. We then calculated the difference in their ∫ΔF/F0∙dt, and tested the significance of the differences obtained from several pairs.To compare the Ca2+ responses between randomly picked certain numbers of GC pairs by the following procedure:2+ responses (∫ΔF/F0∙dt) was then calculated in all pairs.A number (n = 1–17) of GC pairs of dT-positive GCs and dT-negative GCs were randomly selected in a single slice, and the difference in Ca2+ responses from N numbers (N = GC pairs sampled per slice (n) × number of slices) of pairs were obtained, and their average is plotted as a single circle in Additional file Such random selection and calculation were performed in all the slices. As a result, differences in CaThis procedure was repeated 20 times with a certain n, so that 20 different data are plotted for each N.We further performed statistical tests for the specific cases of n = 1 or 17 pairs sampled per slice  and Alexa Fluor 647-conjugated anti-mouse IgG , respectively. Mice subjected to AAV-GABRα6-GFP injection were anesthetized at P21–P23, and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (pH 7.4). The cerebellum was post-fixed with 2% PFA and sagittally sectioned (40 µm) using a vibrating microtome (Leica VT 1200S). Immunohistochemistry was performed by following a protocol described previously . Single Numbers of mice used for the analyses were 3 or 4 per injection time point. For the image analyses, all GC somas, including both GFP labeled and unlabeled GC somas, were detected by utilizing the spot detection function of NIS Elements on the images of GCLs stained with a Kv4.2 antibody, which was used to visualize the GC membranes. The analyses of the GCL were performed on 2 highly magnified images (0.21 × 0.21 mm) per mouse, and their average was taken as representation of each mouse. GCs were determined to be GFP-positive if more than 80% of the detected somas were labeled with GFP. The percentages of GFP-positive GC somas among all GCs were calculated to estimate the expression percentages. The positional information of GFP-labeled GC somas along anterior–posterior axis was used for the analysis of GC soma distribution. To estimate the location of GFP-positive PF bundles, line scan profiles across the Purkinje cell layer and the ML were taken at 5 different areas per mouse, and their average was taken to be the representation of each mouse.2+ responses in whole GCL, by the KS test for the comparison of cumulative distributions, by one-way ANOVA with the Fisher test for the comparison of time to peak and decay time constants in GC groups, and by Mann–Whitney test for the comparison of Ca2+ responses between the presence and absence of bicuculline. Under the hypothesis that the Ca2+ responses in two GC groups are equivalent, the difference in their Ca2+ responses is supposed to be equal to 0. To test whether the Ca2+ responses were different in two GC groups, we therefore used the one-sample Wilcoxon signed rank test and compared the differences in Ca2+ responses with test median 0. To test whether the inhibitors significantly affect the difference in Ca2+ responses, or the Ca2+ responses themselves, we also performed a one-way ANOVA, followed by the post-hoc Fisher test. In addition, a two-way ANOVA was used to compare group data, such as comparison between data calculated using the standardized ∫ΔF/F0∙dt and the standardized peak ΔF/F0. Data are presented as mean ± SEM. Image analyses for Ca2+ responses were performed using NIS Elements and OriginPro software, and those for immunohistochemical images were performed using NIS Elements and ImageJ  software. The statistical analyses were performed using OriginPro software. Exact p values for the datasets in this study are provided in Additional file All sample sizes applied to the statistical test are indicated in the figure legends. Statistical differences were determined by the paired sample t-test for examining the effects of AP5 and CNQX on CaAdditional file 1: Fig. S1. Consistent results of the difference between the dT-positive GCs and the other GCs in analyses using the unstandardized ∫ΔF/F0∙dt or the unstandardized peak ΔF/F0. Fig. S2. Analysis of neighboring GC pairs and the effects of sample numbers. Fig. 3. Time course profiles of the Ca2+ responses in the presence of bicuculline and analyses with 60 other GCs. Fig. S4. Comparison of decay time constants. Fig. S5. The flow chart of how to calculate the Ca2+ responses in individual GCs. Table S1. Estimation of the effect size based on the calculation of percentage of the difference in ∫ΔF/F0•dt between two GC groups in averaged total ∫ΔF/F0•dt upon MF stimulation. Table S2.P values and statistical tests


Since ANXA2 regulation is not well understood, further research to determine the coagulopathy-initiating events in AML and APL is indicated. Moreover, ANXA2 and PDPN MFC assessment as a tool to determine the risk of life-threatening DIC in AML and APL patients should be evaluated.Patients with acute promyelocytic leukemia (APL) often present with potentially life-threatening hemorrhagic diathesis. The underlying pathomechanisms of APL-associated coagulopathy are complex. However, two pathways considered to be APL-specific had been identified: 1) annexin A2 (ANXA2)-associated hyperfibrinolysis and 2) podoplanin (PDPN)-mediated platelet activation and aggregation. In contrast, since disseminated intravascular coagulation (DIC) is far less frequent in patients with non-APL acute myeloid leukemia (AML), the pathophysiology of AML-associated hemorrhagic disorders is not well understood. Furthermore, the potential threat of coagulopathy in non-APL AML patients may be underestimated. Herein, we report a patient with non-APL AML presenting with severe coagulopathy with hyperfibrinolysis. Since his clinical course resembled a prototypical APL-associated hemorrhagic disorder, we hypothesized pathophysiological similarities. Performing multiparametric flow cytometry (MFC) and immunofluorescence imaging (IF) studies, we found the patient’s bone-marrow mononuclear cells (BM-MNC) to express ANXA2 - a biomarker previously thought to be APL-specific. In addition, whole-exome sequencing (WES) on sorted BM-MNC (leukemia-associated immunophenotype (LAIP)1: ANXA ANXA2 gene located on chromosome 15, serves as a cell surface receptor for both, plasminogen (PLG) and tissue-type plasminogen activator (PLAT), accelerating plasmin formation  - previously known as a marker of lymphatic endothelial cells - has been recently identified as a contributing factor to APL-associated hemorrhagic disorders . PDPN, eIn contrast, coagulopathy/hyperfibrinolysis is less frequent in patients with non-APL AML. Therefore, 1) the potential threat of coagulopathy in non-APL AML patients may be underestimated and 2) underlying pathophysiology of AML-associated hemorrhagic disorders is not well understood.9/L , hemoglobin was 4.65 mmol/L , and platelet count was 51,000 x 109/L  with a WBC differential including 27% neutrophils, 25% monocytes, 38% lymphocytes, 1% eosinophils, and 9% myeloblasts. Prothrombin ratio (PR) was 49% , activated partial thromboplastin time (aPTT) 40 s , initial fibrinogen level 1,28 g/L , D-dimer level >4 µg/mL FEU , antithrombin activity 112% , and the International Society on Thrombosis and Haemostasis (ISTH) DIC score was 6  all together indicating overt DIC with ongoing hyperfibrinolysis (KMT2A (KMT2A-PTD) as well as an IDH2 and a SRSF2 mutation .A 64-year-old man (unique patient number (UPN) 1) noticed spontaneous bruising of the arms and legs about six weeks before admission, at the same time progressive fatigue