Paediatric acute myeloid leukaemia (AML) is a heterogeneous disease characterised by genetics and morphology. The introduction of intensive chemotherapy treatments together with patient stratification and supportive therapy has resulted in a moderate improvement in patient prognosis. However, overall survival rates remain unacceptably poor, with only 65% of patients surviving longer than 5 years. Recently age-specific differences in AML have been identified, highlighting the need for tailored treatments for paediatric patients. Combination therapies have the potential to improve patient prognosis, while minimising harmful side-effects. In the laboratory setting, identifying key combinations from large drug libraries can be resource-intensive, prohibiting discovery and translation into the clinic. To minimise redundancy and maximise discovery, we undertook a multiplex screen of 80 apoptotic-inducing agents in paediatric AML pre-clinical models. The screen was designed using an all-pairs testing algorithm, which ensured that all pairs of compounds could be tested, while minimising the number of wells used. We identified a combination of ABT-737, a Bcl-2 family inhibitor and Purvalanol A, a CDK inhibitor, as a potential targeted therapy for AML patients with an MLL rearrangement and an FLT3-ITD. Our approach has the potential to reduce resource-intensity and time associated with the identification of novel combination therapies. Patients with refractory disease have a significantly worse outcome, with the five-year OS rate reducing to 30% for those experiencing early relapses3. To date, our understanding of paediatric AML has been inferred from genomic data collated from adult cases. With the high-throughput sequencing of paediatric samples, it has become apparent that the mutational landscape of paediatric AML differs widely from adult AML, with age-specific alterations more evident5. Mutations in genes such as NRAS, KIT and KRAS are more common in paediatric AML, whilst mutations in DNMT3A and NPM1 are associated with adult cases5. Currently, there are no targeted therapies for paediatric AML, with the standard treatment consisting of Cytarabine plus an anthracycline agent. This has remained largely unchanged for four decades6, with the Food and Drug Administration (FDA)-approval of Gemtuzumab ozogamicin being one of the few breakthroughs for the treatment of patients with relapse/refractory CD33+ AML7. The significant side-effects of these therapies can affect survivors for the rest of their lives, with cardiac dysfunction and later health complications such as subsequent malignancies8. In addition, 10–20% of children and young people do not achieve remission during induction therapy, therefore the identification of novel targeted therapies has the potential to improve treatment outcomes while reducing both side-effects and the risk of refractory disease11. Combination treatments are preferable to monotherapy due to their superior efficacy at targeting tumour growth in an additive or synergistic manner. Combinations of therapies also allow lower doses to be used, therefore reducing potential harmful acute and chronic side effects, whilst also reducing the potential for drug resistance and relapse12. However, the identification of effective combination therapies has its challenges, including the complex screening process, identification of synergistic dose ranges and whether therapies are given in a sequential or simultaneous order for optimal response. These limitations are common to many disease contexts; Tan et al. was the first to propose a multiplex screening approach in order to identify new compound combinations derived from large reference libraries, for the treatment of the human immunodeficiency virus (HIV)13.Acute leukaemia is the most prevalent cancer diagnosed in children, adolescents and young adults (AYA), accounting for approximately 30% of cancer cases. While, acute myeloid leukaemia (AML) makes up only one fifth of all acute leukaemia cases, it accounts for more deaths than the more common acute lymphocytic leukaemia (ALL). This can be attributed to the relatively higher risk of relapse among AML patients, with a corresponding five-year overall survival (OS) rate of 60 to 70% in comparison to 90% in ALL14, can facilitate compound combination testing in paediatric AML. Using a reference library of 80 apoptotic-inducing agents, this novel approach maximised the probability of identifying new compound pairs with therapeutic potential, while minimising replication and redundancy. Thus, we identified the combination of ABT-737, a B-cell lymphoma (BCL)-family inhibitor, and Purvalanol A, a cyclin dependent kinase (CDK)-inhibitor, as a potential targeted therapy for AML patients carrying a mixed lineage leukaemia (MLL) rearrangement and internal tandem duplications of the fms-related tyrosine kinase 3 gene (FLT3-ITD).In this paper we show how an alternative multiplex screening approach, using a design adapted from error-testing in computer sciencehttps://ocg.cancer.gov/programs/target/data-matrix). Analysis of EFS, using Kaplan–Meier estimation and clinically-relevant thresholds, was used to identify good and poor prognostic patients within each of four cytogenetic risk groups, normal cytogenetics, MLL, inv(16) and t , 9.38e−8–1.12e−2 (MLL), 1.43e−7–4.34e−2 (inv(16)) and 3.35e−19–1.66e−3 ).Clinical data corresponding to paediatric AML cases from the TARGET AML initiative were extracted  Fig. . Next, w1 Fig. . 21) Fig. , which wTo validate apoptotic processes as targetable pathways in paediatric AML and select a dose for combination screening, we considered 80 apoptosis-inducing compounds  in three paediatric AML cell lines  at three concentrations  and assessed cell viability at several time-points up to 72 h. Fluorescent readings for each compound were normalised to a DMSO vehicle control and any compound with an RFU value greater than or equal to 2 indicating that cell death had occurred and was considered a successful treatment with anti-leukaemic effects Fig. . Due to N = 80) could be accommodated by using ten (n) compounds per well over 160 wells (m), with 45 possible pairs in each of the 160 wells. Calibration analysis indicated as expected, that with each additional compound, i.e. an increase in n, added to a well, the number of wells required decreased, i.e. m reduced , the advantages of using an increased number of compounds could be observed. Though the rate of increase at which this benefit is accrued begins to slow after n = 3 /2). Using our all-pairs testing algorithm (50 repeated iterations), summarised in Fig. Using the optimum dose of 0.1 µM, three cell lines were each treated with different combinations of ten of the 80 compounds across 160 wells and assessed at 24 h, 48 h and 72 h. In 82 wells, cell viability was reduced across the panel of cell lines at 72 h , against the MV4-11 cell line up to 72 h , to establish whether this combination specifically targeted the MV4-11 cell line or if it had the potential to reduce cell viability in a range of AML subtypes. Interestingly, the data showed that this combination specifically targets cell lines with a FLT3-ITD and an MLL rearrangement including the MV4-11 and MOLM-13 lines, whilst not impacting the viability of the THP-1 cell line, included as a control for the MLL rearrangement or the PL-21 cell line, included to control for the presence of a FLT3-ITD, or any of the other lines used Fig. .Figure 615. Since 2017 the FDA have approved seven  new drugs for treating adult AML, with only one (Mylotarg) for paediatric AML18. In general, the discovery of new treatments for different diseases are financially restrictive, with a high risk of failure—only one in ten compound candidates may reach the clinical testing stage19. The lack of characterisation of disease heterogeneity, such as in paediatric AML can add further obstacles. Four decades ago approximately 30% of children diagnosed with AML survived five years or more20. As a result of improved diagnostic approaches, better risk stratification and more intensive chemotherapy, more patients now achieve complete remission and EFS, with approximately 65% of cases surviving five years or more22. This can come at a considerable cost, the intensification of the chemotherapy regime can leave some paediatric patients with life-long complications or result in death24. In addition, around 30% of patients relapse and one fifth of cases do not show any benefit from current treatments25.The treatment for AML has not changed for decades, cytarabine in combination with anthracyclines26. This may involve a combination of in silico, in vivo and/or in vitro techniques. High-throughput screening of drug libraries, in particular combination screens, have revealed novel drug pairs of therapeutic potential with respect to AML. For example, recent studies have identified synergistic effects of ruxolitinib, a JAK1/2 inhibitor, with venetoclax, a BCL-2 antagonist27 and quinacrine and cytarabin28.Drug repositioning of approved compounds for use in different disease settings can contribute to accelerating the discovery process29. As such, the goal was to determine a minimum solution which covers all possible pairs. Our approach reduced the combinatorial search space, balancing the quantity of reagents, consumables and time required against the ability to detect novel compound pairs.These studies have tended to focus on adult AML, while the paediatric disease has received relatively little attention. This study, therefore, set out to develop a rapid, high-throughput drug combination screening approach to aid in the identification of more personalised treatments for paediatric AML. High-throughput screening of drug combinations can be both time- and resource-intensive. We therefore implemented a two-stage in silico approach to inform the design of the combination screen. First, to select a drug library with the greatest potential, we carried out statistical analysis of the TARGET paediatric AML patient RNA-sequencing data, identifying “apoptosis” as a therapeutically targetable pathway. Next, to identify novel combinations, we generated an algorithm which detected all possible pairings of 80 apoptotic-inducing agents and placed them in groups of 10, across 160 wells. This used an optimisation method in which different subsets of parameters, comprising all pair combinations, examined in parallel, can be used31. The screening technique revealed a ‘novel’ drug pairing, a BCL-2 inhibitor (ABT-737) combined with a CDK2 inhibitor , with therapeutic potential in the MV4-11 cell line with a FLT3-ITD and MLLr. Unfortunately, in the FLT3-ITD AML population MLLr are rare, with only 2% of patients previously presenting with a combination of both32. To our knowledge, this particular drug pair has not been reported previously, however, this was not a novel mechanistic pairing as similar have been investigated by several other groups35. For example, Xie, et al.36 investigated LS-007, a CDK inhibitor, in combination with venetoclax against AML and observed a marked increase in apoptosis across a panel of AML cell lines. Additionally, several groups38 have investigated the inhibition of CDK9 as a way of targeting AML blasts observing down-regulation of survival genes, including MCL1. Furthermore, Venetoclax, another BCL-2 inhibitor, has