CBFB has been reported, but most of them were studied by chromosome and fluorescence in situ hybridization (FISH) analyses. The genomic characteristics of such deletions remain largely undefined, hindering further understanding of the clinical significance of the deletions.Abnormalities of chromosome 16 are found in about 5–8% of acute myeloid leukemia (AML). The AML with inv(16)(p13.1q22) or t  is associated with a high rate of complete remission (CR) and favorable overall survival (OS) when treated with high-dose Cytarabine. At the inversion breakpoints, deletion of 3’MYH11/3’CBFB gene fusion, which were characterized by chromosome, FISH, and single nucleotide polymorphism (SNP) microarray analyses. Both cases have achieved CR for more than three years.We report here two AML cases with inv(16) and deletion of the 5’CBFB in AML with inv(16) is also accompanied with deletion of 5’MYH11 in all the cases studied by SNP microarray, suggesting that 3’CBFB and 5’MYH11 were most likely deleted together as a fusion product of inv(16) instead of occurring separately. In concert with the findings of other published studies of similar patients, our study suggests that deletion of 5’MYH11/3’CBFB in AML with inv(16) may not have negative impact on the prognosis of the disease.Deletion of 3’ CBFB/MYH11 gene fusion [CBL, FLT3, KIT gene mutations, are frequently found in AML with inv(16). These additional changes/mutations may influence the OS positively or negatively [KIT mutations appear to have an increased risk of relapse and shorter survival in adult patients [CBFB, may occur at the inversion breakpoints. Fourteen cases with inv(16) and deletion of 3’CBFB have been reported in literature [3’CBFB deletions were undefined.Abnormalities of chromosome 16 are found in about 5–8% of acute myeloid leukemia (AML) and are one of the three AML defining chromosomal aberrations regardless of blast percentage under the World Health Organization (WHO) classification , 3. The e fusion , 5. Howegatively . For exapatients . Less coterature –19. Howeterature . The genMYH11 at 16p13.1 and 3’CBFB at 16q22. The genomic characteristics of both cases were characterized by chromosome, FISH and SNP microarray analyses. We also reviewed similar cases reported in literature to investigate possible clinical significance of the deletions at the inv(16) breakpoints.Here we reported two AML with inv(16) cases, both carried an additional deletion at one inversion breakpoint involving the fusion between 5’9/L with 80% blasts and a platelet count of 25 × 109/L. He had no hepatosplenomegaly. His bone marrow (BM) aspirate exhibited greater than 90% myelomonoblastic cells, with increased maturing eosinophils. Cytochemical staining was positive for peroxidase and esterase. Flow cytometry showed 76% positive CD34 and 5% positive CD64, but negative CD14, consistent with a diagnosis of AML. The patient started induction chemotherapy and achieved a complete hematological recovery on day 21. He then received autologous stem cell transplant (ASCT) after two cycles of consolidation chemotherapy and has remained in CR for three years.A 24-year-old male presented with intermittent fever and sore throat. A complete blood examination demonstrated a hemoglobin (Hb) count of 70 g/L, a white blood cell (WBC) count of 170 × 109/L and a platelet count of 43 × 109/L. This patient had splenomegaly. His BM aspirate showed 50% myeloid blasts. Flow cytometry showed 50% blasts expressed CD34, CD117, CD13, CD33, HLA-DR, and myeloperoxidase (MPO), consistent with a diagnosis of AML. The patient started induction chemotherapy and achieved a complete hematological recovery. He then received two cycles of consolidation chemotherapy. The patient has been in CR for four years.A 47-year-old male presented with intermittent low-grade fever and progressive fatigue. A complete blood examination demonstrated a Hb count of 92 g/L, a WBC count of 3.5 × 10CBFB dual-color break-apart probe kit  following the manufacturer’s protocol. The results were analyzed using the Leica CytoVision system .Chromosome G-banding was performed following standard techniques on BM aspirate. FISH was carried out with the commercial Genomic DNA was extracted from BM using QIAGEN EZ1 kit . SNP microarray was set up using Illumina CytoSNP-850 K v1.1 BeadChip  and analyzed using BlueFuse Multi (Illumina), based on human genome build GRCh37/hg19.The nomenclature for the chromosome, FISH and array findings is based on the International System for Human Cytogenomic Nomenclature (ISCN) 2016 [CBFB-MYH11. In addition, 4 of these cells showed an extra copy of chromosome 22, which is a common secondary change in this disease. FISH analysis confirmed the inversion but also detected a deletion of 3’CBFB in approximately 95.5% of the interphase cells examined  instead of being deleted separately. More than 10 CBFB-MYH11 fusion transcripts with different sizes due to various genomic breakpoints have been reported [CBFB exons 1–5 fused with MYH11 exons 8–42 in case 1 and CBFB exons 1–4 fused with MYH11 exons 33–42 in case 2. Both are typical gene fusions in AML-associated inv(16), and the 5’CBFB/3’MYH11 fusion genes are believed to be responsible for the disease of the patients [MYH11/3’CBFB fusion genes. Both patients in this study responded to chemotherapy well and achieved CR for multiple years. Three cancer-related genes listed in the Cancer Gene Census (CGC) in the Catalogue of Somatic Mutations in Cancer (COSMIC) were involved in the deleted regions, including MYH11 in 16p, CBFB and CTCF in 16q  or t  results in two fusion genes, 5’CBFB/3’MYH11 and 5’MYH11/3’CBFB. The former is a known pathogenic cause of AML, but the pathogenic effect of the latter is uncertain. We further reviewed reported AML cases with inv(16) and deletion of 3’CBFB  were known to achieve CR with known survival time up to 48 months at the time of report. This small cohort data appears to be in line with the CR rate (86–88%) and the five-year OS rate (50%) in AML with inv(16) [MYH11/3’CBFB fusion gene may play minimal roles in AML pathogenesis. CTCF is thought to be a tumor suppresser gene [CTCF haploinsufficiency destabilized DNA methylation and predisposed mice to cancer [CTCF in AML with inv(16) needs to be further investigated. There are five cases with inv(16) and deletion of 3’CBFB, which either did not achieve CR or relapsed (Table AML with inv16) and deletion of 3’t probes –19. To ot probes , 11. Notreported , 22. In patients . However6 and del inv(16) . Taken tser gene . Kemp eto cancer . However3’CBFB gene is a recurrent finding in AML with inv(16). It most likely represents deletions of the 5’MYH11/3’CBFB gene fusion from the inv(16) as a secondary change following the inversion. The 5’CBFB/3’MYH11 fusion resulting from the inv(16) is a known pathogenic cause of AML, but the 5’MYH11/3’CBFB fusion may play a minimal role in AML pathogenesis. The CTCF gene adjacent to the 3’CBFB was deleted in the current cases, suggesting that deletions of CTCF in AML with inv(16) may not have prognostic significance either. However, potential pathogenic effects of other genes involved in extended deletion regions may not be excluded.Deletion of the


This article introduces a package developed for R (R Core Team,  Joint analysis of multiblock data , which has been widely used for performing data integration from multiple sources in biomedical research, bioinformatics, genomics, and psychology . Based on the sparse SCA framework, Gu and Van Deun  method  data block containing scores of Ik subjects, objects, or experimental conditions on Jk variables. Based on PCA, Xk can be decomposed as followsR components. The component score matrix Tk is of size Ik × R; the matrix of component loadings Pk is of size Jk × R. Ek denotes the residuals. To identify the solution, extra constraints, such as SCA can be regarded as an extension of principal component analysis (PCA). Suppose I1 = ⋯ = Ik = ⋯ = IK = I. SCA requires that the component scores should be the same across all data blocks, and thus for each data blockTTT = I and T1 = ⋯ = Tk = ⋯ = TK = T. Estimates of the model , its component loading vector corresponding to the k th data block is denoted by r th component is referred to as a common component across all data blocks, if at least one loading in the r th component belonging to the k th block is not zero) for all k. Figure r th component is referred to as a distinctive component, if for some k, all loadings in the r th component belonging to the k th block are zero). In Fig. In this article, we give a formal definition of common and distinctive components as follows. For the nent r = ,2,...,R,nent r = ,2,...,R,nent r = ,2,...,R,nent r = ,2,...,R,Pk, as we do here, gives a very clear meaning to the components. Zero loadings suggest that the corresponding variables are not associated to the component, whereas variables with non-zero loadings are associated to the component. These components represent structural sources of variation, and components with linked variables across data blocks represent joint variation while components with non-zero loadings for variables of only one or a few blocks represent specific sources of variation. Unfortunately, the SCA model does not yield such structures of zero loadings : We do not know the specific component structure; that is, for each component, we do not know whether zero loadings are fixed for a single block or for multiple but not all blocks  or whether all blocks contain non-zero loadings . Situation (2): We know the specific component structure ; that is, we know the position of zero loadings defining the distinctive structure but we need to estimate the remaining undefined (non-zero) loadings.Throughout this section, we assume that the total number of components for all data blocks, Building upon sparse principal component analysis . We discuss how to identify R, λL, and λG in the Model Selection section below. T is estimated by T = VUT, where UΣVT is the SVD of PC is the concatenated component loading matrix consisting of K blocks of component loadings Pk, and XC is the concatenated data consisting of K blocks. To estimate Pk, Gu and Van Deun  plays the role of the outcome, I ⊗T of the predictors, and I ⊗T)T(I ⊗T) = I , Eq. x]+ = x, if x > 0; [x]+ = 0, if x ≤ 0. Equation Pk, which we refer to as the block-wise method. Equation We now show that Eq.  problem  is in fa and Lin  and Frie and Lin . To see  and Lin  with resr th component , denoted by Pk, resulting in the component-wise method. In this case, starting from Eq. tr is the r th column in T. Lettr ⊗I)T(tr ⊗I) = I, it can be proven thatAlternatively, the Group Lasso penalty can be imposed on the  and Lin . The firOur experience is that the component-wise method is more useful in practice, because by imposing Group Lasso penalties on each component of each block, the common and distinctive components are directly identified. The block-wise method is useful when users are not sure whether certain data blocks provide any information at all—if not, the entire data blocks can be dropped from analysis. In the remainder of this article, we focus on the use of the component-wise method, but the block-wise method is also mentioned when necessary.PC is updated. PC and T are updated iteratively, and this procedure guarantees that the loss is non-increasing, but local minima rather than the global minimum might be attained. Thus, Algorithm 1  conditions , is not needed. Thus, the minimization problem , and the remaining half of Eq. PC is updated. Because the loss function is biconvex, convergence is thus to a local minimum and the algorithm also requires a multi-start procedure. In addition, one may undo the shrinkage of the non-zero loadings by means of OLS  and fixed values for the tuning parameters for the Lasso and Group Lasso . The regularized SCA model with known component structure  is formulated with a fixed number of R and a fixed value for λL. To help users choose the most suitable model, we present a flow