and night sweats developed. Due to episodes of spontaneous, prolonged gingival hemorrhage the patient presented to his primary care doctor two weeks later, whereupon the patient was referred to our center. On admission, the patient´s white blood cell (WBC) count was 5.3 x 10Following initiation of induction chemotherapy with a standard “7+3” regimen, hemorrhagic diathesis worsened: severe hyperfibrinolysis with diffuse mucosal and post-interventional bleeding occurred. Beside extensive transfusion of platelets, fibrinogen, prothrombin complex concentrate (PCC), and factor XIII, application of tranexamic acid was required to counteract coagulopathy and stabilize fibrinogen levels. About two weeks following admission hemorrhagic diathesis began to resolve and we could deescalate the transfusion regimen Figure 1KMT2A-PTD, the patient was assigned to undergo matched-unrelated hematopoietic cell transplantation (HCT) (Due to 1) primary refractory disease and 2) the adverse prognosis associated with on (HCT) .9/L , hemoglobin was 4.9 mmol/L , and platelet count was 57,000 x 109/L  with a WBC differential including 9% neutrophils, 31% monocytes, 49% lymphocytes, 2% eosinophils, 1% basophils and 8% myeloblasts. Fibrinogen level was <0,4 g/L , D-dimer level >4 µg/mL FEU , PR 39% , aPTT 36 s , antithrombin activity 99% , and ISTH DIC score was 8 , resembling the situation at initial presentation  count was 1.93 x 10entation Figure 1Two months after transplantation, examination of a bone marrow–biopsy specimen revealed complete morphologic and molecular remission and a full donor chimerism. On last follow-up, 12 months after transplantation, the patient is free of complaints and remains in complete remission.via multiparametric flow cytometry (MFC) and immunofluorescence staining of cytospin samples (IF). Furthermore, PDPN expression was examined via IF. Further, whole exome sequencing (WES) was performed on sorted UPN1 BM-MNCs populations: Briefly, DNA - isolated from BM-MNC populations - was quantified using the Qubit dsDNA HS Assay . Library construction was performed from isolated DNA using TruSeq DNA Nano Sample Preparation kits  according to the manufacturer’s instructions and indexed libraries were paired end (2x151 bp) sequenced on Illumina HiSeqX instrument (Illumina). FASTQ files were generated using the BCL2 fastQ pipeline (bcl2fastq 2.19.0.316). Per sample, on average 498M (range 455M-514M) read ends were obtained. HG19 was used as reference genome for bioinformatic analyses. The bioinformatics evaluation was performed using the Biomedical Workbench from CLC (12.0.3) using a customized analysis algorithm with following filters: coverage >/=25, variant allele frequency >/=10%. SNVs were further annotated for their biological effect and filtered based on SNV-quality, minimum number of supporting reads and biological relevance (non-synonymous SNVs). Variant assessment/classification was performed using VarSome, which includes—among others—ClinVar and dbSNP databases, population frequency information from gnomAD and in-silico prediction tools such as FATHMM, SIFT, REVEL and Polyphen2 (KMT2A-PTD and an IDH2 mutation without evidence of DIC (UPN3), and a healthy donor served as positive and negative control, respectively. Here, BM-MNCs were obtained from the Study Alliance Leukemia (SAL) biobank, which has been approved by the Ethics Committee of the TU Dresden (EK98032010). Written informed consent had been obtained from all participants.We determined ANXA2 and PLAT expression on the patient´s (UPN1) bone marrow derived mononucleated cells (BM-MNCs) olyphen2 . BM-MNCslo/midCD45dim) (lo/midCD45dimCD13+CD33+HLA-DR+CD56-CD117+) and high (LAIP2: SSClo/midCD45dimCD13-CD33+HLA-DR+CD56+CD117+) ANXA2 expression, respectively  (data not shown).MFC revealed pronounced ANXA2 expression on patients BM-MNCs  showed neither ANXA2 nor PDPN expression, arguing against a correlation between evidence of KMT2A-PTD and/or IDH2 mutation and presence of DIC. In contrast, as a possible genotype/phenotype association, DiNardo et al. reported two patients diagnosed with AML with evidence of t translocation (KMT2A-MLLT10 rearrangement) presenting with DIC  in larger studies DIC was not reported as frequent event in adult patients diagnosed with t; KMT2A rearranged AML (hi) as compared to LAIP1 (MFC ANXA2lo) we performed whole exome sequencing (WES) on sorted BM-MNCs populations. Here, T-lymphocytes (SSCloCD45brightCD7+) served as a germline-like control. WES identified a total 24 variants  in 21 genes including the previously known IDH2 and SRSF2 mutations: ATXN1, CLIC6, DDX51, GOLGA6L2, HTT, IDH2, MAML2, MUC4, MUC19, MXRA5, NACA, PCLO, PIEZO1, PRG4, PTPRZ1, RBBP6, SLC9B1P1, SRSF2, TRBV7-8, UBXN11, ZNF208 - all variants are reported in detail in hi): CLIC6, DDX51, HTT, MUC4, PIEZO1, PRG4, PTPRZ1. Here, previous studies found mastermind like 2 (MAML2) to be a rare fusion partner of KMT2A in patients with myelodysplastic syndromes and secondary AML as well as acute lymphoblastic leukemia (ALL); mucin 4 (MUC4) expression to be associated with adverse prognosis in AML and piezo type mechanosensitive ion channel component 1 (PIEZO1) frequently mutated in patients with hereditary xerocytosis, hereditary stomatocytosis, respectively  BM-MNCs phenotype p;q23 trannged AML –15. To fectively –18. AlthHerein we report a non-APL AML patient with clinical and pathophysiological APL features. Although ANXA2 mediated hyperfibrinolysis is a hallmark of APL, this hemorrhagic disorder can also occur in other AML subtypes. In contrast, PDPN expression and associated coagulopathy appears to be specific for APL . TherefoManagement of hemostatic complications in acute leukemia: Guidance from the SSC of the ISTH” guideline, frequent evaluation for coagulopathy (complete blood count (CBC), PR, aPTT, fibrinogen level, b.i.d.) was performed; further a standardized score was used to diagnose/reevaluate DIC  - a lysine derivate with antifibrinolytic activity by inhibiting the activation of plasminogen - was administered additionally to prevent major hemorrhage. The evidence supporting the use of antifibrinolytic agents in acute leukemia patients remains limited. Despite anecdotal reports claiming beneficial effects of TA in APL patients, the GIMEMA and PETHEMA studies both did not demonstrate differences in incidence of hemorrhagic death as compared to standard treatment  evaluate ANXA2 MFC assessment as a tool to determine the risk of life-threatening DIC in AML patients, 2) investigate PDPN as a marker of APL/APL-associated coagulopathy, 3) determine the efficacy and safety of antifibrinolytic agents in preventing hemorrhage in people with hematological malignancies, and 4) understand the genetic background and upstream regulation of ANXA2 and PLAT and coagulopathy-initiation in non-APL AML and APL clones, is indicated.The datasets presented in this article are not readily available because ethics restrictions apply. Requests to access the datasets should be directed to the corresponding author.The studies involving human participants were reviewed and approved by TU Dresden ethics committee (EK98032010). The patients/participants provided their written informed consent to participate in this study.LR, FS, and MB conceived the presented idea. LR, FS, LW, and MvB performed research. HA provided BM-MNC. AR and ES helped to perform molecular analysis. SH helped to assess WES data. LR wrote the manuscript with help from FS, UP, SH, JS, MB, and MvB. All authors contributed to the article and approved the submitted version.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.