recently been approved for the treatment of AML as a single agent18 and trials are on-going investigating combinations with either FLT3 inhibitors or demethylating agents, to test its efficacy in the treatment of AML40. These studies are a validation of the strength of the utility of our combination screening approach.Using three cell lines representing paediatric AML, MV4-11, CMK and Kasumi-1 lines, we observed some similarities in response to the same compound combination wells e.g. Well-47, 99 and 155. However, in the majority of cases, differences were observed between cell lines, indicating further a need for personalised medicine approaches in the treatment of paediatric AMLOur approach has the potential to reduce resource-intensity and time in the identification of novel combination therapies. Other groups working on similar mechanistic pairings validates the potential of our approach to identify functional small molecule combinations with a larger cohort of drugs and the use of patient material.https://ocg.cancer.gov/programs/target/data-matrix) and differential gene expression analysis performed. Only patients with mRNA-sequencing data obtained using the Illumina HiSeq 2000 platform were included. DESeq2, the Bioconductor package for R, was used to detect differentially-expressed genes (DEGs) using the raw count data from patients within each of the four subdivisions; t, inv(16), MLL and normal cytogenetics. Using event free survival (EFS), which is the time from study entry until an adverse event  as defined by Bolouri et al.5, Kaplan–Meier curves within each cytogenetic group were plotted using the ‘survival’ package within R. Thresholds which could separate patients within each cytogenetic group into poor and good prognosis using EFS were next selected. Different selection criteria were used due to the cytogenetic risk groups affecting patient outcome. For inv(16) and t cytogenetic groups, poor prognosis patients were defined as those with an EFS of less than one year (365 days), good prognosis was defined as an EFS of greater than five years (1825 days). For patients possessing MLL rearrangements and normal cytogenetics, EFS thresholds of 6 months (183 days) and 2 years (730 days) were used to define poor and good prognosis respectively. Prior to the DEG analysis, to remove any potential confounding, each pair of poor and good prognostic groups were balanced by gender, race, ethnicity, age at diagnosis and treatment protocol, using the R package ‘Matchlt’41. To obtain DEGs, data comparisons were made within each cytogenetic group between patients with poor and good EFS. Unnormalised raw count data was normalised by size factors and results were ordered by increasing adjusted p-value. Next, genes with adjusted p-values less than 0.05 were selected for Ingenuity Pathway Analysis (IPA)  was used to identify targetable pathways based on functional enrichment analysis of the differentially expressed genes determined within each cytogenetic risk group  and t) and the significant (p < 0.05) pathway common to the four groups was investigated. A summary chart of data analysis is shown in Fig. RNA-sequencing and matched clinical data from paediatric AML patients was obtained from The National Cancer Institute’s (NCI) Therapeutically Applicable Research to Generate Effective Treatments (TARGET) data matrix ( 2 years 30 days w43. The CMK cells were derived from a 10-month-old boy with Down's syndrome and Acute megakaryoblastic leukaemia (AML FAB M7 subtype) at relapse45. The Kasumi-1 cell line was derived from a 7-year-old boy with AML FAB M2 (acute myeloblastic leukaemia with maturation) in second relapse after bone marrow transplantation46. The MOLM-13, THP-1, PL-21 and CMS cell lines were used for validation experiments, each representing a case of AML with different cytogenetic and mutational backgrounds. All cell lines were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Germany) except the CMS line which was provided courtesy of Dr Yubin Ge 47.MV4-11, CMK and Kasumi-1 cell lines were used during this study to represent the clonality of AML in paediatric patients. The MV4-11 cell line was derived from a 10-year-old boy with the French-American-British (FAB) M5 subtype of AML (acute monocytic leukemia) at diagnosisEach cell line was maintained in Roswell Park Memorial Institute (RPMI) 1640  supplemented with, either 10% Fetal Bovine Serum  (MV4-11) or 20% FBS (CMK and Kasumi-1 cell lines) and 100 µg/mL Penicillin–Streptomycin . All cell lines were cultured in a humidified incubator at 37 °C supplemented with 5% carbon dioxide.48, an algorithm was designed for application to the multiplex screening context. Given a compound reference library of a defined size (N) and a set number of compounds (n) to be used in each plate well, the algorithm was designed to include all possible pairwise compound combinations, using the least number of wells.Using the principle of all-pairs testing, as used in computer scienceN, however as the size of the compound library increases, a compromise between computational resources and near-optimal solutions must be sought49. The algorithm was therefore designed to dissect the problem into stages with a local optimum solution identified at each stage and repeated occurrences of compounds pairs tolerated. Given a total of N compounds, N × (N − 1)/2 pairs are possible. Likewise, if n compounds are to be used in each well n ×  (n − 1)/2 pairs will be achieved.Exact solutions are possible for smaller values of m wells, the problem can be redefined as minimising m × n ×  (n − 1)/2 so that all possible N ×  (N − 1)/2 pairs are represented. The algorithm considers a full list of compound pairs together with an active list of pairs not yet allocated to a well. At the beginning, both lists are identical. Initial random penalties are allocated to each compound pair. From the full pair list a set of n compound pairs are selected at random and their combined penalty scores evaluated until a set is obtained with a minimised  penalty score. These selected pairs are removed from the active list and the penalty scores increased (full list). This guides the next step of the search process away from previously used pairs. The algorithm stops when all possible pairs (active list) are allocated. Due to the random allocation of initial penalties, in this study the process was restarted 50 times, to determine an overall near-optimal solution. For more information see Supplementary Methods With n ×  n − /2 so thahttps://cran.r-project.org/). Code and an accompanying tutorial are available at https://sourceforge.net/projects/geca/files/musicAL/.The algorithm was implemented in the R statistical programming language (https://www.selleckchem.com/screening/apoptosis-library.html). Compound screening assays were carried out on 96-well black optical bottom plates . Cell lines  were seeded at a density of 4.0 × 104 cells per well. Compounds were diluted with Dimethyl Sulfoxide (DMSO)  and added to cell culture to give final concentrations of 1 µM, 0.1 µM and 0.01 µM for the single agent screen or 0.1 µM for the combination screen. Cells treated with 0.1% DMSO were used as a vehicle control. The plates were then transferred to a humidified incubator at 37 °C supplemented with 5% CO2 for up to a 72 h incubation. Cell viability was assessed at several time-points, 24 h, 48 h and 72 h, with CellTox Green Cytotoxicity assay , added to the cell culture at seeding. Relative fluorescence unit (RFU)  was measured using a Synergy HTX Multi-Mode Microplate reader  to determine cytotoxicity proportional to loss of cell membrane integrity and increased fluorescence. Unless stated otherwise, all assessment of cell viability was performed using CellTox Green Cytotoxicity assay.The Selleckchem Apoptosis compound library was used as the basis for the multiplex screen  from the mean (solid line) of all the data points generated from the 72 h time point per cell line. This is a common method used for identifying active compounds when performing large scale compound and RNA interference screens54.In specific wells of interest, in order to identify potential synergistic pairs, the ten compounds were deconvolved into 45 pairwise combinations used at 0.1 µM. A full list of the compounds in each of the 160 wells is available in Supplementary Table 5 cells per mL of complete media, supplemented with a 1/500 dilution of CellTox Green reagent, and 100 µL added to the wells of a 96 well black optical bottom plate. Alongside each pairwise combination, the single agents were also tested to ensure an effect observed was not due to the action of a single agent. Plates were incubated in a humidified incubator at 37 °C supplemented with 5% CO2 for up to 72 h. Readout of cell viability was achieved using the Synergy HTX Multi-Mode Microplate reader  to detect fluorescence  as above at 24, 48 and 72 h post treatment.Target combinations were next tested in a wider panel of paediatric AML cell lines . In brief cells were seeded at 2 × 10Supplementary Information 1.Supplementary Legends.Supplementary Methods 1.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.


Acute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly. Although complete remission (CR) is achieved for the majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has become of major clinical interest in AML. We utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the protein changes and protein phosphorylation events associated with AML relapse in primary cells from 41 AML patients at time of diagnosis. Patients were defined as relapse-free if they had not relapsed within a five-year clinical follow-up after AML diagnosis. Relapse was associated with increased expression of RNA processing proteins and decreased expression of V-ATPase proteins. We also observed an increase in phosphorylation events catalyzed by cyclin-dependent kinases (CDKs) and casein kinase 2 (CSK2). The biological relevance of the proteome findings was supported by cell proliferation assays using inhibitors of V-ATPase (bafilomycin), CSK2 (CX-4945), CDK4/6 (abemaciclib) and CDK2/7/9 (SNS-032). While bafilomycin preferentially inhibited the cells from relapse patients, the kinase inhibitors were less efficient in these cells. This suggests that therapy against the upregulated kinases could also target the factors inducing their upregulation rather than their activity. This study, therefore, presents markers that could help predict AML relapse and direct therapeutic strategies. Acute myeloid leukemia (AML) is an aggressive and heterogeneous malignancy, the two main subsets being acute promyelocytic leukemia (APL) and the heterogenous group non-APL AML ,2. The ACEBPA, FLT3, NPM1 and TP53) are implemented in the routine prognostic evaluation and risk adapted treatment of AML patients procedures . B. Bg at 4 Image J . StatistThis study describes the proteomics profiles of patient-derived AML cells collected at the first time of diagnosis that differ between patients that are long-term relapse free survivors and patients that relapse. The relapse-associated group displayed decreased expression of V-ATPases and the increased activity of CDKs and CSK2. Furthermore, our results suggest a subset of transcriptional and metabolic regulators that could be considered as predictors of prognosis and therapeutic targets in AML.


