The effect of matrix cracking on the delamination morphology inside carbon fiber reinforced plastics (CFRP) laminates during low-velocity impact (LVI) is an open question. In this paper, the relationship between matrix cracking and delamination is studied by using cross-ply laminates. Several methods, including micrograph, C-scan, and visual inspection, were adopted to characterize the damage after LVI experiments. Based on the experimental results, finite element (FE) models were established to analyze the damage mechanisms. The matrix cracking was predicted by the extended finite element method (XFEM) and the Puck criteria, while the delamination was modeled by cohesive elements. It was revealed that the matrix crack in the bottom ply not only promoted the outward propagation of delamination but also contributed to the narrow delamination beneath the impact location. Multiple matrix cracks occurred in the middle ply. The ones close to the plate center initiated the delamination and prevented large-scale delamination beneath the impact location. For the cracks that were far away, no significant effect on delamination was found. In conclusion, the stress redistribution caused by the crack opening determines the delamination. Carbon fiber reinforced plastics (CFRP) have been widely applied to aeronautical engineering for several decades due to their high specific strength and stiffness . NotwithAlthough researchers have made numerous efforts in the impact mechanisms study and revealed that the delamination is induced by matrix cracking ,3,4, theWith the purpose of analyzing the impact mechanisms and predicting the damage, many finite element (FE) models were proposed by researchers. Since the matrix cracking has a close relationship with the delamination initiation, it is necessary to take their interaction into consideration in FE modeling for an accurate prediction in delamination. In some studies, FE models were established based on continuum damage mechanics (CDM). The effect of matrix damage on stress redistribution was considered by modifying the damage evolution behaviors of CDM ,11. HoweThe extended finite element method (XFEM) proposed by Belyschko and Blacks  is an atThe main goal of this paper is to figure out the influence of matrix cracking on delamination morphology during LVI, especially the undelaminated region beneath the impact location. An FE modeling strategy based on XFEM, the Puck criteria, and cohesive elements is proposed to predict matrix cracking and delamination accurately. The simulations are verified by the experimental results. Cross-ply laminates were utilized in this paper due to their simple damage mechanisms. Although many researchers studied the damage mechanisms by applying cross-ply laminates ,19,20,21Cross-ply laminates tested in this paper were manufactured from carbon fiber reinforced epoxy resin composite IMA/M21 prepregs by an automatic tape-laying technique. The prepregs were supplied by Hexcel Corporation . The stacking sequence was designed to be Impact damage was introduced to the center of each specimen at room temperature by applying a drop-weight impact instrument, which was designed according to ASTM D7136. The impactor used in this test consisted of a hemispherical nose with a diameter of 16 mm. The total weight of the impactor was 5.495 kg. The specimens were placed on a fixture base with a 125 × 75 mm cut-out. Meanwhile, the corners of the specimens were restrained by four clamps. The boundary condition of the impact experiments is presented in After the impact experiments, the impact dent depth was measured with a Syntek micrometer gauge  immediately. The impact damage was inspected by visual inspection and ultrasonic C-scan. A UC-120 C-scanner equipped with a 10 MHz ultrasonic probe was used. Both the C-scanner and probe were products of Physical Acoustics Corporation . To characterize internal impact damage, one specimen in each group was cut along the longitudinal direction. The internal damage was observed with an MV-GED500M industrial camera manufactured by Shenzhen Mindvision Technology CO., LTD .FE models were established in the commercial FE software ABAQUS 6.13 to simulate the impact processes using subroutines UDMGINI and UMAT. As shown in In this study, the Puck criteria proposed by Puck and Schürmann  were empessed as :for σn, To determine the angle lgorithm  was proplgorithm  and Schilgorithm . Four ston (IPI) ; (4) comthan 25° , the numXFEM framework embedded in ABAQUS was used. In ABAQUS, cohesive segments method and phantom nodes are adopted when modeling the moving cracks. Before the crack initiation, the phantom nodes are superposed on the corresponding real nodes. When an element