Indeed, it couples the electrical signal, which pervades the myocardium, with cardiomyocytes contraction. Moreover, alterations in calcium management are the main factors contributing to the mechanical and electrical dysfunction observed in failing hearts. So, simultaneous analysis of the contractile function and intracellular Ca2+ is indispensable to evaluate cardiomyocytes activity. Intracellular Ca2+ variations and fraction shortening are commonly studied with fluorescent Ca2+ indicator dyes associated with microscopy techniques. However, tracking and dealing with multiple files manually is time-consuming and error-prone and often requires expensive apparatus and software. Here, we announce a new, user-friendly image processing and analysis tool, based on ImageJ-Fiji/MATLAB® software, to evaluate the major cardiomyocyte functional parameters. We succeeded in analyzing fractional cell shortening, Ca2+ transient amplitude, and the kinematics/dynamics parameters of mouse isolated adult cardiomyocytes. The proposed method can be applied to evaluate changes in the Ca2+ cycle and contractile behavior in genetically or pharmacologically induced disease models, in drug screening and other common applications to assess mammalian cardiomyocyte functions.Intracellular calcium (Ca The AP spreads between sarcomeres, leading to Ca2+ influx (ICa) through the voltage-gated L-type Ca2+ (LTCC) lining in the T-tubules. Ca2+ ions inflow induces Ca2+ release from the sarcoplasmic reticulum (SR) through the ryanodine receptors (RyRs) through a process known as “Ca2+-induced Ca2+-release” (CICR). This mechanism leads to a 10-fold increase in the free intracellular [Ca2+]i and provides a sufficient amount of Ca2+ to bind troponin-C and promote cardiomyocytes contraction. Force of contraction is dependent on the amount of Ca2+ bound to troponin-C and it will be a function of both the magnitude and duration of the rise of [Ca2+]i. Cardiomyocyte relaxation is induced by rapid Ca2+ reuptake in the SR due to the activation of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2a (SERCA2a) pump. Intracellular Ca2+ signals then include transients, oscillations, and propagating waves [2+]i is finely tuned, so it is not surprising that its abnormalities are associated with cardiac dysfunctions [2+ homeostasis [2+ cycling and the correlated contractile function in isolated cardiomyocytes, widely used as an in vitro model, provides important information regarding their health status. Moreover, the analysis of E-C coupling is used to study the effects of different drugs with potential inotropic or lusitropic effects to confirm their effectiveness [Calcium  are used due to their feasibility and low cost. After time-lapse video acquisition, the temporal Ca2+ signal is evaluated as time-dependent, normalized, mean-in-space fluorescence (f/f0) within a user-defined area or region of interest (ROI); the subsequent analysis is considerably time consuming.[Ca2+ transients, the use of them requires good knowledge of programming. Some software, such as Video Sarcomere Length (VSL) , has a user-friendly interface; however, VSL performs the shortening detection via two length calculation methods, using the fast Fourier transform analysis that requires high-resolution videos, high image magnification, and high-speed camera [2+ signal analysis, but is not cost effective. Thus, the laboratories are often unable to afford the substantial costs involved in purchasing both commercial software and video acquisition equipment.Despite the existence of some computational solutions for the analysis of cardiomyocyte fraction shortening and Cad camera . Other cWe aimed to develop a simple tool that allows the researchers with basic programming skill to measure the contractile parameters of primarily isolated adult cardiomyocytes.https://fiji.sc/, accessed on 7 March 2022) and then analyzed by our custom-made MATLAB® scripts in order to evaluate cell fraction shortening and Ca2+ transient and kinematics/dynamics of primary isolated adult cardiomyocytes . These MATLAB® scripts can perform Ca2+ transients and shortening analysis of cells that are not necessarily positioned horizontally. Our method only requires recorded videos, even at low resolution, captured in wide-field fluorescence and bright-field microscopy, without the requirement of additional equipment. In addition, our custom-made scripts, when compiled and given upon request, can be easily run without a license since MATLAB® software house provides, for free, the runtime which enables the execution of scripts on computers without an installed version of MATLAB® .Therefore, this work aims to describe a new analysis tool that can be used on videos or image stacks loaded in ImageJ-Fiji  and with the ethical guidelines for animal care of the European Community Council (Directive 86/609/ECC). Male C57BL/6N mice (2–4 months old) were obtained from the Charles River Laboratories Italia  and were housed in the DAHFMO, Sapienza University of Rome, under 12 h light/dark cycles, at a constant temperature, with food and water ad libitum.2PO4 1.2 mM, NaHCO3 20 mM, MgSO4 1.2 mM, glucose 5.6 mM, 2,3-Butanedione monoxime (BDM) 10 mM, taurine 20 mM, pH = 7.33). The heart was gently cannulated via the aorta in a Langendorff perfusion apparatus and filled with cannulation buffer. The hearts were perfused initially with a predigestion buffer  and 0.01% protease type XIV  solution with no calcium) and then with digestion buffer  for complete digestion. The heart was cut below the atria into a Petri dish containing 5 mL of digestion buffer when the heart became soft. The minced heart was then transferred into a stopping buffer (12.5 μM CaCl2 and 10% FBS). The isolated myocytes were recovered with a gradient of calcium cannulation buffer to 1 mM CaCl2. Freshly isolated myocytes were washed three times and suspended with beating buffer  to remove BDM before shortening and calcium transient assay.Adult ventricular myocytes (AVMs) were isolated as previously described . BrieflyFreshly isolated AVMs were loaded with Fluo-4 AM  for 15 min. Cells were then placed in the middle of a glass-bottomed dish with 3 mL beating buffer and settled for 5 min. Platinum electrodes were placed near the cell and paced the cell at 1 Hz (with a voltage of 50 V) using the SD9 stimulator  as previously described . The cal2+ transient amplitude were compared between groups by linear regression analysis  with Spearman correlation coefficient. p < 0.05 was defined as statistically significant.All data were expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 6 software . The sample size for each group is shown in the figure legends. The cells came from at least three sets of independent experiments. Differences between two groups  were evaluated by Welch’s t-test. The relationships between τ of decay, τ to peak, fraction shortening, and Ca® software in order to evaluate fraction shortening (To comprehensively study the contractile cycle of adult ventricular myocytes (AVMs), a new simple analysis tool was developed based on ImageJ-Fiji/MATLABortening .0 (pixel) of each cardiomyocyte was measured by ImageJ-Fiji . The movement of each cell tail was evaluated as previously described [1 and MD2, respectively. Subsequently, the cell fraction shortening (%) was calculated as (MD1 + MD2)/(L0/2) /(L0/2) B.2) was calculated as the sum of the tails’ force (each measured as previously reported [2/s2) were calculated as the average of the tails’ parameters  stimulation was used to obtain a positive lusitropic and inotropic effect to check the validity of our method. As reported in 2+ transients of Fluo-4-AM-loaded cardiomyocytes starting from acquired image sequences.The right panel in http://akiramuto.net/archives/148 (accessed on 7 March 2022) and [0) was measured within a user-defined ROI  and , initiatined ROI A, in pary-axis shifts of the signal baseline during the observation time; for this reason, via a custom-made script in MATLAB® , we have detrended the fluorescence signal, obtaining the typical pattern of the Ca2+ transients , which is the state of the art for that kind of signal analysis. In particular, in MATLAB®, there is an implementation of the preceding IEEE standard : on the statistical basis between the various transitions from peaks to baseline, the red crosses are placed where the fluorescence signal is correctly recognized at its 10%, 50%, and 90% for both rise and fall patterns  a combined ImageJ-Fiji/MATLAB2+ cycle is fundamental to determine the physiological consequence of various genetic manipulations and to identify potential therapeutic targets [An easy-to-use method to quantify cardiomyocyte shortening and Ca targets .2+ transients, but often they are not user-friendly, needing specific instrumentation and thus becoming expensive, such as Video Sarcomere Length (VSL)  [2+ handling [Some platforms are available to quantify heart or cardiomyocytes shortening or Ca Canada)  or other Canada) . In parthandling ,29.® scripts, reducing the time consumed in the analysis and providing a comprehensive set of functional parameters from isolated adult cardiomyocytes. Given the fact that the custom scripts can be used for the analysis of videos and time-lapse image sequences, it is natural to consider their use for moving cells , stained with calcium dye or infected with GFP- or RFP-based calcium probes, would be possible. Quantitative and qualitative assessment of Ca2+ transient parameters can be performed in pathological conditions, e.g., in patient-specific cell line carrying arrhythmogenic diseases such as arrhythmogenic right ventricular dysplasia (ARVD) and catecholaminergic polymorphic ventricular tachycardia (CPVT) [Here, we developed and tested straightforward MATLABng cells , left anng cells , right: a (CPVT) .2+ transient analysis is the implicit statistics to identify the pattern of the bi-level (peak-baseline) waveform transitions, discarding the outliers. This signal analysis method is defined in an IEEE standard and represents the state of the art for such signals, it is easy to be implemented in customized scripts and, specifically, there is no need to hypothesize mathematical models of the transient temporal pattern, thus avoiding data fitting and fitting interpretation. In other words, the main information for the transient’s τ is easily obtainable without using mathematical models .2+ transient amplitude and, also, a decrease of the transient time throughout the recovery phase, as expected after β-ARs activation. Moreover, the temporal patterns of the Ca2+ transient and Ca2+ transient amplitude, at basal (untreated) and after isoproterenol stimulation, obtained with our software tool, were identical to those showed by CalTrack, an alternative validated software [The reliability of our tool was verified using isolated adult murine cardiomyocytes, giving expected results  when a ksoftware .2+ transient amplitude, and the kinematics/dynamics parameters with our software tool, providing a real-data-based novel insight into cardiovascular physiology.It is worth noting that isoproterenol stimulation induces biphasic dose-dependent cAMP and PKA activation in cardiomyocytes: at picomolar doses, a transient increase in the initial peak response, and, at nanomolar doses, a saturated and sustained increase has been observed. This dose-dependent response, which dictates PKA substrate-specificity and cardiac contraction response, could be investigated by analyzing fractional cell shortening, CaWe have highlighted the beat-to-beat variability emerging from the transient’s analysis : whereasWe are aware that, to date, the gold standard for such measurement in adult cardiomyocytes is a technology, namely IonOptix . Here we2+ transient, whereas the amplitude is constant and therefore receptive to isoproterenol. Cameras with higher quantum efficiency and FPS will certainly provide new insights in the Ca2+ transient analysis and in the cell shortening. Nevertheless, other published algorithms demonstrated a similar response to a positive inotropic agent, including accelerated Ca2+ decay and time to peak, even though a lower temporal resolution of 25 fps was applied [One limitation is that our temporal resolution  is not optimal regarding the single beat analysis. This is the reason why we observed a discrepancy in the baseline-to-peak upstroke time of the Ca applied .2+ handling and contractile behavior of isolated cardiomyocytes is a valuable tool for the study of cardiovascular pathophysiology. Remarkably, more information will be available for analyzing dose-dependent effects of inotropic drugs  on the aforementioned parameters. This allows greater understanding of how the cardiomyocyte contraction is affected by different diseases, drugs, or genetic manipulations. Our method is an easy and useful tool that generates relevant data regarding the cardiomyocyte functionality. We expect that this tool might help to expand the study of functional features of cardiomyocytes without the need for expensive equipment and software or vast programming expertise.The analysis of the Ca


Experiments have indicated that calcium release is stochastic and regulated locally suggesting the possibility of spatially heterogeneous calcium levels in the cells. This spatial heterogeneity might be important in mediating different signaling pathways. During more than 50 years of computational cell biology, the computational models have been advanced to incorporate more ionic currents, going from deterministic models to stochastic models. While periodic increases in cytoplasmic Ca2+ concentration drive cardiac contraction, aberrant Ca2+ release can underly cardiac arrhythmia. However, the study of the spatial role of calcium ions has been limited due to the computational expense of using a three-dimensional stochastic computational model. In this paper, we introduce a three-dimensional stochastic computational model for rat ventricular myocytes at the whole-cell level that incorporate detailed calcium dynamics, with (1) non-uniform release site placement, (2) non-uniform membrane ionic currents and membrane buffers, (3) stochastic calcium-leak dynamics and (4) non-junctional or rogue ryanodine receptors. The model simulates spark-induced spark activation and spark-induced Ca2+ wave initiation