chart  for all blocks to retain?”, (2) “How to identify the component structure, given R?”, and (3) “Is there a reason  to believe that there is some sparseness within common/distinctive components?” Depending on the answers to the questions, one chooses a model in Fig. The regularized SCA model  with unkart Fig. . Users aetain?”,  “How to R∗ >> R and compute the VAF for each component in each block. According to Schouteden et al. (R such that the VAF for the first R components is clearly higher than for the remaining (R∗− R) components in any block.The RegularizedSCA package provides the VAF method and the PCA-GCA method for identifying the number of components. The VAF method computes the proportion of VAF for each simultaneous component in each data block . However, deciding R for all data blocks by means of the PCA-GCA method requires a second step (discussed below); that is, R is obtained once the component structure is identified.The PCA-GCA method  is performed on the component scores of every two data blocks to identify the number of common components. A common component is identified if the correlation is above a threshold. For example, Smilde et al. , p. 15 uR has been decided by the VAF method. In this case, we let the algorithm identify a suitable component structure for us by identifying λG via cross-validation.The third tool requires the Group Lasso penalty by means of the component-wise method, provided that λL≠ 0, and λL is identified by means of a cross-validation procedure. By giving answers to the three questions, users choose one of the six models in Fig. R is decided by means of the VAF method, and the component structure is identified by means of DISCO-SCA, the recommended model is the regularized SCA model with known structure with λL ≡ 0, if it is believed that there is no sparseness within common/distinctive components . Alternatively, one may first use Model 2 and check whether the cross-validation procedure recommends a very small λL. A very small λL may suggest that there is little support for a sparse model, and therefore one may use Model 1 instead. If one intends to achieve some sparseness within common/distinctive components, then Model 2 is preferred.Once a component structure is identified, one may decide whether there is sparseness within the common/distinctive components; that is, whether the components should look like the first and fourth columns in Fig. K-fold cross-validation procedure for identifying the optimal λL and/or λG. When both λL and λG are used , the algorithm searches through a grid of λL and λG values, and for each pair of λL and λG, K-fold cross-validation is performed. Take 10-fold cross-validation for example, 10% of the cells from the data are replaced with missing values, which are then replaced with the mean across subjects that do not contain missing values. The optimal combination of λL and λG is identified as follows. First, the algorithm computes mean squared prediction errors  denote the mean squared prediction error given λL and λG. Let K estimates of the prediction error associated to λL and λG, denoted by λL or λG is used , the algorithm searches through a sequence of λL or λG, and the optimal λL or λG is also obtained based on the “one standard error rule”. For example, when only λL is used, the algorithm first computes the mean squared prediction errors and looks for the lowest mean squared prediction error, denoted by Note that models 2, 3, 4, and 6 incorporate a e et al. , p. 13.  et al.,  and Witt et al., , and in  et al., .R and identify the same component structure. We advise readers to choose one of the two approaches, depending on their research fields and/or existing research. It is possible to establish a cross-validation procedure for deciding R, λL, and λG altogether in one step . However, such a comprehensive cross-validation procedure is computationally expensive and still too immature to be included in RegularizedSCA, because such a procedure requires an algorithm to search through a three-dimensional grid using a multi-start procedure. Studying the usefulness of such comprehensive procedures is much needed and deserves full attention in a separate article. Recently, Gu and Van Deun R> names(Herring)The “Herring” data consist of two small datasets. The “Herring_ChemPhy” dataset contains physical and chemical changes of 21 salted herring samples in a ripening experiment. The “Herring_Sensory” dataset contains the same 21 samples’ sensory data (such as the smell and the sweetness of the herring). Researchers in chemometrics and food sciences are interested in whether certain physical or chemical changes in herring (such as protein level) are associated with certain sensory characteristics (such as sweetness). Thus, we perform a joint analysis on these two datasets and inspect the association between the two datasets by means of their common and distinctive components.R> ChemPhy <– pre_process(Herring$Herring_ChemPhy)R> Sensory <– pre_process(Herring$Herring_Sensory)R> herring_data − num_var <– cbind(dim(ChemPhy) [2], dim(Sensory)[2])The first step is to pre-process the data by using the function pre_process  and then to concatenate the data. pre_process can automatically handle missing data by using multiple imputation. In addition, when the number of variables in one block is much larger than another block, it is likely that the information in the former block dominates the latter block. We recommend weighting each block by taking into account the number of variables R> summary(vaf)Model 2 states that the number of components R = 10 components in the concatenated data. The VAF function displays the proportion of VAF per block and per component in each block. We primarily focus on the component part. In the first block , the first three components explain most of the information of the block , whereas the remaining components explain much less information. In the second block , the first four components explain most of the information . Thus, taking two blocks together, we may conclude that at most four components are needed for further analysis. As a side note, despite that the fourth component in the first block accounts for a much smaller variance than the first three components, it has to be retained for further analysis because we decide to retain four components for the second block.We have let the function evaluate the proportion of VAF, if there would be R = 4: R> discoresult <– DISCOscaR> summary(discoresult)Next, we use the DISCO-SCA method to identify the component structure, given R = 4). The element in the first row and the first column of the matrix is a “1”, meaning that all the component loadings in the first component in the first block may be non-zero loadings. The element in the first row and the fourth column is a “0”, meaning that all the loadings in the fourth component in the first block may be zero loadings. The remaining elements in the matrix are interpreted in the same way. Thus, the matrix suggests that there are two common components  and two distinctive components .Figure λL, which in RegularizedSCA is realized by the functions structuredSCA and cv_structuredSCA. Note that the former function requires the user to specify a value for λL, whereas the latter function uses K-fold  cross-validation to decide the proper value for λL. Here we use cv_structuredSCA first. cv_structuredSCA  requires the user to specify the component structure, which in this case is generated by the DISCO-SCA method  and target matrix, as follows: R> targetmatrix <– matrix, nrow = 2, ncol = 4)which is simply the matrix in Fig. R> maxLasso <– maxLGlasso$LassoR> set.seed(115)R> results_cvS <– cv_structuredSCA, LassoSequence = seq)R> plot(results_cvS)Thus, in R, we specify the component structure, which we refer to as a λL, denoted by PC ≡0). Thus, sparse results are found when λL is between 0 and Note that in the code above, we use the function maxLGlasso to decide the smallest value for R> results_cvS$LassoRegion,Figure λL = .8922256: R> set.seed(115)R> result_str <– structuredSCA, LASSO = 0.8922256)The Lasso not only puts component loadings exactly to zero but also shrinks each of the non-zero loadings towards zero. Such shrinkage of the non-zero loadings can be undone as follows: R> final_comLoadingS <– undoShrinkageR> summaryThe region is between .8814759 and .9029753, and thus a proper Lasso value could be .8922256, the average of the two. We remind readers that the optimal value lies within this region, meaning that if one choose .9029753, the largest of the two, then the estimated component loading matrix may be sparser than the matrix by using the optimal value. Because, in this example, the algorithm uses a sequence of 200 evenly spaced values, the region generated by the algorithm is very small (.9029753-.8814759 = .0214994). Thus, using either the average value  or the larger value  does not drastically influence the final result. Using the smaller value  may be a safer choice. Alternatively, users may also ask for a Lasso value that is recommended by the algorithm by calling for summary(results_cvS), but we remind readers that the recommended value is the one whose MSPE is closest to  the smallest MSPE plus one standard error. Users who prefer a value whose MSPE is closest to and smaller than the smallest MSPE plus one standard error may consult the full report by using summary. We now re-run the analysis with Now we obtain a component loading matrix with combination of common and distinctive components as defined previously, and meanwhile there is some sparseness within the common and distinctive components. Figure see Fig. , have alR is decided by means of the VAF method (R = 4 for the “Herring” data), the component structure is identified by λG, and the regularized SCA model with λL and λG is used. The 10-fold cross-validation for λL and λG is performed by evaluating the following code: R> set.seed(115)R> results_cv <– cv_sparseSCAModel 4 states that the number of components R> summary(results_cv)we obtain the recommended Lasso tuning parameter (λL = 1.503148) and Group Lasso tuning parameter (λG = .3655355) based on the “one-standard-error” rule. Users may also consult summary to get a full view of results of the cross-validation procedure, which includes, for example, the values of tuning parameters that have been evaluated, and mean squared prediction errors etc. We remind readers that the recommended Lasso and Group Lasso values here are the ones whose MSPE is closest to  the smallest MSPE plus one standard error. Users may also consult summary to identify a pair of Lasso and Group Lasso values whose MSPE is closest to but smaller than the smallest MSPE plus one standard error. We run the final model with the recommended tuning parameters λL = 1.503148, λG = .3655355, and R = 4, and check the estimated component loading matrix R> set.seed(115)R> final_results <– sparseSCABecause both the Lasso and the Group Lasso shrink the non-zero component loadings towards zeros, the shrinkage may be undone as follows: R> final_Loading <- undoShrinkageR> summaryNote that if not specified otherwise, cv_sparseSCA performs 10-fold cross-validation, with a sequence of 20 Lasso tuning parameters and 20 Group Lasso tuning parameters . Users may also use maxLGlasso and specify a sequence of Lasso and Group Lasso tuning parameters by themselves. In addition, the Group Lasso penalty is applied to each component separately, which is the component-wise method mentioned in the Method section. As an aside, the user may also impose the Group Lasso penalty on an entire data block  by calling cv_sparseSCA. By evaluating the following command λL for Model 2 is approximately .89, and that for Model 4 is 1.50. We do not expect that the λLs for the two models are of similar magnitude, because in Model 4, both the Lasso and the Group Lasso cause shrinkage, but in Model 2 the Lasso has to account for all the shrinkage.Figure λL and λG. A possible solution is to provide a series of