Asthma is one of the main intractable diseases recognized by the international medical community. The current widely used bronchodilators for asthma—β2-adrenal receptor agonists—have limited therapeutic effects, necessitating the development of novel antiasthma drugs with increased efficacy and fewer adverse effects. In this study, we investigated the relaxant effects and underlying mechanism of an ethyl acetate extract from dandelion (EAED) on mouse airway smooth muscle.2+ concentration was measured using a calcium imaging system. L-type voltage-dependent calcium channel (VDLCC) and non-selective cationic channel (NSCC) currents were measured by patch-clamp. The lung functions of healthy and asthmatic mouse groups were assessed via the forced oscillation technique.The effects of EAED on agonist-induced precontraction in mouse airway smooth muscle were evaluated with force measurement. Mouse lung slices were used to study the effects of EAED on bronchial smooth muscle. The intracellular Ca2+ concentration in airway smooth muscle cells, blocks VDLCC and NSCC currents, and relieves the respiratory resistance of healthy and asthmatic mice.EAED inhibits acetylcholine-induced sustained contractions of whole airway smooth muscle by inhibiting VDLCCs, NSCCs, and some unknown channels, reduces the agonist-induced increase in the cytosolic free CaEAED may have potential beneficial effects on mitigating asthma attacks. These oscillations are caused by the release of Ca2+ from the intracellular calcium pool and the influx of Ca2+ from the extracellular space i was returned to 2 mM, the extracellular Ca2+ flowed rapidly, the intracellular [Ca2+]i increased, and the TR constricted. This contraction was inhibited by 1 mg/mL EAED. Furthermore, incubation with EAED almost completely abolished the contraction induced by 2 mM Ca2+  in the presence of EAED, ACh did not induce a transient contraction. With the addition of 2 mM Ca2+, only a very weak contraction occurred, which gradually returned to baseline , which leads to extracellular Caa2+ Fig. c. Thus, 2+ in single ASMCs were examined by use of the TILL calcium imaging system. High K+−  dissolved with vehicle or EAED. Under baseline conditions, the four experimental groups studied were indistinguishable with the forced oscillation technique. When the ACh concentration was increased to 25–50 mg/mL, the atomized EAED dissolved with ACh significantly reduced the respiratory resistance of the control and asthma groups compared with the vehicle group Fig. . As expe+- and ACh-induced precontractions in mouse TRs and lung slices by inhibiting L-type Ca2+ channels and additional TRPC3 and/or STIM/Orai channels, respectively. EAED suppressed the cytoplasmic Ca2+ concentration elevation caused by high K+ and ACh. In addition, in an in vivo study, EAED effectively reduced the elevated respiratory resistance, Rrs, induced by ACh in healthy and asthmatic mice.In this study, we found that EAED reduced both high K+ and ACh—were applied to elicit mouse TR precontractions and examine the effects of EAED. The experimental results showed that EAED markedly antagonized both high K+- and ACh-induced TR contractions in mice, with the maximum relaxant efficiency reaching almost 100% . These data demonstrated that EAED possesses relaxant potency against contractions induced by ACh/high K+ stimulation.As mentioned above, β2 adrenergic receptor agonists are often used as first-line bronchodilators to relieve asthma , but the+-induced contraction. The experiments were conducted under 0 and 2 mM extracellular Ca2+ conditions. High K+ caused membrane depolarization, leading to the activation of VDLCCs [+-induced contractions were completely abolished in Ca2+-free medium (Fig. 2+ influx through VDLCCs. Moreover, EAED completely blocked VDLCC currents (Fig. +-induced contraction by blocking VDLCC-mediated Ca2+ influx (Fig. We further investigated the underlying mechanism of the EAED-mediated relaxant effects on high Kf VDLCCs . In our ium Fig. a, suggesnts Fig. a. These lux Fig. a and b.2+ conditions but had no effect on ACh-induced transient contractions under 0 Ca2+ conditions (Figs. 2+ influx, but not intracellular Ca2+ release-induced transient contractions. However, EAED almost entirely eliminated both of these two types of contractions (Fig. 2+ influx-mediated by VDLCCs and the intracellular Ca2+ release.We then explored the pathways involved in EAED-mediated relaxation of ACh-induced contractions. We found that nifedipine partially inhibited ACh-induced sustained contractions under 2 mM Cans Figs. a and 5c.2+ influx [2+ channels and NSCCs, other channels may mediate the ACh-induced contractions further inhibited by EAED (Fig. Moreover, TRPC3 and/or STIM/Orai channels, as NSCCs, also play roles in ACh-induced contractions by mediating Ca+ influx . Our resAED Fig. c. These 2+ dynamics in ASMCs, we further showed that EAED decreased high K+− and ACh-mediated increases in intracellular Ca2+ (Fig. The above experimental results were all obtained using the main trachea of mice. Moreover, our subsequent experiments conducted in lung slices suggested that EAED could also inhibit bronchial smooth muscle contraction Fig. , indicata2+ Fig. . In addia2+ Fig. .2+ concentration in ASMCs, and relieves respiratory resistance in healthy and asthmatic mice. Meanwhile, unknown pathways might also be involved in EAED-mediated relaxation, in addition to VDLCCs and NSCCs. These results suggest that EAED could be a new inhibitor of asthma attacks.In summary, this study demonstrated that EAED inhibits agonist-induced sustained contractions of ASM by inhibiting several types of ion channels, decreases agonist-induced elevation of the cytosolic free Ca


Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L.pumilum system was improved, and the genetic transformation efficiency increased by 5.7–13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development.  Auxin plays a critical role in plant development by controlling cell expansion, polarity, and organ patterning . Auxin iPhyscomitrella patens, Selaginella moellendorfii, Arabidopsis thaliana, and Oryza sativa, respectively [The ABCB subfamily is the second largest ABC protein subfamily in plants with 10, 18, 21, and 22 ABCB members in ectively . Some meectively ,10. ABCBectively  and planectively . For exaectively  and ABCBectively  have higectively , ABCB4 [ectively , and ABCectively  can bindectively . The AUXectively ,18. Howeectively ,16.The structure of PILS proteins (PILS1-PILS7) is similar to PIN proteins, although the former constitute an independently evolved protein family . PILS anAgrobacterium-mediated transformation is a plant transformation method that employs an efficient and low-cost system that has been widely used for stable gene transfer [Agrobacterium is necessary for T-DNA (transfer DNA) transfer into the host cells to integrate T-DNA into the host genome [vir) gene of the TI plasmid determines the transformation ability of Agrobacterium [vir gene requires signals from the host plants, including phenolic derivatives, acidic pH, monosaccharides, and low PO4 [4 [Agrobacterium infection and transformation due to lack of wound response [Lilium has been one of the recalcitrant monocotyledon species for Agrobacterium-mediated transformation until transgenic lily was generated for the first time [Lilium and several Lilium varieties have successfully obtained transgenic plants, such as Lilium × formolongi [Lilium tenuifolium oriental × trumpet “Robina” [Lilium oriental “Sorbonne” [Lilium cv “Manissa” [Lilium [Lilium pumilum DC. Fisch. (L. pumilum) was successfully established in our study [Genetic transformation is an important technical means to study gene function. transfer . In thist genome ; the viracterium ,28,29. A low PO4 ,31. Phenw PO4 [4 . Monocotresponse  and defiresponse . Lilium rst time . Since trmolongi , Lilium “Robina” , Lilium orbonne” , and LilManissa” . However [Lilium . In 2019ur study ; howeverLpABCB21 and LpPILS7 auxin transporters are significantly up-regulated in SE and may play an important regulatory role in L. pumilum SE [L. pumilum developed previously by our team [LpABCB21 and LpPILS7 in L. pumilum SE are confirmed.At present, the regulatory role of auxin transport in somatic embryogenesis (SE) remains unclear. Our previous results demonstrated that the expression levels of milum SE . Moreoveour team  is improLpABCB21 (4086 base pairs (bp)) and LpPILS7 (1461 bp) were inserted into the pRI101-ON vector by double enzymes digestion, and the generated vectors were named OE-LpABCB21 and OE-LpPILS7, respectively (Complete CDSs (coding sequences) of ectively A. The seLpABCB21 and LpPILS7 are shown in LpABCB21 and KO-LpPILS7  was selected in the conserved domain of the CDS. The sgRNA sites in -LpPILS7 B.After two rounds of antibiotic screening, the transformed calli were cultured in somatic embryo induction medium for 30 days. The growth of resistant calli is shown in As shown in LpABCB21 resistant plants were obtained, including 32 plants under Germination II condition (D) and five plants under Germination I condition (A). Thirty-seven OE-LpPILS7 resistant plants were obtained, including 30 plants under Germination II condition (F) and seven plants under Germination I condition (B). Fifty-six KO-LpABCB21 resistant plants were obtained, including 41 plants under Germination II condition (G) and 15 plants under Germination I condition (C), and 63 KO-LpPILS7 resistant plants, including 52 plants under Germination II condition (H) and 11 plants under Germination I condition (D).As shown in NPTII gene fragment (817 bp) and the specific fragment between the vector and the target gene (1105 bp/1004 bp) were successfully cloned, and the positive plants were identified. After verification, 3 OE-LpABCB21 positive lines  and 7 OE-LpPILS7 positive lines  were obtained. All lines except L32 were obtained after the improvement.As shown in LpABCB21 positive plants were obtained; however, five plants were obtained before the improvement. 