and propagation that occur under conditions of calcium overload at the closed-cell condition, but not when Ca2+ levels are normal. This is considered important since the presence of Ca2+ waves contribute to the activation of arrhythmogenic currents.Calcium (Ca In addition, the authors suggested that the high value measured by Wu and Bers did not consider the SR Ca2+ leak during measurement. A recent study by Picht and co-workers supported the previous result in Bers’ group and rejected that claim  in the jSR compared to the network SR, but also the partial depletion of [Ca2+] in the neighboring nSR grid points. The released Ca2+ includes not only the free calcium but also the calcium that binds to CSQ. The amount of Ca2+ that binds to CSQ in the cardiac muscle was estimated to be about 50% , which suggested 70% of Ca2+ was bound to CSQ2 [2+]jSR from 1000 μM to ~100 μM during a Ca2+ spark, as suggested by a previous version of our current model jSR should be long enough to provide an adequate amount of Ca2+ to refill the free [Ca2+]jSR, to neighboring nSR, including Ca2+ to bind to [CSQ]. This, not including the possible rate-limiting effect between the jSR and nSR due to geometrical structure, can be used to explain the long recovery time, given the diffusion constant of [Ca2+]SR at 60 μm2/s. Thus, in the model, we chose DCasr = 60 μm2/s.Another factor for the long recovery time is not only the sharp depletion of jSR at the ith release site is described by the following ordinary differential equation:jsr = Vjsr/Vmyo is the volume fraction.The calcium in the jSR is replenished from the nSR by a diffusive flux , time-second (s). In the spatial model, the forward difference in time and central difference in space derivatives yields:The model is fully stochastic in terms of the channel gating of RyR2s and LCCs. The program was mainly written in Fortran and CUDA Fortran, using the CUDA programming toolkit to run on Nvidia Fermi GPU, with some parts written in C++. The Euler method was used to solve the partial-differential equations (PDEs) of diffusion-reaction, and other ordinary differential equations (ODEs). The adaptive time-step ranging from 10 ns to 1 µs was used. When there is some activity, due to channel gating, the time-step will be reduced for numerical stability. The units in the systems are as follows: transmembrane potential—mV, membrane currents—µA/cmFor a single cell, the Neumann boundary condition is used, i.e., no flux:+, Na+) were kept constant for the duration of the simulation. Similarly, the corresponding currents were also modeled as uniform myo, [Ca2+]SR, and CRU distances in spark triggering. The result was collected based on 1416 simulation cases.The current model has the following key features: (1) The gating of ion channels is modeled stochastically with the RyR2 kinetics fitted to give the proper fidelity of spark generation that matches the experimental data, i.e., ~100 sparks/cell/s at rest. (2) The non-uniform distribution of SL, SR buffers and NCX were incorporated in the model to reflect experimental reality. (3) The dynamics of calcium buffering in the subspace is modeled explicitly, without using fast binding assumption. (4) The ultra-fast MCMC method allows us to obtain a better statistic of spark-induced spark compared to other studies which only enable the examination one, two, or a few pairs of CRUs at a time. The first simulation series explores the role of different factors, e.g., myo = 0.4 µM, it significantly increases the Po,trigger which is now 89.12% for CRU at 0.6 µm apart and is 3.56% for the CRU at 0.8 µm apart. Along with this, we see a reduction in the delay time, which reduces from 6.6 ms for the CRU at 0.6 µm apart in 2+]sr , and in 2+]myo = 0.156 µM, and [Ca2+]nsr = 1.30 mM). In each condition, we measure the probabilities of one CRU triggering the neighboring ones at different distances and the delay upon which condition it occurs under. The main cluster can trigger the neighboring cluster at 0.6 µm apart with high fidelity  and very small delay (<8 ms). This strongly suggests the role of small clusters in bridging the gap between CRUs in generating calcium waves across all tested conditions.Under normal conditions, a sharp transition occurs when the CRU distance goes beyond 0.6 µm  with thein 2+]sr . In part2+]o = 10 mM, a 4-fold increase in Ca2+ spark was observed jSR, not [Ca2+/CSQ]. The multiple forms of CSQ, i.e., dimers and multimers, which can function as Ca2+ buffers to hold more SR Ca2+ during Ca2+ overload have not been modeled. With a fixed amount of CSQ at the jSR, we suggest that when [Ca2+]jSR exceeds a certain amount, the dependency on [Ca2+]jSR saturates. Thus, the lumenal function is revised to match the spark frequency from the experimental results at the Ca2+ overload condition, while also allowing a higher [Ca2+]SR that can trigger a Ca2+ wave. The lumenal function is changed from:max = 1.13 mM being the saturation value of [Ca2+]jSR that was selectedwas to be in agreement with the previous experiment.During Cao,trigger between the two cases is small, as shown in o,trigger is strongly influenced by the Ca2+ diffusing from the neighboring release site. Based on the measurement in The difference in P2+ wave propagation.Experimental studies using high resolution imaging showed that RyR2s are organized in subclusters that can function as single functional CRUs ,89,90,912+ sparks, with spark amplitude and spark size increasing 4.1-fold and 1.7-fold accordingly. These macro Ca2+ sparks also served as the sites of wave initiation (65%), indicating that the macrosparks may represent aborted waves involving propagation between release sites. Our study, which is in agreement with these studies, also suggests that the Ca2+ spark observed is the result of calcium release, not from a single CRU but multiple CRUs, and thus the mass of Ca2+ release from multiple CRUs is considered strong enough to trigger the wave in most cases.In the study by Cheng and co-workers , increas2+ wave propagation is the fire-diffuse-fire model myo, [Ca2+]nsr, different distances and of the presence of satellite RyR2. The second factor contributing wave formation along the longitudinal direction at which the distance between the CRUs is much longer at 1.4–2.2 µm. The question is under what condition can this happen? This is determined by two factors: the number of CRUs activated on one Z-line and the mass of Ca2+ release, which depends critically on the level of Ca2+ overload.The previous section already provided the Po,trigger of the ‘blue’ CRU , it is practically impossible to accurately detect the level of Ca2+ in the jSR. In addition, fluo-5N, the dye being used to estimate [Ca2+]jSR, has the Ca2+ affinity ~400 μM, which is 2–3 times smaller than the diastolic level of [Ca2+]SR. Thus, estimating the level of [Ca2+]SR overload is currently impossible [2+]SR during a Ca2+ overload, it requires detailed modeling of CSQ in multiple forms which is not available in the current model. Therefore, we focus on the number of activated sites. Nevertheless, we were able to see a full and repetitive Ca2+ wave with [Ca2+]SR = 1.7 mM and [Ca2+]myo = 