cross-validation curves conditional on λGs, and therefore for each λG a cross-validation curve is presented. But this solution is problematic for two reasons. First, imagine a sequence of 100 λGs is evaluated by the algorithm, then 100 cross-validation curves have to be provided, making interpretation difficult. Second, based on user feedback, we noticed that users were often confused by this conditional approach.One may notice that, for Model 4, a cross-validation curve like Fig. λL ≡ 0 is used to estimate the component loadings and scores. Because λL ≡ 0, there is no sparseness within the common/distinctive components.Model 5 states that the number of common and distinctive components and correspondingly the component structure are identified by means of the PCA-GCA method. Afterwards, the regularized SCA model with known component structure and To use the PCA-GCA method, we first evaluate the following function:R = 4 components in total in the integrated data.The output  could be “Herring_ChemPhy”: 1 1 1 0 “Herring_Sensory” : 0 1 1 1,whose corresponding target matrix is matrix, nrow = 2, ncol = 4). Because the structures are equivalent, the resulting estimated component scores and loadings are identical, after switching the columns and/or signs of loadings. In other words, both target matrices above can be used. We now use the first target matrix and estimate the final model.whose corresponding target matrix is matrix, nrow = 2, ncol = 4). We emphasize that, because of invariance of the DISCO-SCA solution under permutations of components, the component structure proposed above is one of the many equivalent structures. Another possible component structure, by switching the position of the first and third columns, is R> targetmatrix <– matrix, nrow = 2, ncol = 4)R> set.seed(115)R> result_strModel5 <– structuredSCAR> final_LoadingModel5 <– undoShrinkageR> summaryA screenshot of the estimated loadings can be found in Fig.  set.seed5R> resuR = 10 + 1 = 11. In this case, one may specify the target matrix as follows: Block 1: 1 1 1 1 1 1 1 1 1 1 0 Block 2 : 0 0 0 0 0 0 0 0 0 0 1,provided that R is smaller than the number of subjects. Second, the specification of the target matrix can be easily done by calling the (summary of) DISCOsca function as shown before. When more than two blocks are to be analyzed jointly, we recommend using DISCOsca, because this function can directly analyze more than two data blocks.Before concluding this section, we mention two points about the specification of the target matrix. First, when the PCA-GCA method is used, because one chooses the number of components for each block based on PCA, it is normal to have a different number of components per block. For example, imagine that one block with many variables requires ten components, and another block with, say, two variables that requires one component. We further assume that there is no common component, and therefore To illustrate the usefulness of RegularizedSCA, we present an analysis of empirical data on parent–child relationship, which readers in behavioral sciences are familiar with. We use this example to show how information regarding parent–child relationship can be obtained by examining the components estimated by the model.https://github.com/ZhengguoGu/paperRegularizedSCA/blob/master/ForAuthorsOnly/500FamilyData.Rmd.In psychological, sociological, educational, and medical research, researchers are often interested in the relation between parents’ behavior and children’s behavior  does not provide information regarding the directionality of the dyadic relationship, and thus if directionality is of primary interest, readers are advised to use other methods, such as directional network models.https://cran.r-project.org/web/packages/RegularizedSCA/index.htmlhttps://cran.r-project.org/web/packages/RegularizedSCA/index.html. RegularizedSCA, as far as we know, is the first R package that focuses on data integration from the (regularized) simultaneous component perspective.With an increasing trend in using large datasets coming from multiple sources, data integration tools that yield insights in joint and specific sources of variation and select the important variables therein are of crucial importance. The package proposed here fulfills this need. The functions in this package are flexible, and they cover important methods for identifying common and unique information in datasets. RegularizedSCA is available from the Comprehensive R Archive Network (CRAN) at The Regularized SCA approach to data integration is a fruitful field for future research. At this moment, little is known about which model selection method(s) are suitable for regularized simultaneous component analysis: Cross-validation is a popular choice, but it is known that cross-validation methods tend to retain more variables than needed (Chen & Chen,


Herein, the neuroprotective roles of PF in the models of middle cerebral artery occlusion (MCAO) followed by reperfusion in rats and N-methyl-d-aspartic acid (NMDA)-induced excitotoxicity in primary hippocampal neurons were investigated. Moreover, we attempted to confirm the hypothesis that its protection effect is via the modulation of the Ca2+/CaMKI)/CREB signaling pathway. In this study, PF not only significantly decreased neurological deficit scores and infarct volume in vivo, but also improved neurons’ cell viability, and inhibited neurons’ apoptosis and intracellular Ca2+ concentration in vitro. Furthermore, PF significantly up-regulated p-CREB and p-CaMKII, and down-regulated calmodulin (CaM) in vivo and in vitro. The results indicate that the protective effect of PF on cerebral ischemia reperfusion injury is possible through regulating the Ca2+/CaMKII/CREB signaling pathway.Paeoniflorin (PF) is an active ingredient of Paeoniae Radix which possesses the neuroprotective effect. However, so far, the neuroprotective mechanism of PF has still not been fully uncovered. The Ca Ischemic stroke  remains a leading cause of disability and mortality worldwide ,2. Due t2+/Ca2+/calmodulin-dependent protein kinase II (CaMKII)/cAMP response element-binding (CREB) signaling pathway has been shown to play a crucial role in the intracellular signal transduction pathway involved in cell proliferation, cell survival, inflammation and metabolism [2+ influx in ischemic stroke. Ca2+ influx leads to the elevation of the Ca2+ concentration in neurons which disrupts the balance of ionics, then activates various Ca2+-dependent enzymes  and Ca2+-binding proteins. Ca2+ overload into neurons, induced by ischemic insult, may excessively activate Ca2+/CaM-dependent pathways and lead to irreversible cell damage [2+/calmodulin (CaM) complex combines with CaMKII, CaMKII is activated and further autophosphorylates at Thr286 to render the kinase constitutively active. Several reports have demonstrated that phosphorylation of CaMKII plays an important part in the regulation of ischemia [Ischemic stroke involves the interaction of numerous pathophysiological processes, and concomitantly triggered pathogenetic mechanisms. The Catabolism ,7,8. Accl damage . CaMKII,l damage ,10. CaMKl damage ,10. Whenischemia ,12,13. Pischemia ,15,16. Pischemia .2O2 [+ [25–35 [Paeoniflorin (PF) is a monoterpene glycoside which is isolated from Radix Paeoniae  and is, at the same time, the main active ingredient of Radix Paeoniae. Previous studies have demonstrated that PF exerts a neuroprotective effect in in vivo models of cerebral ischemia ,21,22,232O2 , MPP+ [22O2 [+ , glutama2O2 [+ , Aβ25–35+ [25–35  and lipo+ [25–35 . They ha2+/CaMKII/CREB signaling pathway is involved in the above neuroprotective effect.Thus, as a continuation, the aim of this paper was to demonstrate the neuroprotective effect of PF in vivo and in vitro, and to elucidate whether the CaThe neurological deficit score and the infarct volume of rats were determined after PF treatment for 7 days. As shown in As shown in Western blot analysis A,B reveap < 0.01). By contrast, incubation of cells with different concentrations of PF (100 and 200 μM) alone for 24 h increased the cell viability -2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. After primary hippocampal neurons were exposed to NMDA (200 μM) for 6 h, cell viability was significantly decreased  staining was evaluated by flow cytometric analysis. As demonstrated in 2+ increased significantly after NMDA treatment compared with the control group . PF treatment significantly decreased the intracellular Ca2+ concentration .As shown in 2+/CaMKII/CREB signaling pathway in vitro, we also examined the expression of proteins in primary hippocampal neurons after NMDA-induced excitotoxicity. Consistent with results in vivo, pretreatment of PF changed the levels of CaM, CaMKII, p-CaMKII, CREB and p-CREB  and Ca2+-binding proteins. Ca2+ overload into neurons induced by ischemic insult may excessively activate Ca2+/CaM-dependent pathways and lead to irreversible cell damage [2+ concentration could inhibit the progression of brain injury, providing an extended therapeutic strategy for neuroprotection.Cerebral ischemic injury is a complicated cascade process, which is mainly differentiated into two processes: the initial tissue injury caused by ischemia and the secondary tissue injury inflicted by ischemia reperfusion . The secl damage . Thus, tNeurons are the core components of the central nervous system and can form a neural network through dendrites and axons. Neurons, along with the neural network, are the basis for neurological functions . In addiN-methyl-d-aspartate receptors (NMDAR) of postsynaptic neurons, activates the calcium channel controlled by the receptor, and results in calcium overload. Under pathological conditions, Ca2+ internal flow leads to acute edema in the cell, and secondary cell toxicity ultimately triggers neuron apoptosis. It has been proved that NMDA can result in rising Ca2+ concentration in the cytoplasm and marked elevation in Ca2+ then causes neuronal death [NMDA is an excitatory neurotransmitter, which acts on al death ,32,33. T2+ followed by intracellular Ca2+ overload, and the kinase is activated by autophosphorylation at Thr286; then the CREB protein is activated. CREB is a transcription factor present in numerous tissues, which plays a regulatory role in neuronal survival, precursor proliferation, neurite outgrowth, and neuronal differentiation in neurons [In addition, CaM is activated by combining it with Ca neurons ,15,16.2+. Therefore, we hypothesized that PF may protect against cerebral ischemia via regulating the expression of the Ca2+/CaMKII/CREB signaling pathway. Thus, in the present study, we used MCAO and NMDA-induced excitotoxicity models of ischemic stroke to further investigate the neuroprotective effects of PF as well as the underlying mechanism, by focusing on the Ca2+/CaMKII/CREB signaling pathway, that was closely related to anti-apoptotic signaling. In an in vitro study, we investigated the neuroprotective effects of PF against NMDA-induced neurotoxicity in neurons and verified its neuroprotective effects. Previous studies reported that PF exerts a neuroprotective effect in in vitro models induced by H2O2 [+ [25–35 [Thus, protecting neurons may improve neurological functions which is beneficial to neurodegeneration diseases. In a previous study, paeoniflorin protects against ischemia-induced neurons apoptosis, mainly via regulating the Bcl-2/Bax signal pathway , the Ras by H2O2 , MPP+ [2 H2O2 [+ , glutama H2O2 [+ , Aβ25–35+ [25–35  and lipo+ [25–35 . In line2+/CaMKII/CREB signaling pathway, including Bax, BcL-2, Bad and caspase 3 [Furthermore, in accordance with the hypothesis, in this study, we observed that p-CaMKII, and p-CREB were markedly decreased and these reductions were attenuated by PF treatment in both the MCAO and NMDA-induced excitotoxicity models, but the expression of CaMKII and CREB was not notably changed, suggesting that they exerted their function through the phosphorylation rather than regulation of their protein expression. It was found that PF also regulated downstream proteins along the Caaspase 3 . In conc® medium, B-27® serum-free supplement and GlutaMAX™-I were bought from Life Technologies . Antibodies to CREB, p-CREB, CaMKII, p-CaMKII, CaM and β-actin were bought from Cell Signaling Technology . Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (mouse) IgG were from Xiamen Lulong Biotech Co., Ltd. . Polyvinylidene fluoride membrane was from Merck KGaA . All other reagents were from Beyotime Institute of Biotechnology  unless otherwise stated.PF . All animals were housed in the standard laboratory animal conditions . Furthermore, all experimental protocols and handling procedures of animals were approved by the Ethics Committee of Fujian University of Traditional Chinese Medicine .Rats were fasted 12 h before surgery but were allowed free access to water. All animals were anesthetized with 10% chloral hydrate solution . Then the MCAO model was performed for the induction of focal cerebral ischemia as previously described ,36,37,38Rats that underwent the MCAO were divided randomly into three groups: the MCAO group, the PF group and the sham-operated group. All rats were administrated twice per day for 7 days as follows: rats in the PF group were intraperitoneal injected PF 5 mg/kg; rats in the sham-operated group and MCAO group received the same volume of normal saline.Neurobehavioral deficits of rats were evaluated on a five-point scale as described previously ,36,37,38After neurological evaluation, rats were deeply anesthetized and then were decapitated to take out the brain quickly. Then it was done as described previously ,37,38. Bd-lysine (Sigma) coated 96-well plate or 6-well plate and cultivated in neurobasal® medium (Life Technologies) supplemented with 2% B-27® serum-free supplement (Life technologies) and 0.5 mM GlutaMAX™-I (Life Technologies) at 37 °C in a humidified atmosphere of 5% CO2. Half of the culture medium was replaced every third day.Primary cultures of neurons were obtained from the cerebral hippocampi of newborn SD rats within 24 h. The procedures were the same as described previously ,38. In bThe NMDA-induced excitotoxicity model was performed as in our previous research . After 7The cell viability was assessed by MTT assay. Briefly, after treatment, 10 μL MTT solution (5 mg/mL) was added into each well for an additional 4 h incubation. Then MTT reagent was replaced with DMSO (100 μL per well) carefully to dissolve formazan crystals. Absorbance at 570 nm was measured in a microplate reader . Results were expressed as the percentage of the absorbance of control cells, which was considered as 100%.After treatment, cells were collected and quantitated according to the manufacturer’s protocol. Briefly, cells were resuspended in binding buffer and stained with annexin V/PI for 15 min. Then samples were analyzed by a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm . Apoptotic cells were expressed as a percentage of the total number of cells.2+ concentration was determined by alternating excitation wavelengths of between 340 and 380 nm with emission at 510 nm in a fluorescence spectrophotometer (Infinite M200 Pro). Intracellular Ca2+ concentration was expressed as a percentage of non-treated control.After treatment, the cells were washed with D-PBS and incubated with the complete medium containing 5 μm Fura-2/AM at 37 °C for 45 min. Subsequently, the cells were washed with D-PBS containing 0.2% bovine serum albumin (BSA). Then, the cells were incubated at 37 °C for another 5 min prior to measurement. Intracellular Cag for 15 min at 4 °C. Then, equal amounts of proteins were electrophoresed on 12% density sodium dodecyl sulfate, sodium salt (SDS) acrylamide gels, transferred to a polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk  in tris-buffered saline and tween 20 (TBST) for 2 h, followed by overnight incubation with antibodies to CREB (1:1000), p-CREB (1:1000), CaMKII (1:1000), p-CaMKII (1:1000), CaM (1:500) and β-actin (1:1000). Membranes were then incubated for 2 h at room temperature with HRP-conjugated secondary antibody . Finally, all specific bands were visualized using the electrochemical luminescence (ECL) western detection reagents on Image Lab analysis software . β-actin was used as a loading control. Three repeats of the experiments were performed.After treatment, cells and cortex tissues of ischemic brain were harvested and lysed by radio-immunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (PMSF) on ice; they were then centrifuged at 12,000× p < 0.05 was considered to indicate statistical significance.Data were presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA)  followed by a post hoc Fisher’s least significant difference (LSD) test to evaluate multiple group difference. In summary, the results of this study demonstrated that the neuroprotective effects of PF on cerebral ischemia-reperfusion injury were associated with the improvement of CaMKII and CREB activation, at least in part. The findings may represent a novel mechanism of PF in focal cerebral ischemia-reperfusion injury in rats. Whether other pathways or mechanisms participated in the beneficial effects of PF on ischemic brain injury needs further investigation.


Nasturtium officinale). PEITC has multiple biological effects, including activation of cytoprotective pathways, such as those mediated by the transcription factor nuclear factor erythroid 2 p45‐related factor 2 (NRF2) and the transcription factor heat shock factor 1 (HSF1), and can cause changes in the epigenome. However, at high concentrations, PEITC leads to accumulation of reactive oxygen species and cytoskeletal changes, resulting in cytotoxicity. Underlying these activities is the sulfhydryl reactivity of PEITC with cysteine residues in its protein targets. This chemical reactivity highlights the critical importance of the dose of PEITC for achieving on‐target selectivity, which should be carefully considered in the design of future clinical trials.Cruciferous vegetables are rich sources of glucosinolates which are the biogenic precursor molecules of isothiocyanates (ITCs). The relationship between the consumption of cruciferous vegetables and chemoprotection has been widely documented in epidemiological studies. Phenethyl isothiocyanate (PEITC) occurs as its glucosinolate precursor gluconasturtiin in the cruciferous vegetable watercress ( These phytochemicals are of widespread occurrence in the cruciferous vegetables, the Cruciferae or Brassicaceae family containing hundreds of genera and thousands of species, and about 15 other plant families.Glucosinolates , is physically separated from its substrate glucosinolates, yet may be present in the same cells and tissues..1.147, iNasturtium officinale (watercress) is a cruciferous vegetable that is rich in gluconasturtiin, the aromatic glucosinolate precursor of phenethyl isothiocyanate  are enriched with only one glucosinolate, gluconasturtiin, containing approximately 3% by weight.http://www.clinicaltrials.gov). Due to the presence of the electron withdrawing ITC (N = C = S) group  or reactive nitrogen species  produced by both endogenous and exogenous sources. Oxidative stress is defined as the imbalance between oxidants and antioxidants, where the former are in excess, leading to disturbances of the intracellular redox homeostasis and subsequent damage.At basal conditions, NRF2 is negatively regulated by Kelch‐like (ECH)‐associated protein 1 (KEAP1)NRF2 mediates the transcription of numerous detoxification and antioxidant genes. Examples of some of its targets are NAD(P)H‐quinone oxidoreductase 1 (NQO1), glutathione S‐transferases (GSTs), aldo‐keto reductases (AKRs), aldose reductase (AR), γ‐glutamyl peptidase (GGT), carboxylesterase (CES), heme oxygenase 1 (HO‐1), UDP‐glucuronosyltransferases (UGTs), thioredoxin (TXN), and glutamate‐cysteine ligase catalytic/modifier subunits (GCLC/GCLM).Numerous reports have documented the cytoprotective role of NRF2 activation by PEITC, and these have been recently reviewed.Figure m) concentration of PEITC was attenuated in the KKO MEF cells rescued with either the single mutant C151S or the triple mutant C151S/C273W/C288E KEAP1  concentration of PEITC caused the stabilization of NRF2 to nearly identical levels in all genotypes regardless of the mutation status of KEAP1  in primary peritoneal macrophage cells isolated from wild‐type and KEAP1C151S/C151S knock‐in mice. In close agreement with the data in MEF cells, we found that following treatment with the lowest (1 μm) concentration of PEITC, the stabilization of NRF2 was attenuated in the KEAP1C151S/C151S primary macrophage cells compared to their wild‐type counterparts  in other proteins may react with PEITC. It is interesting to note that the highest concentration of PEITC (7.5 μm) caused a significant reduction in the protein levels of α‐tubulin ,3The HSR is a transcriptional program that is elicited by the transcription factor HSF1 which in normal cells is a cytoprotective mechanism to ensure adaptation and survival during conditions of stress, including exposure to elevated temperatures or proteotoxic chemicals, oxidative stress, heat shock protein 90 (HSP90) inhibition, and proteasome inhibition. We have shown previously that numerous sulfhydryl‐reactive NRF2 activators, are able to activate the HSR through the transcription factor HSF1, although the concentrations required for HSF1 activation are higher than those that activate NRF2.In one proposed mechanism, under basal conditions, the inactive monomeric HSF1 is primarily sequestered in the cytoplasm by the HSP90 multi‐chaperone complex.Table Due to the mounting evidence that the ITCs induce heat shock proteins, we tested a series of mildly electrophilic SFN analogues, and found that sulfoxythiocarbamate alkyne (STCA) modifies cysteines in HSP90 Figure .54 We alPhosphorylation of HSF1 at serine 326 (pS326) is one of the primary and hallmark posttranslational modification (PTM) events that occur during HSF1 activation. Within minutes, PEITC induced the phosphorylation of HSF1 at S326 as well as the activation of the p38 mitogen‐activating protein kinases (MAPKs).14C]‐PEITC and mass spectrometry, PEITC has been found to bind HSP70, which is a component of the HSP90 multi‐chaperone complex.It is currently not known how PEITC inhibits HSP90, and several possibilities could be considered. First, similar to STCA, PEITC may directly modify cysteines in HSP90. Second, PEITC may modify cysteines in HSP70. By use of  PEITC activation of p53 in oral squamous cell carcinoma cell lines with a functional p53 caused an induction of the ataxia telangiectasia mutated serine/threonine kinase‐checkpoint kinase 2 (ATM‐CHK2) pathway as well as the expression of p21. Interestingly, in breast cancer cells expressing mutant p53, PEITC exposure caused the depletion of mutant p53.Inhibition of HSP90 and/or glutathione depletion, leading to the accumulation of ROS, all of which are related to the cysteine reactivity of PEITC, may provide potential mechanisms for the observation that, like other ITCs, high concentrations of PEITC are cytotoxic. PEITC arrests cells at the G2/M phase.Additionally, PEITC induces extrinsic and intrinsic apoptotic pathways in cancer cellsAnother mechanism by which PEITC may promote cell death is through its ability to modify cellular cytoskeletal elements. Human lung cancer cells (A549) treated with radiolabelled PEITC or SFN exhibited binding of the ITCs to tubulin.6PEITC has multiple intracellular targets. PEITC activates the cytoprotective transcription factors NRF2 and HSF1 and modifies the epigenome. At high concentrations, PEITC is cytotoxic. Importantly, the ability to react with cysteines has been implicated in all of the biological activities of PEITC. Hence, the on‐target selectivity of PEITC depends on its concentration. It is thus essential to carefully consider the dose and duration of treatment when designing future clinical trials.The authors declare no conflict of interest.