is cut by a crack, DOFs of phantom nodes are independent of those of real nodes. Consequently, the element is cut into two segments. The separation between these two segments leads to the stress discontinuity in a cracked region. Such separation is governed by the following traction-separation cohesive behavior:The critical energy release rate aw model :(7)Gc=GIwas used . When thTo enhance the numerical convergence, the viscous regularization technique with a small viscosity coefficient (0.0001) was adopted . In a tyIt has been revealed by many researchers that the transverse/parallel shear behavior of composites exhibits high nonlinearity ,35,36. Wbehavior ,40:(8)τ1The constitutive response of cohesive elements was also described by the traction–separation relationship (see Equation (6)), indicating that only three stress components . Symmetric boundary conditions were applied to the symmetric planes. Interactions between parts were all defined as surface-to-surface contact. The contact between plies after delamination was also taken into consideration. Friction coefficients of all contact pairs were set as 0.3. Composite plies were modeled with linear solid elements (C3D8) to improve the crack calculation accuracy. Two interlaminar interfaces were simulated with cohesive elements (COH3D8). For composite plies, a fine mesh with 0.3 mm for the in-plane size was used near the impact location. The element size increased to 2 mm at the edges of the laminate. To improve the accuracy of modeling delamination shape, the size of cohesive elements was determined to be 0.2 mm in the prospective delaminated regions. These mesh sizes satisfied the mesh convergence according to the preliminary simulations. The total number of elements was 95,178.The material properties were listed in Caminero  were ado Mortell  indicate” effect . This wa” effect  and stre” effect . In addi” effect ,48 and f” effect ,46. In tAccording to the study of Chen , a matriIn this section, the experimental results, mainly the damage modes and their sizes, are presented firstly. During the experiments, these damage sizes, including the delamination sizes and angles of matrix crack, were measured by the image analysis of C-scan data and micrograph. Later, the damage modes and sizes are used for qualitative and quantitative verification of the modeling strategy. As revealed by the Puck criteria, the angle of a matrix crack is determined uniquely by the stress state. Hence, the numerical results of crack angles can be used for the verification of the stress calculation. After the simulations are verified, the stress distributions and damage formation in the simulations are used for the damage mechanisms analysis with the aid of the failure theories mentioned in As revealed by the C-scan and micrograph of the internal damage, delamination only existed at the lower interface. The typical delamination detected by C-scan from the lower surface is displayed in For each specimen, the outline of the delamination was idealized, as in A micrograph was used for revealing the internal damage at different locations see a,b. In tDelamination extended along the longitudinal direction with a long distance, including the region beneath the impact location. Due to the permanent deformation of the bottom ply after delamination and debris between the delaminated surfaces , the gapMultiple matrix cracks started at the lower interface and propagated towards the impact location. All of them were confined in the middle ply. When the impact energy increased to 12 J, matrix crushed in the middle ply beneath the impact location. Although the numerical results showed that both the tensile transverse stress For each impact case, the crack that was far from the impact location usually propagated with a larger inclination compared to the crack close to the impact location. The angles of the farthest crack for three impact energy levels  were 58.0°, 61.2°, and 64.6°, respectively. As for the closet crack, its angles were 53.6°, 51.7°, and 50.7° for the three impact cases. Probably related to the randomness of defects, the crack spacing varied from 2 to 7 mm without regularity.The typical external impact damage was presented in e debris  and permSince the damage modes and their symmetry remained unchanged in all impact cases in the experiments, the modeling strategy proposed in this paper was reasonable.The numerical results verified the ability of the modeling strategy to capture the main damage modes and their sizes, i.e., the delamination at the lower interface and matrix cracks. Although isolated artificial delamination occurred near the NDR, it was neglected in the discussions about damage mechanisms, since it