24 KO-LpPILS7 positive plants were obtained; however, only 5 plants were obtained before the improvement.As shown in LpABCB21 and KO-LpPILS7 positive plants were purified and sequenced; the mutation modes are shown in LpABCB21 positive plants, and no mutations occurred in line L29 at the sgRNA1 site. Mutations occurred in six of the 28 KO-LpPILS7 positive plants, and heterozygous mutations occurred in lines L10 and L28 at the sgRNA3 site.To identify the mutation modes of the mutant lines, the cloned PCR fragments from KO-LpABCB21 and KO-LpPILS7 mutant lines had frame shift mutations, protein mutations between dual sgRNA, and amino acid mutations in dual sgRNA (LpABCB21 line L18 had insertion of a nonpolar leucine (+L) and replacement of polar asparagine with nonpolar alanine (N→A), and line L29 had replacement of polar negatively charged glutamate with polar glutamine (E→Q). KO-LpPILS7 mutant line L5 had replacement of polar threonine with nonpolar proline (T→P), and two polar negatively charged aspartic acid residues (-DD) were missing.Analysis of the changes in the protein sequences after mutations indicated that the protein sequences of KO-al sgRNA C,F. ThreLpABCB21 and LpPILS7 are membrane transporters, the hydrophilicity, hydrophobicity, and transmembrane domains of the mutant protein sequences were estimated by prediction. The results showed that hydrophilicity and hydrophobicity of the proteins were considerably affected by the frame shift mutation and protein mutations between dual sgRNA ; however, these properties were affected to a lesser extent by the amino acid mutation in dual sgRNA  (LpABCB21 protein sequence consists of 11 transmembrane domains (A). The frame shift mutation resulted in deletion of the third to eleventh transmembrane domains . The protein mutation between dual sgRNA caused deletion of the third transmembrane domain; the distribution of amino acid residues to the inner and outer membrane prior to the missing transmembrane domain was also affected (F). The transmembrane domain of the amino acid mutant with mutation in dual sgRNA was identical to that of the wild-type protein (G and H). The wild-type LpPILS7 protein sequence consists of 10 transmembrane domains (I). The frame shift mutations resulted in deletion of the third to tenth transmembrane domains (K and M) or the fifth to tenth transmembrane domains (J and L). The protein mutation between dual sgRNA resulted in deletion of the fourth transmembrane domain (N). The transmembrane domain of the amino acid mutant with mutation in dual sgRNA was identical to that of the wild-type protein (O)  . The wiltein (O) .LpABCB21 was significantly increased by 2.0–9.6-fold in 3 OE-LpABCB21 lines compared with that in wild-type plants  was the highest, and the adventitious bud induction rate was 79.2% (19/24) (LpPILS7 mainly regulated late SE and had little effect on early SE. Only a few lines can advance (OE1: L30) or delay (KO1: L10 and L40) the emergence of somatic embryos by 1 day. Somatic embryo induction efficiency increased by approximately 10–30% when LpPILS7 expression levels increased by 2.0–9.8-fold, while LpPILS7 mutations decreased somatic embryo induction efficiency by approximately 10–60%  A. LpPILSy 10–60% D. AlthouAgrobacterium virulence in in vitro tissue culture [Agrobacterium growth is 7.0 [vir gene is favored under the acidic conditions [vir gene is barely induced at neutral pH; the expression level of the vir gene is increased by 1.2–2.0-fold when pH drops from 7.0 to 5.5 [Agrobacterium tumefaciens and plant cells, the expression level of the vir gene changes depending on pH and reaches the maximum at pH 5.1 [vir gene decreases when pH increases from 5.1 to 5.6 [Solanum lycopersicum) cotyledon [Agrobacterium-mediated genetic transformation. The number of resistant calli increased when pH of the re-suspension solution and co-culture medium decreased from 5.8 to 5.0 indicating that pH 5.0 is more suitable for the genetic transformation of L. pumilum than pH 5.8. However, a pronounced decrease in pH has an inhibitory effect on genetic transformation [L. pumilum was compared only under the conditions with pH 5.0 and pH 5.8 in this experiment, additional improvement in the genetic transformation efficiency of Lilium should consider optimization of pH of the transformation process.The pH of the medium was reported to be crucial for  culture . The opth is 7.0 , but opth is 7.0 ,44. In tnditions , while t0 to 5.5 . In the t pH 5.1 ; the exp1 to 5.6 . Genetic1 to 5.6 ,45. The otyledon . Our stuormation . Since t2  can promote the induction of embryogenic calli (EC) and the germination of somatic embryos. Addition of 10 mM CaCl2 increased the germination rate of somatic embryos by 51.79% [2 from 0.44 g/L to 1.32 g/L can improve the germination coefficient of somatic embryos. However, the role of CaCl2 in SE is genotype- and variety-dependent [2 regulation of the germination of somatic embryo requires additional investigation.Calcium ions are the major second messenger in plants, and the calcium signaling pathway has been extensively studied ,48. Recey 51.79% . The resependent . TherefoLpABCB21 may also influence auxin transport rate. Several studies have shown that SE is more easily induced when the explants contain high auxin levels [LpABCB21 may shorten the time required for auxin to reach the threshold of SE to induce an additional advance in the emergence time of somatic embryo. However, if the auxin content is so high that it exceeded a certain threshold, the continued growth of somatic embryo is inhibited. Bielach found that cytokinin application on lateral root primordia delays the appearance of maximum auxin levels and lateral root growth but has no effect on the formation of the lateral root [LpABCB21 mutant lines, and there was no significant influence on the induction efficiency of somatic embryos. It is not clear whether this is the result of functionally redundancy of ABCB4 with ABCB21 during SE or if AUX/LAX1 and ABCB21 coordinate to regulate the exogenous auxin influx.Previous studies have shown that ABCB21 and ABCB4 are bidirectional auxin transport carriers, which control the direction of auxin transport depending on auxin concentration to regulate the auxin level in the cells ,16. Recen levels ,51,52, wn levels . These cral root . ABCB21 LpPILS7 and LpABCB21 involved in auxin regulation may be similar; however, the proteins are associated with different patterns of auxin regulation. The reasons for this difference may be due to two factors. First, PILS protein is located at the ER and controls the accumulation of auxin in the cells. Second, the function of PILS protein mainly involves regulation of auxin signal through binding or releasing auxin and availability of auxin in the nucleus [LpPILS7-overexpressimg and mutant lines induce somatic embryos, the effect on the emergence time of somatic embryos was very small. This phenomenon may be explained by very high rate of auxin transport [LpABCB21, is not affected. Our previous study demonstrated that LpPILS7 expression level is increased in the light culture, and somatic embryo induction efficiency in the light culture is better than that in the dark culture [LpPILS7 can promote L. pumilum SE. PILS protein was suggested to bind auxin to limit nucleus auxin signaling [The function of PILS proteins in plant development is unclear; it is currently believed that PILS proteins can limit the rate of auxin signal transduction and response to external stimuli, such as light and temperature, to regulate auxin-dependent development ,22. Our  nucleus ,22; howe nucleus ,17. Howeransport ; hence,  culture . This exignaling ,22. PILSignaling . The resL. pumilum improved by decreasing the pH of re-suspension solution and co-culture medium to 5.0 and increasing the CaCl2 concentration of germination medium to 1.32 g/L; these results are important for subsequent functional genomics studies and molecular breeding in Lilium. Our findings confirm that LpABCB21 and LpPILS7 have important regulatory roles in L. pumilum SE and contribute to determination of the molecular mechanism of Lilium SE.In conclusion, this study describes a more efficient method of genetic transformation of L. pumilum EC were used as material. EC were obtained according to a previously described method [−2 s−1 provided by cool white fluorescent light bulbs .d method . CultureAgrobacterium tumefaciens EHA105 harboring the helper plasmid pVS1-SAH2, CRISPR/Cas9 knockout vector pG3H-U3Ub, and template pCBC-DT1T2.2 were donated by Professor Chen Qijun from China Agricultural University.Plant overexpression vector pRI101-ON was purchased from Takara Biomedical Technology Co., Ltd. . Primers with restriction enzyme cutting sites were designed using the Primer 5 software according to the sequences of the target genes and overexpression vectors. The sgRNA was designed according to the PAM/NGG domain on the sequences of the target genes. The sgRNA requires the content of GC% to be higher than 40%, absence of four consecutive thymines, and continuous pairing between sgRNA and target gene less than 8 bp. The primers for overexpression and knockout vector construction are listed in LpABCB21 and LpPILS7 were used as templates to clone the CDS of LpABCB21 and LpPILS7. The reaction system volume was 50 μL containing 1 μL of the LpABCB21/LpPILS7 plasmids, 1 μL of LpABCB21-F/LpPILS7-F (10 μM), 1 μL of LpABCB21-R/LpPILS7-R (10 μM), 22 μL of ddH2O, and 25 μL of PrimeSTAR Max DNA polymerase . The incubation was at 98 °C for 1 min;  × 30 cycles; and 72 °C for 1 min. The pBCB-MT1T2.2 plasmid was used as a template to clone the sgRNA expression cassettes. The reaction system volume was 50 μL containing 1 μL of the pBCB-MT1T2.2 plasmids; 1 μL of LpABCB21-BsF/LpPILS7-BsF (10 μM); 1 μL of LpABCB21-BsR/LpPILS7-BsR (10 μM); 1 μL of LpABCB21-F0/LpPILS7-F0 (1 μM); 1 μL of LpABCB21-R0/ LpPILS7-R0 (1 μM); 20 μL of ddH2O; and 25 μL of PrimeSTAR Max DNA polymerase. The incubation was at 98 °C for 1 min;  × 30 cycles; and 72 °C for 1 min. After the PCR product was purified, the CDS of the target genes and the sgRNA expression cassettes were inserted into pRI101-ON and pG3H-U3Ub, respectively, by enzymatic ligation.Previously, sequencing verified plasmids of Agrobacterium expressing the vectors was grown in a liquid medium until the OD600 value reached 0.6. Collected bacteria were subjected to Re-suspension I in the same volume to suspend the bacteria. After addition of 100 µmol AS, the EC that had been precultured in the pre-cultivation medium for 10 days were infected for 15 min. The EC were inoculated into Co-cultivation I for culture for 3 days in the dark, and then inoculated into Selection I and Selection II media for 2 weeks. Finally, transformed EC were inoculated into Germination I for germination. The results of preliminary experiments . The expression levels in the overexpression plants were detected according to the previously described method [http://www.cbs.dtu.dk/services/TMHMM/) was used to analyze the transmembrane domains.The genomic DNA of the leaves was extracted and used as a template for PCR identification. The primers for identification of transgenic plants are listed in d method . Specifid method . Specifi2 and inoculated in the somatic embryo induction medium for dark culture. The wild-type plants were used as the control. The emergence time of somatic embryos was estimated. The results of somatic embryo induction were assessed after culture for 50 days.The scales of the overexpression lines and mutant lines were used as explants. The scales were cut into small pieces of approximately 0.5 cm


In the protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, three isoforms of centrin have been identified. While increasing information is now available that links the function of centrins with defined parasite biological processes, knowledge is still limited on the metal-binding and structural properties of these proteins. Herein, using biophysical and structural approaches, we explored the Ca2+ binding abilities and the subsequent effects of Ca2+ on the structure of a conserved (TgCEN1) and a more divergent (TgCEN2) centrin isoform from T. gondii. Our data showed that TgCEN1 and TgCEN2 possess diverse molecular features, suggesting that they play nonredundant roles in parasite physiology. TgCEN1 binds two Ca2+ ions with high/medium affinity, while TgCEN2 binds one Ca2+ with low affinity. TgCEN1 undergoes significant Ca2+-dependent conformational changes that expose hydrophobic patches, supporting a role as a Ca2+ sensor in toxoplasma. In contrast, Ca2+ binding has a subtle influence on conformational features of TgCEN2 without resulting in hydrophobic exposure, suggesting a different Ca2+ relay mode for this isoform. Furthermore, TgCEN1 displays a Ca2+-dependent ability to self-assemble, while TgCEN2 did not. We discuss our findings in the context of Ca2+ signaling in toxoplasma.Centrins are calcium (Ca However, only a small percentage of total centrins (~10%) is restricted to MTOCs, and the remaining percentages are distributed more diffusely in other organelles and cellular compartments [Saccharomyces cerevisiae a single centrin is responsible for such functional diversity, various centrin isoforms have been identified in other organisms, comprising higher plants and mammals [Centrins are calcium  state to a holo (open) state causing solvent exposure of a hydrophobic region that is responsible for molecular recognition and interaction with cellular targets. A major