Twenty-six healthy young adults performed four office tasks  and transitions between these tasks while sitting on a dynamic and on a static office chair. For all task-transitions, chair sway was higher in the dynamic condition (p < 0.05). Muscle activation changes were small with lower mean activity of the left obliquus internus during hand writing (p = 0.07), lower mean activity of the right erector spinae during the task-transition calling to hand writing (p = 0.036), and higher mean activity of the left erector spinae during the task-transition reading to calling (p = 0.07) on the dynamic chair. These results indicate that an increased BioSwing® chair sway only selectively alters trunk muscle activation. Adjustments of chair properties  are recommended.Employing dynamic office chairs might increase the physical (micro-) activity during prolonged office sitting. We investigated whether a dynamic BioSwing Physical inactivity is considered an independent risk factor for many non-communicable diseases and has been entitled as biggest public health problem of the 21st century . Hence, ® chair  enables two-dimensional swinging of the seating area. These reflective micro-motions of the trunk induced by the body are believed to beneficially affect trunk motion and muscle activity, yet to date evidence of this mechanism is lacking. Moreover, typical office activities do not only comprise individual tasks but also transitions between tasks. It has been shown that the muscle activation level during sitting is task-dependent [® chair increases sway of the chair seat. Secondly, we tested the hypothesis that a greater sway of the chair induces relevant changes in trunk muscle activation during typical office tasks  and their task-transitions. Such “micro-activity” of the trunk may be particularly important when prolonged sitting cannot be avoided or when habits cannot be changed.Besides leisure time PA opportunities, work places involving mainly sitting activities  represent a challenging but also promising setting for encouraging PA. In recent years, researchers have focused on active breaks, cycling desks and standing desks for promoting occupational PA ,5,6. Howependent . Task trependent . Thus, i2) participated in this study. Written informed consent was given prior to the start of the study. Participants were blinded the chair conditions and no information on the chairs were provided. Inclusion criteria were age between 18 and 55 years of age, working most of the time in sitting position , and proficiency in German language for correct and reliable introductions of the tasks. Exclusion criteria were pain, restrictions in sitting position and pregnancy. All participants were right handed and used their right hand for both writing and calling.Twenty-six healthy young adults . We implemented a single randomized crossover-design for sitting on two different Haider Bioswing office chairs . Both chFor each chair condition, participants were asked to complete four different tasks in randomized order: reading on a computer; typing on a keyboard; writing by hand; and a phone call using a mobile phone. Each task was performed for 3.5 min followed by a 30-s task-transition period where the participants were instructed for the new task. The same sequence of the four tasks was performed twice on each chair resulting in a total sitting time of 32 min per chair. The order of the chair conditions was randomly assigned. There was a 10-min break between testing sessions on the two chairs. Trunk muscle activity and movement of the chair were measured during the middle 30 s of the task and during the 30-s task-transition using surface electromyography and reflective markers .All tests were performed under standardized conditions in an indoor laboratory. Chair height was adjusted for each participant to ensure full contact of the feet with the floor and a knee angle of 90° and was identical between chairs.For every participant, identical instructions for the tasks and task transitions were used. The task “typing on a keyboard” consisted of writing a text about the participant’s current workplace . During the task “reading on a computer”, participants were always asked to first open a text file and then read a text about office work. The task “writing by hand” consisted of writing a text about short-term and long-term goals and their implementation. For the task “calling”, participants were instructed to enter the number on their mobile phone and to hold the phone to their ear while talking about the participants’ field of work and work tasks.Chair motion and upper body position were measured using an 8-camera motion analysis system . Reflective markers were placed at the base of the left and right armrest representing the chair center  and on tAfter shaving and cleaning the skin with alcohol, six wireless surface electromyography (EMG) electrodes  were placed bilaterally on the erector spinae, obliquus internus abdominis and rectus abdominis muscles according to the guidelines of the European Surface Electromyographic Society (SENIAM) ,12.To remove electrocardiogram (ECG) contamination in the EMG signal of the trunk muscles, an independent component analysis based ECG filter was used . All EMGt-tests. Effect sizes were estimated using Cohen’s d [Differences in the length of path of the chair center and mean muscle activation levels between the two chair conditions were analyzed using paired ohen’s d . Calcula® Excel spreadsheet  [The change scores between both chairs were calculated as the 90% confidence intervals according to the magnitude-based inference approach . A practWA, USA) .On average, the length of path of the chair center for the dynamic chair was between 100 and 400% longer for the individual tasks and around 200% greater for the task transitions compared to the static chair with a high between subject variability . For thep = 0.041, effect size = 0.349). For the other tasks and all task transitions, there were no significant differences in the length of path between the chairs . For the transition from calling to hand writing, the mean intensity of the right erector spinae was likely lower for the dynamic than the static chair . Moreover, there was a possibly lower mean intensity of the right erector spinae during the transition from reading to hand writing and typing to hand writing, of the left obliquus internus during the transition from calling to hand writing, of the left rectus abdominis during the transition from reading to typing, and of the right rectus abdominis during the transition from calling to hand writing for the dynamic chair compared to the static chair. All observed effect sizes were small  the path length of the chair center was significantly longer for the dynamic chair during calling and all task transitions and (b) muscle activation during all tasks did not differ between the chairs but showed small to notable changes during the task transitions reading to calling, calling to writing and typing to writing. Specifically, for the transition between reading and calling bilateral erector spinae muscle activation was higher for the dynamic chair than for the static chair but effect sizes were small and MBI revealed only a trivial effect. For the transitions between calling and writing and between typing and writing the right erector spinae muscle activation was lower for the dynamic chair than for the static chair with very small effect sizes and trivial MBI.The aim of this study was to investigate whether a BioSwingThe longer path length of the chair center for the dynamic than for the static chair confirms that the springs facilitate motion of the seat. This is comparable to previous results with a different dynamic office chair that showed greater chair movements (measured on the top of the backrest) when performing office tasks such as typing on a keyboard and placing files in folders while sitting on a dynamic compared to a conventional chair . During ® chair used in our study has a backrest, the observed changes in muscle activation are likely attributable to the dynamic swinging system.Previous studies on trunk muscle activation during sitting on different chairs reported different changes in muscle activation levels depending on the design of the tested chairs and measured muscles. No differences in lumbar and thoracic erector spinae activation were reported for reading a book, word-processing and computer-aided design between sitting on a chair with fixed seat and backrest, a chair which allowed independent sagittal plane rotation of the seat and back rest and a chair which allowed a rotation in a fixed ratio between the seat and back rest . Similar® chair in our study. In contrast to these previous studies [Generally, muscle activation levels during seated office tasks were small  in all studies ,9. Altho studies , our stu studies ,9.Contrary to our initial hypothesis, we observed mainly possible or likely lower trunk muscle activity during task transitions while sitting on the dynamic compared to the static office chair. This result is in contrast to a previous study that showed an increase in the thoracic erector spinae muscle activation for sitting on an unstable ball compared to a conventional chair  but agreThe tested dynamic and static chairs in this study were identical except of the swinging mechanism being enabled for the dynamic and disabled for the static chair. Moreover, participants were blinded to the differences between chairs before the experiment to reduce potential conscious adaptions in sitting posture and movements during the measurements. To reflect common working situations, we not only investigated muscle activations during the performance of single tasks but also during task transitions. The randomization of the order of the tasks resulted in 12 possible task transitions with not every participant performing the same task transitions. To increase the number of observations per task transition, both directions of the task transition  were pooled for the analysis. This might have increased the variability in muscle activation patterns but should have little effects on mean muscle intensity over the entire 30-s measurement. Nevertheless, it is possible that the data pooling reduced the observed differences and resulted in smaller differences between chair conditions for task transitions. In this study, we tested the experimental chairs in 26 subjects. Similar results would have to be confirmed in a larger population. Moreover, the study population consisted of healthy young adults, and hence it is not clear if older adults or persons with back problems would experience similar differences. Nonetheless, the data presented in this study represents reference data for future studies on the effect of office chair design on trunk muscle activity chair motion.The increase in the path length of the chair center of the dynamic chair indicates changes in the chair motion of the dynamic chair compared to those of the static chair. However, the observed altered chair motion was associated with only small differences in trunk muscle activation levels for sitting on the dynamic chair compared to the static chair. In addition to previous studies, not only office tasks but also tasks transitions were investigated and it was seen that there are more changes in trunk muscle activation levels during these task transitions than for the individual tasks. Hence, investigating task transitions might be more sensitive in assessing effects of dynamic chairs on muscle activation.Greater changes in muscle activation levels may be achieved when removing the stabilizing effect of placing the feet on the floor by adding a foot rest facilitating swinging of the whole body. The effect of the dynamic chair on chair motion and muscle activity levels may depend on the stiffness of the swinging elements, and it may be necessary to adjust the stiffness of the swinging elements to the user’s body weight. Nonetheless, from an epidemiological perspective even small changes in muscle activity during office tasks and task transition may be meaningful in the long run considering that many professionals perform mainly seated tasks during their work life over decades.