was only induced by the limitations of cohesive elements and had no influence on the damage mechanisms.The comparison in The FE modeling strategy could also predict the delamination sizes successfully since the differences in most sizes between the experimental and numerical results were acceptably small see . In the In FE models, two kinds of matrix cracks were also observed, including multiple shear cracks in the middle ply and a tensile crack in the bottom ply see . The comSimilar to the experimental results, most shear cracks propagated upwards linearly with different inclines and stopped in the middle ply. For simplification, these cracks are hereinafter referred to as Crack A’, B’, C’, and D’ according to their locations. Crack A’ was the one closest to the impact location. As mentioned in Since the models were capable of simulating the main damage modes and their trends, it was reasonable to analyze the damage mechanisms based on the numerical results.The damage processes for all models were similar. Hence, the process of Model B is presented as an illustrative example. The propagation of matrix cracking and delamination is shown in The tensile crack in the bottom ply was the first damage. Induced by the tensile Consequently, as illustrated in For the tensile crack, the stress discontinuity induced damage to the cohesive elements at the lower interface. As the impactor moved downwards, the stress jump caused by COD kept increasing and further encouraged the delamination. The crack opening also degraded the bending stiffness of the laminates, which, in turn, facilitated the crack growth. Hence, the delamination propagation was accelerated. At the bottom line of the MDR, delamination propagated towards the impact location was dominated by the shear deformation in the X-Y plane. For a cohesive element in this region, the shear strain (2.0%) was much larger than the normal strain (0.2%) before its deletion. Beneath the impact location, the crack opening induced the large deformation at the lower interface, including the normal deformation and shear deformation in the Y-Z plane. The deformation initiated the narrow delamination in this region. These can be proved by the failure strain of a cohesive element in this region: 2.1%  and 1.5% (shear strain in the Y-Z plane).The stress distributions of a path were studied to evaluate the effects of Crack A’. As illustrated in In general, Crack A’ initiated the MDR and promoted its propagation in the Outer Zone by the crest of laminate ,52. SincIn FE models, artificial delamination existed at the upper interface and near the NDR at the lower interface see . It was As mentioned above, only three stress components (Furthermore, the through-thickness compression was believed to delay the delamination ,54,55, wConsequently, the cohesive elements near the transverse symmetric plane failed due to the overestimated shear stress, although their failure was supposed to be suppressed by the high-level compression induced by the impactor and stress release caused by Crack A’.The tensile crack is induced by The tensile crack determines the outline and sizes of delamination by the stress concentration. In addition, the stress release by the opening of the shear cracks forms the two-lobe morphology of delamination.The effect of the tensile crack indicates that the delamination extension is related to the global bending stiffness of a laminate. The undelaminated region is only dependent on the impactor geometry and the local stiffness of a laminate, since the distance between two lobes is determined by the contact between the impactor and laminate.In this investigation, the relationship between matrix cracking and delamination in cross-ply laminates during LVI has been studied. Several experimental methods, including C-scan, micrograph, and visual inspection, were adopted to characterize the impact damage. XFEM-based FE models were established to analyze the damage mechanisms. The main conclusions are summarized as follows:Overall, the stress redistribution effect caused by CODs of the matrix cracks determines the shapes and sizes of the delamination.Finally, this study not only provides an effective approach for impact damage modeling but also highlights the significance of modeling the shear cracks accurately since they determine the undelaminated region, which plays an important role in CAI behaviors. Based on the damage mechanisms of this study, more accurate FE models will be established in future work to analyze the impact damage of realistic stacking sequences used in aeronautical engineering. Moreover, a further study is needed to investigate the effect of the through-thickness compression on the impact damage.