difference between CaM and centrins is the presence of an extended unstructured 20–25 residues at the N-terminal domain of centrins that may have specialized a biological function in self-assembly and subcellular localization of the protein. Similar to calmodulin (CaM), centrins generally fold into two independent domains, the N-lobe and the C-lobe, each of which harbors a pair of characteristic helix-loop-helix structures, called the EF-hands, which constitute highly specific potential Cag2+ ions ,17. The Toxoplasma gondii, a protozoan parasite of the phylum Apicomplexa that causes toxoplasmosis in all warm-blooded animals, including humans, three centrin isoforms have been identified, namely centrin 1 (TgCEN1), centrin 2 (TgCEN2), and centrin 3 (TgCEN3) [T. gondii and how they are affected by Ca2+. This could represent crucial information to better understand how these proteins participate in relevant cellular processes and whether they, as EF-hand containing proteins, are part of Ca2+ signaling pathways that control parasite physiology.In (TgCEN3) ,19,20, s(TgCEN3) ,22. Howe2+ ion in determining the structural properties of the proteins and to understand the metal binding, thermodynamic, and structural differences between the two isoforms. TgCEN3 was not included in the analysis because its recombinant production resulted in an insoluble protein and, unfortunately, our efforts to solubilize it have been unsuccessful to date. In the present study, using an array of complementary biophysical and biochemical approaches, we investigated the molecular properties of the recombinant proteins TgCEN1 and TgCEN2 with the specific aim to elucidate the role of the CaThe obtained results might have far-reaching consequences for the functional understanding of the mechanism of action of TgCEN1 and TgCEN2, paving the way for subsequent strategies of parasite disruption through targeting of centrins in treating toxoplasmosis.E. coli cells made competent by the CaCl2 method. TgCEN1 lacking the first 21 residues  and TgCEN2 lacking the first 22 amino acids  were generated by site specific mutagenesis on the pET21a-TgCEN1 and pET21a-TgCEN2 wild type constructs, respectively, using the QuikChange® site-directed mutagenesis kit , according to the manufacturer’s recommendations for multiple-site deletion in a single step. The G43A, G79A and G152A TgCEN1-C and the G44A, G153A TgCEN2-C single-site variants were produced by site specific mutagenesis on the wild type constructs using the QuikChange® site-directed mutagenesis kit. The primer sequences used are listed in Synthetic genes  corresponding to the complete cDNA of TgCEN1 (TGME49_247230) and TgCEN2 (TGME49_250340) with a tag of six His at the N-terminal were cloned into a pET21a expression vector using NdeI and BamHI as restriction sites. The recombinant vectors were transformed into DH5α-derived E. coli Rosetta (DE3) expression host cells. Each culture was grown at 37 °C to OD600 of 0.6 and induced with 0.5 mM IPTG  at 24 °C for 18 h. The cells were harvested by centrifugation (5000 g for 15 min at 4 °C) and resuspended in 50 mM Tris-HCl, 150 mM KCl, 10 mM imidazole, pH 7.5 in the presence of an EDTA-free protease inhibitor. After sonication and centrifugation, the supernatant of lysed cells was collected and applied to a Ni-chelating column equilibrated with 50 mM Tris-HCl, 150 mM KCl, 10 mM imidazole, pH 7.5. Protein elution was performed using a linear gradient from 10 to 500 mM imidazole. Purified centrins were extensively dialyzed in 50 mM Tris-HCl, 150 mM KCl, pH 7.5 buffer to remove imidazole. The homogeneity and purity of the protein variants were verified by SDS-PAGE and protein concentration was determined using the Bradford assay and/or biuret reaction [The verified plasmids for full-length, N-terminal deletion, and site-specific variants of centrins were transformed into reaction ,24. The purification and all of the following experiments with TgCEN1 were performed by adding 0.5 mM DTT to buffers due to the presence of a cysteine residue in the protein.15N labeled proteins were prepared in the same manner, except that they were expressed in M9 minimal medium enriched with 1 g/L 15NH4Cl as the sole source of nitrogen.2 or 2 mM EGTA, as described [Purified recombinant proteins were assayed by native PAGE (12%) and SDS-PAGE (15%) after a 30-min reaction at 25 °C with 2 mM CaClescribed ,25.2O pH 7.5 on a Bruker Avance III spectrometer, operating at 600.13 MHz proton Larmor frequency and equipped with a cryogenic probe. 1H-15N HSQC (heteronuclear single quantum coherence) spectra were collected by a standard pulse sequence. The instrument data matrix consisted of 2 K  × 256  complex points, spectral windows of 9615.38 Hz (1H) × 2432.71 Hz (15N), 4 or 8 transients, and 1.2 s relaxation delay. NMR data were processed with Topspin 3.6 (Bruker).Nuclear magnetic resonance (NMR) experiments were performed at 25 °C with approximately 0.4 mM protein samples in 50 mM Tris-HCl, 50 mM KCl, 10% D2 or 2 mM EGTA, at a protein concentration of 0.2 mg/mL at 25 °C using a 0.1 cm quartz cuvette. An average of five accumulations were measured, using a scan rate of 50 nm/min. Spectra were corrected by subtraction of the buffer signal. Thermal unfolding profiles were obtained by monitoring the CD signal at 222 of apo- and Ca2+-bound proteins over a temperature range from 15 °C to 90 °C at a heating rate of 1.5 °C/min. Mean values ± standard error of the mean (SEM) of melting temperature were obtained from triplicate experiments.Purified proteins were analyzed by far-UV CD spectroscopy (wavelength range 200–250 nm) on a Jasco J-1500 spectropolarimeter equipped with temperature control of the sample compartment. The spectra were measured in 50 mM Tris-HCl, 150 mM KCl pH 7.5 in the presence of 2 mM CaCl2, or 2 mM EGTA at 25 °C. At different time intervals , 10 μL aliquots of proteolysis mixtures were boiled (in the presence of reducing Laemmli buffer) to quench the reaction and analyzed by SDS-PAGE. Proteins (0.5 mg/mL) were treated with trypsin (1:500 w/w) in 50 mM Tris-HCl, 150 mM KCl pH 7.5 after the addition of 2 mM CaCl2+-bound proteins (2 mg/mL) in solution was determined by size exclusion chromatography using a Superdex 75 Increase 10/300 GL column  in 50 mM Tris-HCl, 150 mM KCl pH 7.5. A calibration curve was constructed using the following standard proteins : cytochrome c (12 kDa); myoglobin (17 kDa); carbonic anhydrase (29 kDa); ovalbumin (43 kDa) and albumin bovine serum (66 kDa) as described [The apparent molecular mass of the purified apo- and Caescribed ,27. The 2. The emission spectra were collected from 400 nm to 650 nm after excitation at 380 nm. All measurements were performed in triplicate and the mean values ± SEM are given.Fluorescence emission measurements using 1-anilino-8-naphthalenesulfonic acid (ANS) were conducted on a Jasco FP8200 spectrofluorometer as previously described . A fixed2 in 33 aliquots (1.5 µL each) at 300 s intervals at 25 °C. A first injection of 0.5 μL was made and then the first data point was removed from data fitting. Decalcified ITC buffer  was prepared by processing with Chelex 100 resin  [2 solution into the buffer without any protein (buffer blank titration) was performed but did not show significant heat-related changes in the recording cell. Data was analyzed using Origin-based software. One-site and two-site binding site models were fitted to the data. Of these attempted binding models, it was found that the two-site model provided the best fit for TgCEN1-C data while for TgCEN2-C the best fit was obtained using the one-site model, as determined by the reduced chi squared value. Mean values ± SEM of at least three independent titrations on different protein preparations were reported.Isothermal titration calorimetry (ITC) measurements were conducted on TA Instrument Nano ITC. A typical experiment was carried out on a protein solution (0.2 mL of apo-proteins at 70–100 µM) by adding 2 mM CaClCA, USA) ,30. The The self-assembly propensity of centrins (20 μM) was monitored by turbidimetry experiments measuring the change in the absorbance at 340 nm as a function of time at 37 °C. All spectroscopic measurements were performed using a Jasco V-560 spectrophotometer with 1-cm path length quartz cuvettes in 20 mM Tris-HCl pH 7.5 and different KCl concentrations. 2 in 20 mM Tris-HCl pH 7.5 and different KCl concentrations. All of the buffers were filtered immediately before each measurement in order to eliminate any impurities. The increase in the mean count rate, a parameter that represents the average scattering intensity during the measurement and directly reports on aggregated species present in solution, was followed.Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano S instrument  equipped with a Peltier temperature controller by using disposable 12.5 × 45 mm cells with a stopper. The self-assembly kinetics of centrins (10 μM) were determined at 37 °C before and after addition of 1 mM CaClH. sapiens (HsCEN). The level of homology of TgCEN1 to the other centrin sequences revealed ~50% identity to TgCEN2, ~60% identity to TgCEN3, and ~70% identity to human centrins HsCEN1 and HsCEN2. TgCEN2 displayed a lower level of conservation, reaching only ~46% identity to TgCEN3 and ~50% identity to HsCEN1 and HsCEN2  profiles of potential Ca2+-binding sites, while TgCEN2 is predicted to contain only two (EF-1 and EF-4) potential Ca2+-binding sites [2+. Moreover, in TgCEN2 the EF-2 lacks crucial residues required for Ca2+ binding. A glycine at position 5 of the loop in EF-2 cannot chelate Ca2+, which is thus expected to preclude physiological Ca2+ binding to EF-2. In addition, the presence of a proline at position 7 could prevent the stabilization of negative charges in the Ca2+ coordination sphere. Of note, the replacement of the conserved glutamate residue with an aspartate at position 12 in EF-4 of TgCEN2 can result in a dramatic drop in Ca2+ affinity, as demonstrated for other Ca2+ binding proteins [2+ binding site.TgCEN1 and TgCEN2 consist of eight and nine α-helices, respectively, as predicted by Jpred 4 online  and bothng sites  . In particular, this N-terminal string  was analyzed by using GeneSilico MetaDisorder Service  and resu2+, which is a hallmark of Ca2+ binding proteins. A shift in mobility (more evident in the case of TgCEN1-C) was also seen under native conditions for both proteins, even if in the opposite direction . Thus, all the qualitative and quantitative analyses of the present study were performed using the deleted recombinant proteins . TgCEN1-C and TgCEN2-C were observed as single bands in SDS-PAGE  correspoirection , as repo2+ binding proteins on the basis of EF-hand motifs and Ca2+-dependent electrophoretic mobility shift, we carried out a set of experiments to show whether these proteins could actually bind Ca2+, what effects the metal ion had on their structure, and to identify structural and thermodynamic differences between the two isoforms. Since TgCEN1-C and TgCEN2-C were predicted to be Ca2+ binding to TgCEN1-C and TgCEN2-C was monitored by heteronuclear two-dimensional 1H-15N HSQC NMR spectroscopy  of the EF-hand Ca2+-binding loop, as a result of Ca2+ binding [2+-binding sites and to monitor protein folding. The emergence of two downfield shifted signals in holo-TgCEN1-C and one downfield shifted peak in holo-TgCEN2-C spectra recorded under in vitro conditions suggested the presence of at least two and one functional Ca2+-binding sites in TgCEN1-C and TgCEN2-C, respectively. Similar conclusions were reached by analyzing the NMR spectra of full-length TgCEN2 in the apo- and holo-states even if, as stated above, the spectra generally suffered from low resolution and low quality (2+ (even at low concentrations) to full-length TgCEN1 