0.156 μM. This seems to provide a “safety factor” where normal [Ca2+]SR cannot produce Ca2+ activation of adjacent sites to form waves.Simulations with myo = 0.156 µM, [Ca2+]sr = 1.7 mM, and the statistics were collected based on 144 trial cases.Izu and co-workers used a 5–7 supercluster . Each su2+ is from a single release site, the velocity is 30–45 µm/s. Using 9 activated CRUs, the wave velocity in the simulation setting, as shown in o,trigger increased but also the wave speed was 74 µm/s (for Z-line 2.0 µm apart) and 100 µm/s (for Z-line 1.8 µm apart). This suggested that the wave velocity is strongly dependent on the mass of calcium release, i.e., the number of activated CRUs on one Z-line and the distance to the CRU in the next Z-line and suggests an important role of intermediate RyR2 clusters.We calculated the propagation velocity as shown in the case of spark-induced spark, when the source of Ca2+]o from 2 mM to 15 mM, the wave speed increases from 33 µm/s to 87 µm/s; while at [Ca2+] = 3 mM, the wave speed was 33 µm/s at 23 °C and 74 ± 19 µm/s at 30 °C. Another experiment in rats confirmed the diversity in spatiotemporal properties of Ca2+ waves, modulated by the Ca2+-loading state [2+]i. In addition, we showed the important role of off-Z-lines CRUs in wave propagation. A whole-cell simulation of Ca2+ wave is shown in 2+ waves in experiments under Ca2+ overload conditions, as shown in 2+ changes accompanying the Ca2+ waves at three example times during the simulation. Note that the fall in the network SR calcium occurs slightly behind the Ca2+ wave front as diffusion in the myoplasm is the key driver of wave propagation due to CICR. It is important to note that the irregular spacing of the release sites was essential for wave propagation because in the model with uniform site placement, waves did not propagate beyond a few sarcomeres.The results presented strengthen the positive amplitude-velocity relationship that has been suggested by previous experiments ,96. The ng state  with dif2+ wave was tested, as shown in 2+ waves can propagate farther because less Ca2+ will be sequestered at the release site. When SERCA is reduced, the waves can propagate farther because there is more Ca2+ available for diffusion  reducing SERCA during Ca2+ waves, (2) by using a different SR Ca2+ diffusion constant, however the result follows CICR. As we pointed out, the Ca2+ velocity depends not only on the CRU distance but also the role of intermediate CRU’s. Thus, it is possible that the increase/decrease in Ca2+ wave velocity is the result of the presence of an intermediate RyR2 cluster or a longer CRU distance. Another possible explanation is that with SERCA blocking, the number of activated CRUs on one Z-line can be smaller, thus reducing the mass Ca2+ release, which in turn reduces the Ca2+ wave velocity. We thus reject the hypothesis that the Ca2+ diffusion constant is higher on the SR side.Keller and co-workers  proposed2+-sensitive RyR2’s compatible with experimental measurements, the model has successfully explained the difference in wave speed, the mechanism and conditions at which Ca2+ waves are initiated. In an earlier 2D model, Ca2+-bound fluorescence had not declined to the baseline level after the wave [2+ waves, without any change to RyR2 channel Ca2+-sensitivity and the SERCA pump. The model is the first of its kind at this level of detail and promises to be able to provide a further understanding of different pathophysiological conditions, such as T-tubule remodeling or diverse channel mutations.Using the Cathe wave . The mod2+ dynamics simulates realistic spark frequency, dynamics, and FWHM. This is the first model to produce realistic FWHM with physiologically realistic parameters. The model suggests that the robustness of Ca2+ wave propagation depends on Ca2+ release site placement, the density of RyR2 clusters, the presence of non-junctional or rogue RyR2 channels, and the level of the SR Ca2+ load. The model shows that repetitive Ca2+ waves can be produced under Ca2+ overload conditions, similar to those observed in recorded experiments.Our spatiotemporal model of Ca


This may involve the insertion of an insertion sequence (IS) element into a “stress-induced DNA duplex destabilization” (SIDD) region upstream of the operon promoter, although other types of mutations can also activate the bgl operon. Here, we show that increased expression of the bglG gene, encoding a well-characterized transcriptional antiterminator, dramatically increases the frequency of both IS-mediated and IS-independent Bgl+ mutations occurring on salicin- and arbutin-containing agar plates. Both mutation rates increased with increasing levels of bglG expression but IS-mediated mutations were more prevalent at lower BglG levels. Mutations depended on the presence of both BglG and an aromatic β-glucoside, and bglG expression did not influence IS insertion in other IS-activated operons tested. The N-terminal mRNA-binding domain of BglG was essential for mutational activation, and alteration of BglG’s binding site in the mRNA nearly abolished Bgl+ mutant appearances. Increased bglG expression promoted residual bgl operon expression in parallel with the increases in mutation rates. Possible mechanisms are proposed explaining how BglG enhances the frequencies of bgl operon activating mutations.The cryptic β-glucoside GFB ( Thus,Escherichia coli (E. coli) glycerol FK (glpFK) operon  × 1000  × 1000 .E. coli bgl operon promoter represent a novel form of directed mutation. IS insertion is absolutely dependent on the presence of the bglG gene product, BglG. Analyses revealed the requirements for this novel function of BglG. Moreover, IS insertion occurs only in the presence of an external aromatic beta-glucoside substrate, thus showing that IS insertion is dependent on environmental conditions. A molecular mechanism for the regulation of this directed mutation is proposed.We here provide evidence that several Insertion Sequence (IS) elements when inserted upstream of the


In humans, uterine luminal epithelium functions as a biosensor gauging the embryo quality and transmitting this information to the underlying endometrial stromal cells. This quality control ensures that only high quality embryos are implanted, while aberrant ones are rejected. The mechanisms of the embryo-uterine mucosa crosstalk remain incompletely understood. Trypsin, a serine protease secreted by the blastocyst, has been implicated in the cross-signaling. Here we address the mechanisms by which trypsin triggers the intracellular calcium signaling in uterine epithelium. We found that protease-activated G-protein coupled receptors are the main mechanism mediating the effects of trypsin in human uterine epithelium. In addition, trypsin activates the epithelial sodium channels thus increasing the intracellular Na Further embryonic development critically depends on implantation of the hatched blastocyst into the maternal endometrium. The main purpose of implantation is to ensure that the blastocyst firmly anchors into the endometrial stroma, which will support the developing embryo and allow the process of placentation to begin (2+ ([Ca2+]i) transients that lead to prostaglandin E2 (PGE2) release, phosphorylation of the transcription factor CREB and up-regulation of cyclooxygenase 2 (COX-2) required for prostaglandin synthesis from arachidonic acid (+ channel (ENaC) in trypsin-induced [Ca2+]i transients. Similarly to other epithelia  of the L-type Ca2+ channel blocker nifedipine. At these concentrations, nifedipine may affect other ion channels/transporters in addition to the voltage-gated Ca2+ channel blockade. Indeed, a direct inhibition of alveolar ENaC by high doses of voltage-gated Ca2+ channel blockers has been described in the literature  involvement in trypsin-induced Ca2+ signaling in the primary human uterine epithelium and in the Ishikawa cell line.Pregnancy is a complex and highly coordinated physiological process that includes implantation, decidualization, placentation and birth of offspring through the process of parturition . The ferto begin . Implantto begin . In contto begin . Under tto begin . Our preto begin . Experimnic acid . This stpithelia , trypsinchannels . Howeverterature . Most lisiveness . HoweverTM cannula, starting from the uterine fundus and moving downward to the internal cervical ostium. The biopsy was collected in a labeled Bijoux tube containing 5 ml of cell culture medium and transferred to the lab for processing as quickly as possible. The endometrial epithelial cells were isolated from the endometrial biopsies using collagenase and DNase digestion as described in detail elsewhere . Endometrial biopsies were obtained from four women attending the Implantation Clinic at University Hospitals Coventry and Warwickshire NHS Trust. All research was undertaken with full ethical approval and with written informed consent obtained from all participants in accordance with The Declaration of Helsinki 2000. Biopsies were taken during the secretory phase of the menstrual cycle using an Endocelllsewhere . After s2+]i responses to trypsin application similar to those observed in Ishikawa cells, an uterine epithelial cancer cell line. Given this similarity between the primary human endometrial epithelial cells and the Ishikawa cell line, subsequent mechanistic experiments were performed on Ishikawa cells. The Ishikawa cell line was obtained from Sigma-Aldrich and cultured in glass-bottomed 35 mm Petri dishes using a cell culture method similar to that described by Schmidt and colleagues  responses to trypsin, a ratiometric Ca2+-sensitive indicator Fura-2 was employed. The cells were washed with physiological saline solution (PSS) of the following composition (in mmol/l): 135 NaCl, 4 KCl, 1.2 MgCl2, 2 CaCl2, 5 HEPES and 10 Glucose. The pH of this solution was adjusted to 7.4 with NaOH. The Fura-2 indicator was loaded into cells as acetoxymethyl ester dissolved in DMSO and diluted with PSS to a final concentration of 2.5 μM. The loading solution also contained 0.05% of non-ionic detergent Pluronic F127 to aid the dye dispersal and 2 mM probenecid to facilitate dye retention in the cytoplasm. After 60 min loading at room temperature the cells were washed with PSS and incubated on the bench while protected from ambient light for another 30 min to ensure complete de-esterification of the dye. After that, the Petri dish with Fura-2 loaded cells was mounted on the stage of an inverted microscope  and superfused with pre-warmed to 35°C PSS. Trypsin and other drugs were applied to cells within the field of view using a gravity-fed multi-tube preheater . A fast monochromator  provided Fura-2 excitation alternating between 360 ± 12 and 380 ± 12 nm. The resulting Fura-2 fluorescence was separated from the excitation light by a 495 nm dichromatic mirror  and recorded at a wavelength above 510 nm. Time series of fluorescence images were collected at 1 ratio-pair per second using a front-illuminated EMCCD camera  controlled by the WinFluor software package . The camera exposure was set to 50 ms and the cells were not illuminated for the rest of the cycle time. This substantially reduced the photo-bleaching of Fura-2 during long-lasting experiments. The same software was used for the extraction of fluorescence intensities and calculation of Fura-2 ratio representing the [Ca2+]i dynamics.For imaging intracellular calcium  were drawn around each cell within the field of view, the mean intensity values were extracted from the ROIs and the ratio between fluorescence intensities exited at 360 and 380 nm were calculated in WinFluor. The technical graphing and data analysis software package IgorPro 7.0.8.1  was used for statistical analyses and preparation of figures. The data were obtained from at least three different cultures. Where appropriate, statistical comparisons were made using unpaired t-test. When multiple comparisons were made, a 1-way ANOVA followed by Dunnet’s post hoc test was used.Background fluorescence was calculated from areas devoid of cells and subtracted from each time series. To quantify the cellular [Ca2+]i responses in primary uterine epithelial cells and in the Ishikawa cell line. 2+]i responses to the application of 5 nM trypsin dissolved in PSS. In the majority of cells, trypsin at this concentration triggered a rapid transient rise in [Ca2+]i followed by a long-lasting oscillation . We elected to use 5–10 nM trypsin in all the subsequent experiments.We reasoned that the concentrations of the embryo-secreted protease are unlikely to reach a micro-molar level around the uterine epithelium during human embryo implantation. We therefore tested a low-nanomolar concentration range of trypsin for its effectiveness at triggering the [Caillation . In abou2+]i dynamics were very similar in the primary epithelium and in Ishikawa cells, bar for the occasional irregular spontaneous activity sometimes observed in primary epithelium before the application of trypsin but never in the cell line . On the other hand, pretreatment of the cells with FSLLRY-NH2, a PAR-2 inhibiting peptide, abolished both components of the [Ca2+]i response to trypsin. That is, not only the [Ca2+]i oscillations were abolished but the initial [Ca2+]i transient was eliminated as well .As illustrated in mplitude . This wamplitude . Pre-inc trypsin . There w2+]i signaling in human uterine epithelial cells. Our main findings are: (i) the effects of trypsin application are similar in primary human uterine epithelium and in the Ishikawa cell line; and (ii) the peptide agonists of PAR1 and PAR2 can mimic the effects of low concentrations of trypsin on uterine epithelial [Ca2+]i dynamics while the PAR inhibitor peptide eliminates this effect. These results are compatible with the idea that the proteinase activated receptors play a major role in the trypsin-induced endometrial [Ca2+]i signaling. This is in contrast to the mechanism proposed for implantation in mice, where ENaC and the L-type Ca2+ channels are thought to be pivotal players  chloride channels in uterine fluid secretion and reabsorption has long been recognized as an important determinant of uterine closure, a mechanism by which embryos are held in contact with the uterine epithelium before initiation of implantation . The patients/participants provided their written informed consent to participate in this study.AS and JB conceived and planned the study. JB collected endometrial biopsies and provided reagents and other materials. AS performed the experiments, analyzed the data, and wrote the manuscript. Both authors reviewed and approved the final version of the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