The ability to detect residual levels of leukemic blasts  has already been integrated in the daily routine for treatment of patients with chronic myeloid and acute lymphoblastic leukemia. In acute myeloid leukemia (AML), a variety of mostly retrospective studies have shown that individuals in AML remission who tested positive for MRD at specific time-points or had increasing MRD levels are at significantly higher risk of relapse and death compared to MRD-negative patients. However, these studies differ with respect to the “MRD-target”, time-point of MRD determination, material analyzed, and method applied. How this probably very valuable MRD information in individual patients may be adapted in the daily clinical routine, e.g., to separate patients who need more aggressive therapies from those who may be spared additional—potentially toxic—therapies is still a work-in-progress. With the exception of MRD assessment in acute promyelocytic leukemia (APL), the lack of randomized, prospective trials renders MRD-based decisions and clinical implications in AML a difficult task. As of today, we still do not have proof that early intervention in MRD-positive AML patients would improve outcomes, although this is very likely. In this article, we review the current knowledge on non-APL AML MRD assessment and possible clinical consequences. MRD-)” as the optimal response category for AML patients [NPM1 mutations in NPM1-mutated AML. Here, we review commonly available MRD detection methods in non-APL AML together with their clinical applicability and current practical consequences in daily routines.Acute myeloid leukemia (AML) is a highly heterogeneous and aggressive neoplasia that is characterized by clonal expansion of myeloid precursors. Over the past few decades, a growing understanding of the molecular landscape and the associated AML biology allowed improved risk stratification at diagnosis. These findings are also reflected in the current World Health Organization (WHO) and European LeukemiaNet (ELN) classifications for AML patients ,2. Unforpatients ,10. Whilpatients ,14,15,162 in patients with aberrant cytogenetics. Already this approach allowed risk stratification in remission samples and paved the way for today’s MRD assessments [After achieving first lasting remissions in AML in the 1960s, for many decades, cytomorphologic assessment with a sensitivity of around 1:20 and high interobserver variability remained the only method to evaluate treatment responses. Later, fluorescence in situ hybridization (FISH) allowed detection of leukemic cells with a sensitivity of up to 1:10essments ,5,6. CurMFC uses fluorescence labeled antibodies to mark antigens on leukemic  cells. It has been integrated in the standard diagnostic work up at AML diagnosis where it allows the differentiation of AML from other diseases, e.g., lymphatic leukemia, but also to define a leukemia-associated immunophenotype (LAIP). This LAIP consists of distinct antigen expression patterns, is unique for the individual AML at diagnosis, and can be tracked as MRD in follow-up samples. A second strategy that is used for MFC-based MRD detection is the “different from normal” (DfN) approach. Here, aberrant immunophenotypic maturation patterns are searched for in AML remission samples. The latter approach does not have an obligatory need for diagnostic material and is also useful to identify new or changed immunophenotypic aberrancies, e.g., due to clonal evolution. Today, the ELN recommends a combination of both approaches; however, as the LAIP also—in most cases—represents a DfN approach, the differences between both methods will most likely diminish over time when larger antigen panels (minimum of eight colors) are used . A clearCBFB-MYH11 in inv(16)(p13q22) or t and RUNX1-RUNX1T1 in t), as well as recurrent mutations, such as NPM1 mutations. While there is only one known transcript in RUNX1-RUNX1T1, the three most common breakpoints in CBFB-MYH11 AML [NPM1-mutated AML [−3 for MLLT3-KMT2A [−6 for NPM1 mutated AML [WT1) is overexpressed in a large proportion of AML patients and WT1 expression levels have been repeatedly published as potential MRD markers at various time points [WT1 is also expressed in peripheral blood (PB) and bone marrow (BM) of healthy individuals, this physiological background limits the assay’s sensitivity. Thus, the ELN recommends using this marker only in combination or if no other specific marker is available [BAALC) [MN1) [WT1 expression. Another limitation of qRT-PCR remains the need of control-target serial dilutions, which can be overcome by modern methods such as ddPCR assays that also allow a more sensitive MRD detection than qRT-PCR without the need of reference standard urves [qRT-PCR represents an established MRD evaluation method that also provides a high sensitivity, relative user-independence, and has—at least to some extent—undergone standardization processes ,31. HereX1T1 in t;21 ,40, and C) [MN1) ,42 have rd urves ,44. As trd urves .CBFB-MYH11 and RUNX1-RUNX1T1 allows risk stratification in CBF-AML patients achieving a CR [Due to their excellent response rate to intensive chemotherapy of around 90% and high salvage rate of around 60% in relapsing patients, chemotherapy is usually the chosen consolidation option in CBF-AML. Despite lower assay sensitivity, already 20 years ago, it has been known that assessment of ing a CR ,47. Sinc5ABL1 copies, after only one cycle of intensive chemotherapy, MRD copy numbers of <100 in BM or <10 in PB for CBFB-MYH11 AML [RUNX1-RUNX1T1 AML have been shown to associate with lower relapse risk and longer OS. However, there are also studies not finding a prognostic significance at this early time-point [ABL1) [RUNX1-RUNX1T1 AML at this time-point [5ABL1 copies, after three cycles of chemotherapy, MRD copy numbers of <10 in PB for CBFB-MYH11 [RUNX1-RUNX1T1 AML [RUNX1-RUNX1T1, BM MRD-negativity at the end of treatment was found to associate with OS and disease-free survival (DFS) or relapse probability in some studies [CBFB-MYH11 AML and 4.9 and 4.5 months for BM and PB, respectively, in RUNX1-RUNX1T1 AML [Normalized to 10YH11 AML  and <500YH11 AML  or >2.5 YH11 AML  BM MRD rme-point ,50. Aftet [ABL1) ,49,51 asFB-MYH11  and <500X1T1 AML  were pro studies  while an studies . In an a studies , the medX1T1 AML ,52. The X1T1 AML ,51. HoweX1T1 AML ,52,53. HNPM1 mutations are present in approximately 30% of AML patients and are probably among the most evaluated MRD markers in AML, despite a limited comparability due to heterogeneity in the applied assays, tissue used, and time-point of assessment [NPM1 mutations are present at relapse in nearly all initially NPM1-positive patients makes this mutation a reliable MRD marker [NPM1 MRD analysis in aplasia after induction therapy seems to provide only limited prognostic information [NPM1 transcript levels, as well as still detectable [NPM1 transcripts or levels >0.01%  [NPM1 wild type copies) [NPM1 transcript levels after the second induction therapy to be highly informative for outcome, but also that MRD analysis was superior to diagnostic molecular genetic markers assessed by NGS [NPM1 transcript levels and remission duration. Using an arbitrary cut-off of 200 mutated NPM1/104ABL1, all patients exceeding this cut relapsed with a median time to relapse of 2.6 months (range 0.4–23.6 months) [NPM1 MRD prior to an allogeneic HSCT was also shown to provide prognostic information independently of the used conditioning regimen in a variety of retrospective studies [NPM1mutatedFLT3-ITDpositive patients with a >4 log reduction of NPM1 MRD—but not those with a <4 log reduction—did not benefit from allogeneic HSCT in first CR [NPM1 MRD assessment may have the potential to identify patients who benefit from more intensive treatment strategies, which will be further discussed below. Finally, Shayegi et al. observed that, during follow-up, the applied treatment may influence the optimal NPM1 MRD threshold, as patients whose MRD increased above 1% BM NPM1/ABL1 after chemotherapy, but 10% after allogeneic HSCT had shorter OS and DFS [sessment ,54. The D marker ,54. Whilormation , for allormation  or <3 loormation ,56 reductectable  NPM1 trato ABL1)  or >0.1  copies)  were pred by NGS . Krönke  months) . As some studies ,58,59. Ifirst CR . Thus, N and DFS .DNMT3A, TET2 and ASXL1) can reflect pre-leukemic clones rather than residual AML [A growing understanding of the genomic landscape in AML led to considerable interest in developing MRD tests using NGS to quantify somatic mutations during disease course. The sensitivity of NGS assays depends on the DNA quality and quantity, as well as the coverage of the sequenced genes. Other limitations arise from clonal heterogeneity with potential outgrowth of small subclones at AML relapse, the intrinsic error rates of NGS platforms, and that detection of mutations is highly dependent on a sufficient depth of sequencing and bioinformative algorithms for mutation calling . Addressdual AML ,65,66. Sdual AML ,67. Nevedual AML . Despitedual AML ,70 as wedual AML ,68,71.NPM1-mutated AML [WT1 based MRD, where PB seems to be superior to BM analyses due to a higher physiologic WT1 expression in the latter [One major question relates to the optimal specimen for MRD analyses, since the specimen may have great influence on MRD sensitivity irrespective of the applied methodology. In general, 20 mL PB (or more with white blood counts below 1 Gpt/l) or 5–10 mL BM  are needed for reliable MRD evaluation . On the ated AML ,15,59 orated AML . This inated AML , especiaated AML . As a ree latter .NRAS, KRAS, PTPN11, and KIT associated with worse outcomes, but most likely are later, subclonal events which might lack the high sensitivity wanted for a robust MRD marker [CEBPA, RUNX1 or GATA2 [DNMT3A, TET2, ASXL1 [JAK2 [WT1, BAALC or MN1 may be of help to assess an MRD status in individuals lacking targetable specific aberrations [With the introduction of NGS technology, in nearly all individuals, molecular markers may be identified and potentially used for MRD assessment ,77. HoweD marker . Additioor GATA2 . As the or GATA2 ,82. Furt2, ASXL1 , or JAK2L1 [JAK2  may alsorrations ,40,41,42rrations  or multirrations  may alloRUNX1-RUNX1T1 and especially CBFB-MYH1 have been reported to be slower than most other AML subtypes. Here, a molecular relapse can precede hematologic relapse for up to one year [NPM1 mutated AML seem to be highly variable and dependent on the molecular context, as faster relapses were linked to the presence of a FLT3-ITD with a median time to relapse of 3.5 and 6.5 months for FLT3-ITD-positive and FLT3-ITD-negative patients, respectively [In general, MRD can be assessed at either “early timepoints“ during therapy cycles to assess kinetics of treatment responses or at “later timepoints“ e.g., after completion of therapy to detect impending morphologic relapse . There aone year ,87. In cectively . The recectively . Additioectively . During ectively .Similar to the variety of assessable MRD time-points, there is a large heterogeneity regarding the optimal thresholds to define MRD-positive and -negative patients. Different studies made it clear that repetitive detection of high MRD levels, as well as rising MRD levels after molecular remission can reliably predict frank relapse ,48. CurrThe repeatedly reported close association between MRD test results and clinical outcome has led to considerable interest in using MRD information for clinical decision-making in everyday therapies. Until today, no randomized