caused spectral overlap and a progressive loss of protein signal and resolution (data not shown), indicating that the protein may undergo strong aggregation and/or self-assembly in the presence of the ion , EF-2 (G79A) and EF-4 (G152A) in TgCEN1-C, and EF-1 (G44A) and EF-4 (G153A) in TgCEN2-C. By comparing 1H-15N HSQC spectra of wild type proteins to those of protein variants, we found that the two downfield shifted peaks in the spectrum of holo-TgCEN1-C correspond to G43 and G79, while the one in the spectrum of holo-TgCEN2-C belongs to the G44 residue  CD spectroscopy. The CD spectra of TgCEN1-C and TgCEN2-C displayed two negative peaks with local minima around 208–209 and 221–222 nm typical of proteins containing a mainly α-helical structure   B. In bot2+ to both apo-centrins induced no significant changes in the far UV CD spectra  that was ~30 °C higher than apo-TgCEN1-C (Tm = 51 ± 1 °C). Addition of Ca2+ increased the thermal stability for both proteins even if to a different extent. Indeed, holo-TgCEN1-C displayed a Tm = 60 ± 1 °C, while holo-TgCEN2-C did not reach a plateau, thus it was not possible to determine the inflexion point for the estimation of the Tm. Notably, the presence of a CD signal even at 90 °C independent of the presence of EGTA or Ca2+ suggests that the two centrins preserve a partially folded structure even at this high temperature.Additionally, far-UV CD was used to investigate the thermal stability of TgCEN1-C and TgCEN2-C by following the CD signal at 222 nm C,D. Apo-2+ binding, we performed limited proteolysis and size exclusion chromatography (SEC) experiments of TgCEN1-C and TgCEN2-C in the presence and absence of Ca2+. As further ways to investigate conformational changes occurring upon Ca2+. In the case of apo-TgCEN1-C, the band representing the intact protein disappeared within 60-min and lower molecular mass bands were evident after a 1-min reaction. Upon binding of Ca2+ ions, TgCEN1-C underwent a change of susceptibility to trypsin, becoming more resistant than its apo-form to proteolytic digestion, since the band corresponding to the intact protein was evident following a 2-h reaction. On the other hand, parallel experiments performed with TgCEN2-C showed that in the presence or absence of Ca2+, the banding patterns are quite similar, indicating that trypsin has a similar access to the apo- and holo-conformation of the protein. It is worthwhile underlining that the apo-proteins are affected by proteolysis in a different way. Apo-TgCEN1-C is more easily digested by trypsin and is entirely hydrolyzed to lower molecular mass peptides, while apo-TgCEN2-C is almost completely resistant to proteolytic attack, as illustrated by the presence of the intact protein even after a 2-h reaction. Collective data from digestion correlate well with the results obtained from NMR and CD spectroscopy, providing evidence that significant differences exist within the structural conformations of the two centrin isoforms. Apo-TgCEN1-C is likely more flexible and less stable than apo-TgCEN2-C and Ca2+ binding has a significant effect on the conformation of TgCEN1-C, while it has only a subtle impact on the conformational properties of TgCEN2-C.2+. As shown in 2+-sensor proteins is largely based on alteration in their solvation properties and not just on their molecular weight. Indeed, many Ca2+ sensors, such as CaM and CaM-like proteins from different organisms [We next performed SEC of TgCEN1-C and TgCEN2-C in the presence of EGTA or Carganisms , have at2+ is likely to induce conformational variations of TgCEN1-C toward a slightly more elongated shape, while no major effects are observed in the hydrated shape of TgCEN2-C. Considering the retention time, Ca2+ free and Ca2+ loaded centrins. When ANS interacts with hydrophobic regions on proteins, it displays a blue shifted emission and an increase in emission intensity. As shown in 2+ binds to TgCEN1-C, a large blue-shift (24 ± 3 nm) in the maximum emission wavelength of the probe associated with an evident increase in maximum intensity (2.4 ± 0.3) was observed, likely due to a marked increase in hydrophobic exposure. Notably, there were some modifications in ANS fluorescence even in the presence of EGTA, suggesting that apo-TgCEN1-C also exhibits hydrophobic pockets, which are, however, smaller than those in the holo-protein. On the other hand, only a small blue-shift in emission wavelength (6 ± 1 nm) and a non-significant increase in ANS fluorescence (1.2 ± 0.1) were observed for TgCEN2-C in the presence of Ca2+ . Representative isotherms for Ca2+ binding to isolated TgCEN1-C and TgCEN2-C are shown in We attempted to measure the affinity for Ca2+ binding showed exothermic profiles. The TgCEN1-C data best fit a model that predicted two sets of sites for Ca2+ binding due to a two phases curve: one high-affinity site (Kd1 of ~2.1 µM) and a second site with lower affinity (Kd2 of ~26 µM). On the other hand, the Ca2+-binding isotherm for TgCEN2-C exhibited a classical sigmodal shape and the best fit was obtained using a simple one site model, which results in the binding of a single Ca2+ ion  with low affinity (Kd of ~63 μM). These ITC results are consistent with NMR experiments, supporting the binding of Ca2+ ions to EF-1 and EF-2 sites in TgCEN1-C and to EF-1 in TgCEN2-C.Both thermograms of TgCEN1-C and TgCEN2-C for Ca2+-binding sites with different affinity in TgCEN1-C was clearly supported by NMR spectra. Ca2+ was titrated into apo-TgCEN1-C and 1H-15N HSQC spectra were recorded at each step. As visible in 2+-bound G43 (EF-1), while the peak of G79 (EF-2) appeared only at higher Ca2+:protein ratios. These observations prompted us to assign the dissociation constants determined from ITC Kd1 to EF-1 (higher affinity Ca2+-binding site), and Kd2 to EF-2 (lower affinity Ca2+-binding site).Of note, the presence of two Ca2+ addition and the role of the N-terminal 20–25 residue extension in mediating this process, phenomena already described for many other centrins [2+, no changes in light scattering were observed for any of the variants. However, upon addition of 1 mM Ca2+, full-length TgCEN1 exhibited a considerable increase in turbidity , while TgCEN2 binds only one Ca2+ ion through EF-1 with low affinity (Kd of ~63 µM). Thus, both isoforms likely have only a subset of sites that are active. In both cases Mg2+ has no effect on these Ca2+ sites, which can be considered Ca2+ specific. Interestingly, centrins display large variance in their Ca2+ sensing abilities. Chlamydomonas reinhardtii centrin possesses two N-terminal high affinity Ca2+ binding sites and one site with lower affinity in the C-terminus [2+ with high affinity in the N-terminal lobe and two Ca2+ ions with low affinity in the C-terminal domain [2+ binding site located in EF-4 [Arabidopsis thaliana CEN2 binds four Ca2+ ions, two with high affinity at EF-1 and EF-2 and two with lower affinity at EF-3 and EF-4 [Trypanosoma brucei binds to Ca2+ with high affinity through its EF-3 and EF-4 in the C-terminal domain [2+ sites among centrin proteins. It was noteworthy that EF-4 in both TgCEN1 and TgCEN2 was non-functional in terms of Ca2+ binding, even if it does conform to the EF hand consensus sequence. This finding is reminiscent of the diversities between predicted and experimentally measured binding sites observed for other Ca2+ binding proteins [2+-binding affinity and selectivity in many Ca2+ binding proteins [2+ binding site. However, we cannot exclude the possibility that the Ca2+-binding characteristics (affinity and number of active binding sites) of TgCEN1 and TgCEN2 are likely undervalued in our in vitro conditions and other factors such as the presence of putative interactors can significantly affect the in vivo Ca2+ sensing abilities of the two isoforms. For instance, it has been suggested that in the case of the Ca2+-dependent HsCEN2–melittin interaction, a non-functional EF-hand in the N-terminal domain of the protein may be activated to bind Ca2+ when a complex is formed [Toxoplasma centrins are associated with different cytoskeletal structures , but theterminus . Yeast cterminus . HsCEN3 l domain . HsCEN2  in EF-4 . Arabidol domain . Thus, tproteins ,41,54,55proteins ,17,33, cs formed .2+ sensors in terms of Ca2+-induced conformational changes, including, upon the addition of Ca2+: (i) a significant increase in the elliptic signal likely due to changes in the interhelical angles within the EF-hand domains; (ii) considerable changes in the 1H-15N HSQC NMR spectra with the protein that adopts a more stable tertiary conformation; and iii) exposure of hydrophobic patches as indicated by ANS probe. It should be noted that for proteins that possess self-assembly properties, Ca2+-induced changes in ANS may not be due to conformational change, but rather could be due to binding between molecules in the assembly process. However, here we reported ANS experiments only for the truncated versions of toxoplasma centrins (TgCEN1-C and TgCEN2-C), which showed no detectable turbidity changes, and thus no tendency to self-assemble upon addition of Ca2+ (2+ even for full-length TgCEN1 (the only protein variant that exhibited a considerable increase in turbidity in our experiments)  . Moreoveriments) . These o0–100 nM , as for 2+ sensor, TgCEN1 is supposed to interact and regulate downstream proteins. Unfortunately, information about the possible biological roles of TgCEN1 is still missing. In this scenario, the Ca2+ dependent ability of TgCEN1 to self-assemble observed in this work may account for crucial biological functions of the protein. Ca2+-dependent self-assembly seems to be a peculiar property of centrins and many cell biology studies have shown that centrins are crucial structural components of Ca2+-sensitive contractile filament systems. TgCEN1 is predominately localized to the highly regular structure of centrioles [Therefore, as a Cantrioles , suggestntrioles , both io2+ sensor, binding of Ca2+ to TgCEN2 did not cause a large conformational change, which is a hallmark of Ca2+ sensors, as demonstrated by NMR, CD, and ANS analysis. The NMR spectral fingerprint of Ca2+-bound TgCEN2 is distinct from that of the apo-state, although the differences are more consistent with only localized conformational rearrangements, likely within the single functional Ca2+ site. The very small changes in secondary structure, as indicated by CD spectra, do not preclude per se that Ca2+ binding induces conformational variations. However, following Ca2+ binding, no hydrophobic surfaces are exposed in TgCEN2-C, as shown by ANS fluorescence. Thus, although Ca2+ binds to TgCEN2-C, it does not drive the conformational change with the exposure of a hydrophobic region that is crucial for hydrophobic interaction with target proteins. Moreover, ITC experiments indicated a single Ca2+ binding site with a dissociation constant of ~63 μM in TgCEN2-C. Functional EF-hands are usually paired  and possess affinity in the low micromolar range [2+ sensor domain can function even if it has only one site capable of binding Ca2+. There are many examples of functional Ca2+ sensor domains that have only one EF-hand that is able to bind Ca2+, including some previously studied centrins [2+ binding protein, it is fundamental to consider the difference between affinity for Ca2+ by itself versus its affinity for Ca2+ in the presence of physiological targets. Indeed, binding partners can have an enormous effect on measurements of Ca2+ affinities since the two binding events are energetically coupled. Consequently, vast increases in Ca2+ affinities were often observed when the measurements are made in the presence of a partner protein or even just the interaction motif peptide, as in the case of yeast, plant and green algal centrins [2+ affinity when