With the progress of globalization, international mobility increases, greatly facilitating cross-border transmission of respiratory infectious diseases (RIDs). This study aimed to analyze the epidemiological characteristics and factors influencing imported RIDs, with the goal of providing evidence to support adoption of high-tech, intelligent methods to early find imported RIDs and prevent their spread in China.We obtained data of imported RIDs cases from 2014 to 2018 from the Inbound Sentinel Network of Customs and the National Notifiable Diseases Reporting System in China. We analyzed spatial, temporal, and population distribution characteristics of the imported RIDs. We developed an index to describe seasonality. Pearson correlation coefficients were used to examine associations between independent variables and imported cases. Data analyses and visualizations were conducted with R software.From a total of 1 409 265 253 inbound travelers, 31 732 (2.25/100 000) imported RIDs cases were reported. RIDs cases were imported from 142 countries and five continents. The incidence of imported RIDs was nearly 5 times higher in 2018 (2.81/100 000) than in 2014 (0.58/100 000). Among foreigners, incidence rates were higher among males (5.32/100 000), 0–14-year-olds (15.15/100 000), and cases originating in Oceania (11.10/100 000). The vast majority (90.3%) of imported RIDs were influenza, with seasonality consistent with annual seasonality of influenza. The spatial distribution of imported RIDs was different between Chinese citizens and foreigners. Increases in inbound travel volume and the number of influenza cases in source countries were associated with the number of imported RIDs.Our study documented importation of RIDs into China from 142 countries. Inbound travel poses a significant risks bringing important RIDs to China. It is urgent to strengthen surveillance at customs of inbound travelers and establish an intelligent surveillance and early warning system to prevent importation of RIDs to China for preventing further spread within China. Globalization, increasing air travel, and greater population mobility facilitate inter-regional spread of infectious diseases, including emerging infectious diseases (EIDs) , 2. In 2Since the twentieth century, EIDs, especially severe acute respiratory infectious diseases (RIDs), occur constantly and have had an enormous impact on human health and social development . Rapid dOver recent decades, several international studies evaluated the relationship between population mobility and spread of infectious diseases –12. Two We obtained imported RIDs surveillance data and inbound traveler data, reported between January 1, 2014 and December 31, 2018, from the entry–exit sentinel network of customs (EESNC), which consists of 272 quarantine sentry points in the 31 provinces of mainland China , and frohttps://www.who.int/tools/flunet) . Twenty-seven types of RIDs were detected, including influenza (90.3%), rhinovirus (3.2%), and others (6.5%) , with entry along the developed coastal border areas of China. Most RIDs imported by Chinese citizens were imported from Thailand (14.1%), the Republic of Korea (7.9%), Japan (7.2%), and Singapore (4.2%). Most RIDs imported by foreign nationals were imported from the Republic of Korea (12.8%), Japan (10.4%), Vietnam (7.6%), and the Philippines (5.9%). Guangdong Province had the highest number of imported cases, followed by Jiangsu Province, and Shanghai  0.547 to 0.896]. A linear relationship was observed from the scatter plot, and a best-fit line was calculated using the least-squares method. By simple linear regression, the slope of the regression line was 0.441, implying that the average annual increase of imported RIDs cases was 0.441 per 10 000 foreigners entering China. The simple linear regression model explained 58.3% of the variance (adjusted R2 = 0.583); the regression model was statistically significant, F = 34.5, P < 0.001 (Fig. The Pearson correlation coefficient between the number of imported cases and inbound travel volume was 0.775 [95% confidence interval (The number of imported influenza cases increased with increases of influenza reports from source countries. Importation epidemiologic characteristics varied by continent. Asian countries imported the most influenza cases to China, but the number of influenza cases reported by Asian countries was relatively low Fig. .Fig. 5ScRespiratory infectious diseases have a huge impact on the health of people around the world. With increasing globalization, social, economic, cultural, and population exchanges across countries worldwide are increasing, and the prevention and control of RID cross-border transmission is facing significant challenges , 20. We Other researchers have found differences in the incidence of travel-related infectious diseases by gender and age group , 22. TheWe observed seasonal fluctuation of imported RIDs, and such seasonality may be affected by several factors—for example, international travel on holidays and seasonality of RIDs in originating areas . There iTravel volume is a key factor influencing the number of imported RIDs cases , 33. PasThe number of imported cases is associated with the number of reported cases in source countries, which is likely related to the prevalence of the disease and population of the source country . In geneIn order to assess and predict the risk of importation, investigators have developed statistical models based on data related to importation, such as international flights information, the epidemiology of selected diseases, and demographic information of source countries , 42. GivWe believe that there are additional factors affecting the epidemiology of imported RIDs that are in need of further exploration. For example, vaccination status influences imported RIDs. Environmental changes in temperature, humidity, air pollution, and sun exposure may influence RIDs spread . Travel Imported respiratory infectious diseases incidence increased from 2014 to 2018. Gender, age, continent, inbound passenger volume, and the number of reported cases in source countries were associated with the incidence of imported RIDs. With annual increases in international travel, the potential risk of RIDs spread to China rises accordingly. Therefore, it is urgent to strengthen surveillance at customs for inbound travelers and establish an intelligent surveillance and warning system for imported RIDs to early find and prevent the RIDs from spreading to China. The health and safety of people around the world will be more effectively protected if infectious diseases are controlled in their countries of origin. It is therefore vital for the international community to support prevention and control of important RIDs for countries having limited resources.