studies have been conducted to validate the common suggestion that initiation or intensification of therapies in MRD-positive patients will reduce the risk of hematologic relapse and improve OS in AML patients. The question remains as to whether survival will be truly improved if an individual receives pre-emptive treatment with “only“ a positive MRD test result compared to subjects with frank morphologic relapse . AdditioNPM1-based MRD in PB and defined MRD-negativity as a >4 log reduction. While patients with positive MRD had higher relapse risk and shorter OS, a subanalysis in ELN intermediate risk patients also suggested a favorable impact of allogeneic HSCT on OS in MRD-positive individuals. DFS and OS were similar for MRD-negative patients irrespective of the form of consolidation , as well as for MRD-positive patients undergoing allogeneic HSCT. On the other hand, MRD-positive patients consolidated with chemotherapy had significantly shorter DFS and OS [Allogeneic HSCT is considered to reduce relapse risk at the cost of higher non-relapse mortality and, thus, recommended for patients with higher disease risk in first CR or after relapse. In general, an improvement of DFS of >10% has been adopted to justify the decision towards an allogeneic HSCT . As prevS and OS . VendittS and OS  publisheS and OS ,71. A poS and OS . The GvLS and OS ,104. TakRUNX1/RUNX1T1 or NPM1 mutation levels. With an acceptable safety profile, azacitidine prevented hematological relapse in 51% of patients. In the remaining patients, relapse could be delayed to a median time of 422 days [The data on AML patients with molecular relapse or persisting MRD receiving therapeutic intervention are still limited, but experiences from ALL therapy—where MRD triggered treatment with blinatumumab improves outcomes—showed promising results . Also in422 days , which w422 days ,75,82. A422 days . Despite422 days , IDH2 [1422 days , FLT3-IT422 days  and/or F422 days , surface422 days , or drug422 days  have beeA lot of progress has been made in introducing MRD analyses in the clinical routine, and MRD assessment is already recommended by the ELN for some AML subgroups . Results


In addition, our studies confirmed that the designed oligonucleotide would induce irreversible damages to Btopo I and without any effects occur to eukaryotic topoisomerase I. It is our hope that the results provided in these studies could provide a novel way to inhibit Btopo I.Bacterial topoisomerase I (Btopo I) was defined as potential target for discovery of new antibacterial compounds. Various oligonucleotides containing bulge structure were designed and synthesised as inhibitors to Btopo I in this investigation. The results of this study demonstrated that the designed oligonucleotides display high inhibitory efficiency on the activity of Btopo I and the inhibitory effect could be modulated by the amount of bulge DNA bases. The most efficient one among them showed an IC However, Wang and co-worker have discovered that Btopo I can relax positive supercoiled DNA which containing a single-stranded loopReagents: Plasmid DNA pUC19 and bacterial topoisomerase I (E. coli topoisomerase I) were purchased from New England Biolabs . Eukaryotic topoisomerase I was purchased from Takara Bio . Single strand oligonucleotides were purified by HPLC and provided by Shanghai Generay Biotech Co., Ltd (China).Preparations of 84 bp duplex oligonucleotide: In a solution containing two single strand oligonucleotides, 50 mM NaCl was kept at 90 °C for 10 min followed by allowing it to cool down to room temperature over a period of 1 h.Reactions of Btopo I with pUC 19 and designed duplex oligonucleotide: A mixture containing 50 mM potassium acetate, 20 mM Tris-acetate, 100 µg/ml bovine serum albumin (BSA), 10 mM magnesium acetate, 250 ng pUC 19, 1 U of Btopo I, and each designed duplex oligonucleotide was prepared, respectively, and further incubated at 37 °C for 30 min. After incubation, the product was analysed by 1% agarose gel electrophoresis. The DNA bands were captured using Gel Documentation System .Reactions of eukaryotic topoisomerase I with pUC 19 and designed duplex oligonucleotide: A mixture containing 72 mM potassium chloride, 35 mM Tris-acetate (pH 8.0), 5 mM magnesium chloride, 5 mM dithiothreitol (DTT), 5 mM spermidine, 0.01% BSA, 250 ng pUC 19, 1 U of eukaryotic topoisomerase I, and each designed duplex oligonucleotide was prepared, respectively, and further incubated at 37 °C for 30 min. After incubation, the product was analysed by 1% agarose gel electrophoresis. The DNA bands were captured using Gel Documentation System .B-0, respectively, annealing with others 94-mer single strand 1, 2, 3, 4, 5, 6 to obtain bulge oligonucleotide B-1–1, B-1–2, B-1–3, B-1–4, B-1–5, B-1–10 containing different length of single strand loop. Six 84 bp duplex oligonucleotides have the same base composition, but including different numbers of bulge base. In order to ensure duplex oligonucleotides with same base composition, from 1 to 6 . These low micromolars IC50 suggest that the bulge oligonucleotides could indeed reduce the activity of Btopo I and act as potent inhibitor of this enzyme. Furthermore, consistent with the phenomenon in 50 decrease inversely to the increasing length of the single-stranded loop (bulge loop). B-1–1 containing least bulge base (shortest single-stranded loop) displays the minimum inhibitory effect and the largest IC50 as 233.2 nM, approximate five times as that of the most bulge base duplex oligonucleotide, B-1–10. The above situations indicate that duplex oligonucleotides with more bulge base exhibit effective suppression on Btopo I activity. Strong suppression of Btopo I activity in presence of more bulge base duplex may result from the preferential binding of the enzyme to the bulge loop. The results from these studies revealed that the length of bulge loop in designed oligonucleotides is crucial factor for the inhibitory action of Btopo I and the activities of this enzyme can be modulated by the length of free single strand in duplex oligonucleotides.The correlation between the inhibitory efficiency and the quantity of bulge base is further explored by determining the ICB-1–10 was applied to pUC 19 relaxing assay alone and the time of relaxing assay was lengthened. As shown in B-1–10, almost all pUC 19 became relaxed status (lane 7) even after the reaction time reach 3.5 h and an extra 250 ng pUC 19 was load in the reaction mixture. It is speculate base on the above results that Btopo I nick the single-stranded loop and then not be able to splicing between these slice fragments properly due to the bugle base without corresponding base for complementation, resulting in irreversible damages to the enzyme. The above observations indicate that bulge DNA could serve as an irreversible inhibitor. In addition, with the purpose to identify bulge oligonucleotides as specific inhibitor for Btopo I, eukaryotic topoisomerase I  was utilised to study the inhibition of bugle oligonucleotides to the enzyme. As seen in In order to further exploring whether bulge DNA could induce irreversible inactivation to Btopo I, new assay was designed to determine the inhibitory irreversibility of bulge DNA. In new assay, oligonucleotide B-1–10 (IC50 was 63.1 nM). Moreover, the data of assay illustrated that more potent inhibition could be found with appropriate prolonging of bulge loop structure. The results based on our research suggested that these serious of designed bulge oligonucleotides could serve as a kind of irreversible inhibitor to Btopo I. In addition, the designed substrates would possess specific targeting on Btopo I and have no any reaction with eukaryotic topoisomerase I. The investigation here demonstrates the possibility of bulge oligonucleotide to become a new therapeutic agents targeting Btopo I. Although oligonucleotide is difficult to penetrate into bacteria in current condition, we believe that it has possible to facilitate the transfection of oligonucleotide into bacteria accompany with the advancement of nano-technology and pharmaceutics. It is our expectation that the outcomes provided in these investigations could benefit the future design and optimisation of some new DNA-based therapeutical agents which target Btopo I for antibacterial treatment.In conclusion, various types of duplex oligonucleotides with bulge structure were designed and synthesised in this studies, which exhibited high inhibitory efficiency on the activity of Btopo I, especially No potential conflict of interest was reported by the authors.


As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer’s disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the presenilin-1-associated γ-secretase, leading to dysregulation of Ca2+ homeostasis. In this report, we investigated expression levels of STIM1 in brain tissues  of pathologically confirmed Alzheimer’s disease patients, and observed that STIM1 protein expression level decreased with the progression of neurodegeneration. To study the role of STIM1 in neurodegeneration, a strategy was designed to knock-out the expression of STIM1 gene in the SH-SY5Y neuroblastoma cell line by CRISPR/Cas9-mediated genome editing, as an in vitro model to examine the phenotype of STIM1-deficient neuronal cells. It was proved that, while STIM1 is not required for the differentiation of SH-SY5Y cells, it is absolutely essential for cell survival in differentiating cells. Differentiated STIM1-KO cells showed a significant decrease of mitochondrial respiratory chain complex I activity, mitochondrial inner membrane depolarization, reduced mitochondrial free Ca2+ concentration, and higher levels of senescence as compared with wild-type cells. In parallel, STIM1-KO cells showed a potentiated Ca2+ entry in response to depolarization, which was sensitive to nifedipine, pointing to L-type voltage-operated Ca2+ channels as mediators of the upregulated Ca2+ entry. The stable knocking-down of CACNA1C transcripts restored mitochondrial function, increased mitochondrial Ca2+ levels, and dropped senescence to basal levels, demonstrating the essential role of the upregulation of voltage-operated Ca2+ entry through Cav1.2 channels in STIM1-deficient SH-SY5Y cell death.STIM1 is an endoplasmic reticulum protein with a role in CaSTIM1 protein expression decreases with the progression of neurodegeneration in Alzheimer’s disease.STIM1 is essential for cell viability in differentiated SH-SY5Y cells.2+ entry-dependent cell death.STIM1 deficiency triggers voltage-regulated CaMitochondrial dysfunction and senescence are features of STIM1-deficient differentiated cells.The online version of this article (10.1007/s00109-018-1677-y) contains supplementary material, which is available to authorized users. The Ca2+-sensitive EF-hand domain at the N-terminus rules the conformational change in STIM1 when the intraluminal Ca2+ concentration drops below a threshold, and this change triggers the clustering of STIM1 in plasma membrane-ER juxtapositions where it binds store-operated Ca2+ channels in order to activate the Ca2+ influx pathway termed store-operated Ca2+ entry (SOCE) , with an essential role in Caiewed in , migratiiewed in –8.2+ stores i) was measured in fura-2-AM-loaded cells as described elsewhere [Cytosolic-free calcium concentration . SOCE was measured by monitoring the increase of the [Ca2+]i after the addition of 2 mM CaCl2 to the Tg-containing medium.Depletion of Ca2+]i upon depolarizing conditions was measured in Ca2+-containing HBSS assay medium (1.26 mM CaCl2). To