measured for the isolated protein, such as for TgCEN2, may well be fully functional. Moreover, it is possible that the Ca2+ binding properties of TgCEN2 can be tuned differentially by diverse target proteins.While TgCEN1 behaves biochemically as a Caar range . Howevercentrins ,13,53,57centrins ,58. Thus2+ buffering protein. In fact, exposure of hydrophobic surfaces would be unfavorable for these proteins because it may limit their stability and Ca2+ affinity. However, the low affinity for Ca2+ suggests that TgCEN2 is unlikely to work as a Ca2+-buffer protein, since Ca2+-buffering proteins usually have a very high affinity for Ca2+ to efficiently chelate the ion .2+ buffering protein nor as a typical Ca2+ sensor protein. It is not known how large the impact of Ca2+ on protein structure has to be in order to be physiologically relevant and we cannot exclude the possibility that a different Ca2+-relay mode and different ways of interaction with target proteins exist that do not follow the paradigm of a Ca2+-dependent switch with exposure of hydrophobic surfaces. Notably, as demonstrated by limited proteolysis and CD denaturation experiments, TgCEN2 is very stable even in the apo-form, suggesting the possibility that apo-TgCEN2 might also bind to a potential target and that this interaction may play a significant role in the Ca2+ signaling pathway. Thus, the mechanism of action of TgCEN2 is not clear since it behaves neither as a Ca2+ regulated processes. TgCEN2 may not be involved in the crucial cellular process requiring self-assembly of centrins. Accordingly, TgCEN2, in addition to centrioles, is also targeted to the apical  and basal ends  of the parasite, in addition to annuli located at the lower part of the apical cap within the membrane cortex. This differential distribution of TgCEN2 points to versatile functions of TgCEN2 in the four distinct structures. Experimental evidence based on gradual depletion and knockdown of TgCEN2 revealed that this protein is involved in several Ca2+-mediated processes, namely microneme secretion, motility, host cell attachment, invasion, and egress [2+, TgCEN2 and TgCEN2-binding targets will be crucial to unravel the exact Ca2+-sensing properties, mode of action and biological function of this centrin isoform.Moreover, TgCEN2 does not display the tendency to self-assemble, underlining the molecular differences with TgCEN1, and thus indirectly highlighting different roles in Cad egress . In addid egress ,22. In t2+ and TgCEN1 and TgCEN2, with each centrin isoform displaying a precise detection mode for Ca2+. Toxoplasma occupies a unique phylogenetic position since it is an early branching eukaryote, and therefore, the characterization of the molecular properties, mechanism of action, and functional specialization of parasitic centrins and, more in general of Ca2+ binding proteins, can provide useful information about the early evolution of Ca2+ signaling machinery. Moreover, it may be relevant in the discovery of promising druggable targets to treat toxoplasmosis.In summary, our results suggest the existence of a sophisticated interplay between Ca


Delamination is one of the main problems that occur when machining fiber-reinforced composite materials. In this work, Types I and II of delamination are studied separately in edge trimming of basalt fiber reinforced plastic (BFRP). For this purpose, one-dimensional and area delamination parameters are defined. One-dimensional parameters (Wa and Wb) allow to know average fibers length while the analysis of area delamination parameters (Sd) allow to evaluate delamination density. To study delamination, different tests are carried out modifying cutting parameters  and material characteristics (fiber volume fraction and fiber orientation). Laminates with a lower fiber volume fraction do not present delamination. Attending to one-dimensional parameters it can be concluded that Type II delamination is more important than Type I and that a high depth of cut generates higher values of delamination parameters. An analysis of variance (ANOVA) is performed to study area parameters. Although delamination has a random nature, for each depth of cut, more influence variables in area delamination are firstly, feed per tooth and secondly, cutting speed. In aeronautical, naval and automotive sectors, application of fiber-reinforced polymeric matrix composite materials is increasing, mainly due to their lightness, high mechanical resistance and resistance to corrosion and high temperatures. Manufacturing processes of fiber-reinforced composite materials allow to obtain parts with a geometry close to the desired one. After curing, machining processes are required to eliminate excess of material and to achieve the required shapes and tolerances. However, fiber-reinforced composite materials behave differently in machining than metals, due to their heterogeneous nature and anisotropy .One of the main problems that appear when machining fiber-reinforced composite materials is delamination. Delamination consists of a separation of the fabric layers from the composite material due to the force exerted by the cutting tool during machining process. This defect affects both the dimensional accuracy and the subsequent joining between parts . It can In the literature, the most studied factors to evaluate delamination produced after milling are cutting parameters, fiber orientation angle and cutting tool characteristics  ,5,6,7,8.In milling process, delamination has been mainly studied in grooving and in edge trimming operations. In grooving, a “delamination factor” is defined, similar to the one used in drilling. Instead of referencing the delamination measurement to the hole diameter, reference is made to the groove width . As in dTo evaluate delamination in milling, in both grooving  and edgeGenerally, delamination occurs mostly in one of the layers of the part, the upper or the lower layer, and most factors defined above only evaluate this effect in one of the layers. However, delamination factors have also been defined to jointly evaluate this defect in both layers . In thisIn this paper different parameters are defined to evaluate the two most important types of delamination, Type I and Type II, in edge trimming of BFRP. Basalt fiber is completely inert, non-toxic and good thermal and electrical insulator, and has properties that make it performs better than glass fiber and slightly worse than carbon fiber . All parDifferent tests are carried modifying cutting parameters  and material characteristics (fiber volume fraction and fiber orientation) at different cutting times to evaluate delamination.Delamination parameters are classified in one-dimensional parameters and area parameters. One-dimensional parameters (Wa and Wb) allow to evaluate the length of the fibers that protrudes or goes inward from the trimming edge, considering free spaces between yarns, and without considering these free spaces. These parameters allow to quantify the delamination in all experiments at different cutting times. Anyway, area parameters are also needed to evaluate density delamination. All these parameters allow to study delamination, but considering that delamination has a random nature. Additionally, an ANOVA study has been performed to quantify the effect of cutting conditions and fiber orientation angle in area parameters.Parts to be machining are rectangular laminates (420 mm × 260 mm) of fiber-reinforced polymer matrix with a thickness of 3.4 mm manufactured by resin transfer molding (RTM). Reinforcement chosen is a bi-directional long basalt fiber fabric , with stEdge trimming operation (down milling) is conducted in a milling machine , with a Mitsubishi Materials milling tool holder of diameter 25 mm and two exchangeable uncoated carbide cutting inserts . To avoi16 orthogonal array was selected to determine combination of factor levels to use for each experiment  and material characteristics (fiber volume fraction and fiber orientation). Design of experiments is based in Taguchi’s method for five variables at two levels . An L16 periment . Each exA cutting tool with new inserts was machining without coolant for 80 min. Stopping times  are made to measure delamination parameters. In test 14, due to high cutting conditions, a great flank wear occurred, and it was not possible to reach a cutting time of 80 min.Delamination measurement has been made capturing wick images of the upper face of laminates  in an evAn image is taken of the machined edge of the laminate, and for each wick (i) the following three parameters are captured by means of a cutout and its histogram: surface (Sdi) for each type of delamination; maximum value (hmax i) and invaded edge length (li) .The values obtained by measuring wick to wick delamination from the captured images of each experiment are recorded in Excel data sheets, for subsequent analysis and calculation of delamination parameters. Laminates with a low fiber volume (Fv 40%) do not present delamination, so they will not be evaluated. However, in laminates manufactured with high fiber volume (Fv 60%) delamination always appears on the edge, regardless of the cutting time. In all laminates, delamination on the upper face is greater than on the lower face. In order to evaluate delamination only upper face will be measured.Laminates with 45° and 90° fiber orientation are evaluated. In 45° fiber orientation laminates all warp yarns are over the fill yarn, thereby all wicks present delamination. In 90° fiber orientation, different delamination cases are studied , becauseTo analyze delamination, warp yarns over the fill yarn have been identified with a number. If warp yarns are under the fill yarn they have been identified with a number plus ‘.In the evaluation of delamination, average length of the fibers that protrudes and goes inward from the laminate are measured. To quantify these values, one-dimensional parameters (Wa and Wb) are defined. Wa parameter allows to classify the tests attending at delamination magnitude, distributing delaminated surface in the evaluated length (L). Wb parameter calculates delamination magnitude, but not considering free space between wicks. On the other hand, in order to compare all experiments, a delamination factor (Fdel) is defined as the ratio between Wb value and the maximum theoretical possible value. This factor allows to classify experiment attending to the maximum possible theoretical delamination value. As this maximum value depends on the selected depth of cut, the effect of this variable is mitigated.One-dimensional parameters do not define correctly delamination density. Therefore, it is also necessary to define area parameters (Sdi). As happens in one-dimensional parameters, to compare between experiments an area delamination factor (FSdi) is defined as the ratio between the Sdi and the maximum theoretical possible delaminated area for each experiment.These parameters allow to know the average length of fibers that protrude in Type II delamination and the average length of fibers that are inward from the trimmed edge in Type I delamination.The “medium delamination” parameter (Wa) is defined as the height of an equivalent rectangle of delaminated area with basis the evaluated length (L) . StudiedMedium delamination parameter (Wa) does not allow a real measure of delamination length because all delaminated surface is related to studied length (L). Relating delaminated area to only delaminated fibers length  of parameters Wa and Wb is the depth of cut. In Type I delamination, this maximum value is the yarn measure (3 mm) in 90° fiber orientation tests, and the yarn diagonal (4.24 mm) in 45° fiber orientation tests . DelaminIn each experiment, data are taken at different cutting times. As cutting time increases, tool