trigger plasma membrane depolarization, HBSS with 90 mM KCl + 5 mM CaCl2 was added to cells for 1 min, and then the cells were returned to the initial Ca2+-containing HBSS medium with 5.33 mM KCl. When required, Ca2+ channel inhibitors were added from a stock solution dissolved in water or DMSO. Calibration of the fura-2 ratio signal was performed by adding 5 μM ionomycin + 5 mM EGTA to cells in Ca2+-free HBSS (to assess Rmin), followed by 5 μM ionomycin + 5 mM Ca2+ (Rmax). [Ca2+]i was calculated as [Ca2+]i = (R-Rmin)/Rmax-R) × Kd × (Sf2/Sb2), where Sf2 and Sb2 correspond to the emission of fluorescence when the dye is excited at 380 nm under Ca2+-free and Ca2+-saturating conditions m) was measured as described elsewhere [2+]m was assessed by measuring CFP, YFP, and FRET efficiency between the two channels. Excitation and emission fluorescence wavelengths were selected with the dual CFP/YFP-2 × 2 M-B filter set (Semrock). All measurements were performed at 36–37 °C in Ca2+-containing HBSS for 4–5 min. Spectral unmixing  was performed by determining the bleedthrough coefficients as described in [ROI − FRETbackground) / (CFPROI − CFPbakground) was converted to [Ca2+]m as described in i in response to 90 mM KCl  through retroviral infection of STIM1-KO cells. This long-term silencing of CACNA1C gene transcripts reduced Ca2+ entry in response to depolarization  in differentiated STIM1-KO cells fell back to the background level after silencing Cav1.2 expression . However, the [Ca2+]i in STIM1-KO cells with silenced CACNA1C (54.3 ± 5.9 nM) was significantly lower than that observed for wild-type cells (80.6 ± 4.9 nM), suggesting that the increase of [Ca2+]m was not significantly influenced by the steady-state value of the [Ca2+]i.To test whether the upregulation of this Caion Fig. b, with tion Fig. c. Moreovion Fig. c. CACNA1lls Fig. d, and [Clls Fig. e. The in2+ transport through L-type VOCCs underlies mitochondrial dysfunction and senescence during differentiation of SH-SY5Y cells in the absence of STIM1.Altogether, these results confirmed that the upregulation of Ca2+ dysregulation in the pathogenesis of familial AD. However, the more frequent sporadic late-onset AD is genetically complex, and therefore the lack of suitable model animals makes the study of the involvement of Ca2+ signaling in this form of the disease more challenging.Early-onset familial Alzheimer’s disease-3 (AD3) is caused by heterozygous mutation in the PSEN1 gene on chromosome 14q24 . PSEN1 and PSEN2 are important determinants of γ-secretase activity, which is responsible for the proteolytic cleavage of the amyloid precursor protein (APP). Moreover, the transmembrane domain of STIM1 is also a target for the PSEN1-associated γ-secretase , and it 2+ signaling and Ca2+ homeostasis dysregulation in neurological diseases. In this regard, such genome editing tools as CRISPR/Cas9 provide the opportunity to knockout specific genes with a minimal impact on the genome, i.e., without triggering genome instability. To this end, we used a nickase version of the Cas9 nuclease, a strategy that reduces the number of off-targets because it requires two independent guide RNAs to define the target sequence to be edited. This approach, which had been successfully developed in our laboratory for other cell types [In this work, we report for the first time that a decrease of STIM1 content is associated with neurodegeneration in sporadic AD human brain. Thus, this type of study of cell behaviour with decreased STIM1 levels is an experimental approach to understanding the role of Call types , 32, res2+ dysregulation triggered by the absence of STIM1 in SH-SY5Y cells, similarly to what it has been described for fibroblasts from patients with loss-of-function mutations in ORAI1 or STIM1 loci [2+ also regulates the activity of three enzymatic reactions of the citric acid cycle: isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and pyruvate dehydrogenase, which is activated by the Ca2+-sensitive pyruvate dehydrogenase phosphatase . Here, hod-2-AM . Howevercitation . Here, w2+ channels, such as the voltage-operated Ca2+ channels (VOCCs), Cav1.2 or Cav3.1, but STIM1 has also been implicated in the regulation of the glutamate ionotropic receptor AMPA type subunits 1/2 [2+ entry mainly by inhibition of the Cav1.2 channel and triggering its internalization [v1.2, and predict an augmented Ca2+ entry through L-type VOCCs in those (patho-)physiological conditions that develop with decreasing levels of STIM1. This appears to be the case in AD according to recent findings with familial AD patient fibroblasts [2+ channel current density in these specific neurons, suggesting a contribution of VOCCs to the selective vulnerability of CA1 neurons to tau pathology, and a possible role to neuronal degeneration in AD patients [v1.2 expression in vitro and inhibiting Ca2+ influx [It is also important to consider that the increasing levels of STIM1 observed during differentiation were not accompanied by any increase in SOCE see Fig. b, c. On nits 1/2 , and thenits 1/2 . With relization , 13. Ourroblasts  and the roblasts . In thispatients . Accordipatients , furtherpatients . Similarpatients , and isr+ influx , support+ influx ).2+ entry through VOCCs. The silencing of a specific gene transcript (CACNA1C for Cav1.2 channel) prevents the senescence triggered by the absence of STIM1, restores the basal levels of mitochondrial inner membrane potential and the mitochondrial respiratory complex I activity in STIM1-deficient cells, fully supporting a causal link between STIM1 reduction and cell death. Altogether, the results prove that Cav1.2 upregulation is essential to trigger cell death in STIM1-deficient cells, a proposal that fits well with the protective effect of dihydropyridines against neurodegeneration in AD patients.Because a continuing decline of STIM1 levels is observed in aged neurons , and als2+ channels, the observation that there is a significant loss of STIM1 in hippocampal human tissue from AD patients supports a role for Ca2+ transport and signaling dysregulation in AD. Our results demonstrate that one of the most important downstream targets for the absence of STIM1 in differentiated neuroblastoma cells is the upregulation of Ca2+ influx through Cav1.2, which fully explains the Ca2+ dysregulation observed in hippocampal neurons from model animals as well as the protective effect of VOCCs inhibition in AD patients. In this regard, STIM1-KO cells constitute an in vitro model with which to study the pathogenesis of the disease, and will be useful to help understand the role of STIM1 in the modulation of additional channels and pathways involved in neurodegeneration, with a possible impact on the search for new therapeutic targets.Because STIM1 is a key regulator of CaESM 1(PNG 2673 kb)


Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn2+ surplus. Using cells expressing the Ca2+-sensitive photoprotein aequorin we found that Mn2+ augmented the Ca2+ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn2+-high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn2+ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn2+ activate TRPY1 in the response to oxidative stress.Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The  Saccharomyces cerevisiae has been constantly used as a model eukaryote to study the calcium-dependent response to various types of external stresses, which include salt [S. cerevisiae cells respond to such stresses by a sudden increase in cytosolic Ca2+—denoted henceforth [Ca2+]cyt—following the stimulus-dependent opening of Ca2+ channels situated in the plasma membrane and/or in internal compartments. Abrupt increase in [Ca2+]cyt represents a versatile and universally used mechanism which triggers either cell survival/adaptation or cell death [S. cerevisiae the stress-dependent rise in [Ca2+]cyt can be a consequence of Ca2+ influx via the Cch1/Mid1 channel on the plasma membrane [2+ into the cytosol through the vacuole-located Ca2+ channel TRPY1 [2+]cyt is restored through the action of Ca2+ pumps and exchangers [2+-ATPase Pmc1 [2+/H+ exchanger Vcx1 [2+]cyt into the vacuole, while Pmr1, the secretory Ca2+-ATPase, pumps [Ca2+]cyt into endoplasmic reticulum (ER) and Golgi along with Ca2+ extrusion from the cell cyt elevation under hyperosmotic shock [2+ [2+]cyt has its origin exclusively from the cell’s exterior, with the Cch1/Mid1 channel solely responsible for the majority of Ca2+ entry, and with no contribution of vacuolar Ca2+ [2+]cyt waves which pool both external and vacuolar Ca2+ [2+]cyt under oxidative stress and TRPY1 depends on [Ca2+]cyt elevation for maximal gating, in a process known as Ca2+-induced Ca2+ release cyt cyt elevation induced by H2O2 exposure  and GdCa2+]cyt . For thi cytosol . The celexposure a. Remarkock HOS, b,c. Surpat 10 µM c, a non-2+ influence on elevating [Ca2+]cyt under H2O2 was the result of TRPY1 stimulation by Mn2+. To test this possibility, we determined the effect of Mn2+ on the Ca2+-mediated response to H2O2 of cells completely lacking TRPY1. It was noticed that in WT cells expressing reconstituted aequorin, the H2O2-induced Ca2+-dependent luminescence was significantly increased by cell pre-incubation with 10 µM Mn2+, indicating that in the case of WT cells too, Mn2+ potentiates the Ca2+-dependent response to oxidative stress  with a luminescence curve (2+ (PMR1 encodes the major Golgi/ER membrane P-type ATPase ion pump responsible for transporting Ca2+ and Mn2+ into the Golgi apparatus cyt elevation—neither under low (0.05–1 mM) nor under high  surplus . The Mn2+ concentrations found to augment the H2O2-induced stimulation of TRPY1 were within the physiological limits (10–50 µM) and far below the concentration that would induce a hyperosmotic shock, explaining why the TRPY1 was not extra stimulated by Mn2+ under hyperosmotic stress  failed to alleviate the haploinsufficiency showed by trpy1Δ/TRPY1 strain. That TRPY1 haploinsufficiency in LMeMM is rescued by Mn2+ can be explained in three ways: (1) the supplemental Mn2+ simply counteracts the deficiency of essential metals of the LMeMM, providing the necessary amount of cations  that support cell fitness; (2) Mn2+ stimulates TRPY1 activity by increasing the Ca2+ release to the cytososl, and consequently by stimulating other components involved in maintaining the cell fitness; (3) Mn2+ generates reactive oxygen species (ROS) by a Fenton-like reaction augmenting the oxidative stress and indirectly stimulating TRPY1. The fact that surplus Mn2+ augments the TRPY1-related increase in [Ca2+]cyt under oxidative stress clearly correlates with the Mn2+ cytosolic level, as the strain lacking the high-affinity plasma membrane Mn2+ transporter Smf1 exhibited only a modest increase in [Ca2+]cyt under H2O2, when compared with WT (smf1Δ was sensitive to H2O2 cyt waves, the cell can be led towards adaptation, survival (sometimes with growth arrest or slow growth) or death [trpy1Δ/trpy1Δ cells are probably more fit because they are never “bothered” by periodic nuisance caused by Ca2+ release from the vacuole; on the other hand, trpy1Δ/TRPY1 cells need to find the right balance in Ca2+ gating while depending on only one gene copy, and sometimes extra help—external Ca2+ carried by Cch1/Mid1, mechanical force [2+—may contribute to find the most suitable path to be followed. TRPY1 of rosmotic  and oxidrosmotic . Startinc stress b. It was release ,51. In t of Ca2+ ; this wo with WT c. In thi to H2O2  probablyimits 10– µM and for death ,20,21,23al force ,51 or ev