wear increases and delamination should be greater, increasing values of different parameters and factors. Delamination values present great variation at different cutting times, without an increasing or decreasing trend as tool wear increases. In almost all experiments, Type I and Type II delamination appear at the same places of the trimming edge. Data from all experiments at all cutting times are analyzing, except for test 14, because in this test tool has reached a rapid flank wear and parameters values are excessive high. Values obtained for Type II delamination are much higher than values for Type I delamination. Depth of cut is one important parameter to evaluate delamination. In the test with 90° fiber orientation, Wa value is usually higher than Wa’. Due to delamination values present great variation at different cutting times, without an increasing or decreasing trend, a range is established to delamination parameters and factors in each experiment .In order to quantify density delamination in every experiment, values obtained for Wb and Wb’ parameters in 90° fiber orientation cannot be added. For this reason, delaminated areas measurement has been carried out. In 90° orientation fiber laminates, these areas are compared in the warp yarn over and under fill yarn.As in one-dimensional parameters, area delamination factors (FSd) are also defined. In these parameters delaminated area is related to the maximum possible delamination area:A range is established to delamination variation for FSdi and FSdi’ in each experiment for Type I and II delamination .An analysis of variance (ANOVA) has been performed to quantify the effect of cutting conditions and fiber orientation angle on the area delamination factors (FSdi + FSdi’). For analyzing the significant effect of the factors on the responses, F test with a level significance of 0.05 has been used. The influence of each factor can be represented using a Box-whisker diagram . The meaThis paper defines and evaluates different delamination parameters for Type I and II delamination in edge trimming of basalt fiber reinforced plastics (BFRP). All parameters defined and evaluated in this work can also be applied to carbon or glass fiber reinforced plastics. For this purpose, different tests are carried out modifying cutting parameters  and material characteristics (fiber volume fraction and fiber orientation). Delamination values have been obtained at different cutting times in order to find a relationship between delamination and cutting time, although most authors do not take delamination values at different cutting times.Delamination parameters are classified in parameters that evaluate lengths and parameters that evaluate areas. Parameters that evaluate lengths give an average of protrude fibers length (for Type II delamination) and an average of inward fibers length (for Type I delamination). Two parameters are defined in this category (Wa and Wb). Wa considers free spaces between yarns, but Wb does not consider these free spaces. A delamination factor (Fdel) is also defined in order to compare delamination of each experiment with the maximum possible delamination value. On the other hand, parameters evaluating areas (Sd) are needed to quantify density delamination in the machining edge. Additionally, as in one-dimensional parameters, a factor (Fsd) is defined to compare area delamination.Delamination at the top layer of the laminate only appears with a 60% fiber volume fraction. BFRP consists of a bi-directional long basalt fiber fabric, therefore delamination has been measured in warp yarns over and under fill yarn in 90° fiber orientation laminates. In most of 90° fiber orientation measurements, delamination measured in warp yarns over fill yarn is bigger than one measured in warp yarn under fill yarn, it can be concluded that delamination of the warp yarn below the fill yarn is negligible.Delamination parameters and factors are calculated at different cutting times. Delamination values present great variation at different cutting times, without an increasing or decreasing trend as tool wear is increasing. It is due to the random nature of delamination. At every cutting time, the distance of the warp yarn from the trimmed edge until next dip below the crossing fill yarn (Xd) is changing, observing a uniform or sine wave delamination.Analyzing one-dimensional parameters, it can be concluded that Type II delamination is more important than Type I and that a high depth of cut generates higher values of delamination parameters. Fdel factor can be used to compare between experiments with the same depth of cut, but not with different values of depth of cut, as maximum possible area depends on the selected depth of cut.An analysis of variance (ANOVA) is performed to study area parameters. Although delamination has a random nature, for each depth of cut, more influence variables in area delamination are firstly, feed per tooth and secondly, cutting speed. It is expected that for a feed per tooth of 0.1 mm, delamination is smaller than for 0.4 mm, because at smaller feeds cutting edges impact on the fabric wick a greater number of times.


Mechanoelectrical feedback is an important factor in the pathophysiology of atrial fibrillation (AF). Ectopic electrical activity originating from pulmonary vein (PV) myocardial sleeves has been found to trigger and maintain paroxysmal AF. Dilated PVs by high stretching force may activate mechanoelectrical feedback, which induces calcium overload and produces afterdepolarization. These results, in turn, increase PV arrhythmogenesis and contribute to initiation of AF. Paracrine factors, effectors of the renin‐angiotensin system, membranous channels, or cytoskeleton of PV myocytes may modulate PV arrhythmogenesis directly through mechanoelectrical feedback or indirectly through endocardial/myocardial cross‐talk. The purpose of this review is to present laboratory and translational relevance of mechanoelectrical feedback in PV arrhythmogenesis. Targeting mechanoelectrical feedback in PV arrhythmogenesis may shed light on potential opportunities and clinical concerns of AF treatment. Mechanoelectrical feedback is an important factor in the pathophysiology of atrial fibrillation. Paracrine factors, effectors of the renin‐angiotensin system, membranous channels or cytoskeleton of pulmonary vein (PV) myocytes may modulate PV arrhythmogenesis directly through mechanoelectrical feedback or indirectly through endocardial/myocardial cross‐talk. Stretch may induce Ca2+ overload and produce afterdepolarizations in cardiomyocytes.2+ sources. Stretch modifies cellular Ca2+ homeostasis by increasing the Ca2+ influx via stretch‐activated channels+ influx, followed by extrusion of Na+ accompanied by Ca2+ influx via sodium/calcium exchanger (NCX).2+ current (ICa‐L), have been reported to possess mechanosensitivity.2+ cycling with decreased ICa‐L, which contributes to increased sarcoplasmic reticulum Ca2+ content despite unchanged or lower Ca2+ reuptake by sarcoplasmic reticulum Ca2+‐ATPase (SERCA) and increased diastolic Ca2+ leak.The spontaneous activity of PV cardiomyocytes is mainly regulated by Cacurrent ICa‐L, havcurrent ICa‐L, havAcute stretch has been associated with conduction slowing and complex signal formation in the PV‐LA junction in human studies, and prolongation of refractory period in animal studies.2+ release is also a strongly coupled form of mechanoelectrical feedback in cardiomyocytes.2+ from the myofilaments propagates through neighboring tissue by a combination of mechanical transduction, Ca2+ diffusion, and Ca2+‐induced Ca2+ release. This triggered detachment of Ca2+ from troponin C has been hypothesized leading toward extra‐systoles.2+ release by modulating actin filaments during structural remodeling may contribute to AF genesis.The effects of altered stretch on PV cardiomyocytes not only influence the internal machinery of cells, in part, via stretch‐activated channels,33.12+ overload.2+ transient and produces spontaneous Ca2+ sparks during diastole in myocytes contracting against a higher preload or afterload.PVs contain endothelium and smooth muscle, which may produce nitric oxide (NO) through the enzyme eNOS or iNOS. Previous studies have shown that NO has important regulatory effects on the cardiovascular system.2+ and potassium (K+) currents, and decreases PV spontaneous activity while increasing resting diastolic tension.Endothelin appears to act as a mediator in the pathogenesis of hypertension and its complication resulting from mechanical loading. The major site of generation of endothelin‐1 is in endothelial cells.2+‐activated K+ (SK) channels plays an important role in maintaining vessel relaxation,Previous studies indicated small‐conductance Ca3.2The renin‐angiotensin (Ang) system (RAS) plays a critical role in the pathophysiology of AF, which contributes to onset and maintenance of AF in paced animal models.Mechanical stretch of cultured rat cardiac myocytes caused a rapid secretion of Ang II,ICa‐L, but increased reverse‐mode NCX current, which reduces Cai2+ transients, and SR Ca2+ content in PV cardiomyocytes. Aliskiren decreased PV diastolic tension, and reduced PV firing rate in a concentration‐dependent manner.Renin‐angiotensin system inhibition could be achieved by renin inhibitors, the first rate‐limiting step in RAS, which are thought to be more efficient than other inhibitors such as angiotensin‐converting enzyme or Ang II receptor antagonists. The renin inhibitor enalkiren has more effect on vasodilatation renal response compared to captopril.ICa‐L, late sodium current (INa‐Late), and NCX current in PV cardiomyocytes.Cai2+ transients and SR Ca2+ content, and prevent Cai2+ overload. The effects of Ang‐(1‐7) may reduce the PV spontaneous electric activity and triggered activity, which suggests the anti‐AF potential of Ang‐(1‐7).Ang‐(1‐7) opposes the molecular and cellular effects of Ang II.3.3Stretch‐activated ion channel blockers were shown to inhibit stretch‐induced changes in AP and afterdepolarizations,3.42+ influx via stretch‐activated channels which are tightly regulated by the actin cytoskeleton.2+‐activated K+ channel current, as well as ICa‐L, INa‐Late, and NCX current, which may reduce the Ca2+i transient amplitudes and the SR Ca2+ content. The decrease in ICa‐L, INa‐Late, and NCX current by Lat‐B may be related to the modulation of stretch‐induced changes, which may reduce PV arrhythmogenesis.2+ regulation may contribute to decreased PV spontaneous electrical activity and attenuated stretch‐induced PV arrhythmogenesis.Cells lose their arrhythmic effects on cardiomyocytes after pharmacological ablation of α‐smooth muscle actin‐containing stress fibers.42+ homeostasis and mechanoelectrical feedback because of increasing PV vascular tone through the enhanced contractility from its surrounding myocardium,+ channels, or inhibits phosphodiesterase III.Conditions accompanied with mechanical stretch of the heart such as aging,52+ handling, cytoskeletal modulation and RAS regulation, caused by PV dilatation are expected to be improved after successful ablation. However, plasma levels of renin and Ang II remain higher 6 months after catheter ablation.Catheter ablation has been established as a mainstay for the treatment of AF.62+ handling characteristics of mechanoelectrical feedback. Multiple stretch‐activated mechanisms play important roles in PV mechanoelectrical feedback, which may contribute to the arrhythmogenic substrate in pathology. This should prompt the exploration of these mechanisms as potential novel therapeutic targets (Figure Pulmonary vein arrhythmogenesis is crucially dependent on the electrophysiological outcomes and Cas Figure . ExcessiThe authors declare no conflict of interest for this article.