Using the bioluminescent Ca2+ reporter EGFP-aequorin we observed that the changes in Ca2+ homeostasis occurring during the switch from proliferation to quiescence are controlled through store-operated channels (SOC) since inhibition of SOC drives proliferating GSLCs to quiescence. We showed that this switch is characterized by an increased capacity of GSLCs’ mitochondria to capture Ca2+ and by a dramatic and reversible change of mitochondrial morphology from a tubular to a donut shape. Our data suggest that the remodelling of the Ca2+ homeostasis and the reshaping of mitochondria might favours quiescent GSLCs’ survival and their aggressiveness in glioblastoma.Quiescence is a reversible cell-cycle arrest which allows cancer stem-like cells to evade killing following therapies. Here, we show that proliferating glioblastoma stem-like cells (GSLCs) can be induced and maintained in a quiescent state by lowering the extracellular pH. Through RNAseq analysis we identified Ca According to the cancer stem cell model, the GBM lethality is due to a small sub-population of tumour cells with stem-like properties, called Glioblastoma Stem-Like Cells (GSLCs). The GSLCs have been further characterized as slow-cycling or relatively quiescent cells2, identified in vivo in a mouse model of glioblastoma3 and in human glioblastoma tumors4. These quiescent GSLCs are highly resistant to TMZ treatment5.Multiform glioblastoma (GBM) is the most aggressive brain tumours with very poor prognosis. Despite a combination of surgical resection, radiotherapy and temozolomide (TMZ)-based chemotherapy, more than 90% of the patients show recurrence and the mean survival period rarely exceeds 2 yearsCCNA2, CCNB1 and CCNE2) and the upregulation of genes classified as tumour suppressors, including the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the G0/G1 switch gene 2 (G0S2)7. These data also show that quiescence is a G0 phase and not a prolonged G1 phase8. Furthermore, quiescence is actively regulated by signals provided by the stem cell microenvironment. In glioblastoma tumours, quiescent stem-like tumour cells are found close to necrotic tissues, in specific niches characterized by an hypoxic9 and acidic microenvironment11. The role of the microenvironment in the control of GSLCs quiescence is still poorly understood.Quiescence is a cell-cycle arrest state which differs from the one observed in differentiation or senescence by the fact that it is reversible. Transcriptional profiling data reveals that quiescent stem cells are characterized by a common gene signature with the down-regulation of genes associated with cell-cycle progression  the number of TG1 and TG1_C1 cells increased by about 4-fold while at pH 6.5, proliferation rapidly stopped and by day 5 the number of cells was not significantly different from day 0  to 6.5 does not induce cell death , indicating that cells have stopped proliferating  CCNB1 (cyclin B1) down-regulated in quiescent cells8 and (iii) G0S2 (G0/G1 Switch 2 gene), encoding a cytosolic protein which promotes quiescence of hematopoietic stem cells7. As expected, the transition from proliferative to quiescent GSLCs is associated with the decrease in the mRNA level for CCNB1 and the increase in mRNA level for CDKN1A and G0S2  of TG1 and TG1_C1 cells grown under proliferating conditions and induced to quiescence by either non-renewal of the medium during 9 days or by acidification of pHnes Fig. .Figure 22+ signaling contributes to cancer hallmarks such as excessive proliferation, survival or resistance to cell death18, we focused on genes belonging to the Ca2+ signaling toolkit19. A set of 105 genes expressed in GSLCs cells were unveiled  or down-regulated  under quiescent conditions. Moreover, the mitochondrial Ca2+ uniporter (MCU), two of its modulators (MICU1 and MICU2) and the voltage-dependent anion channel of the outer mitochondrial membrane (VDAC1), responsible for the transfer of Ca2+ from the endoplasmic reticulum (ER) to the mitochondria were down-regulated in quiescent cells whereas the mitochondrial Na+/Ca2+ exchanger (SLC8B1) was up-regulated  is a major mechanism of Ca2+ entry in non-excitable cells. SOCE controls cell proliferation in numerous cell types including cancer cells24. To examine SOCE between proliferating and quiescent TG1 cells, Ca2+ measurements were performed using EGFP-Aequorin targeted to the cytosol (CytGA)  to inhibit intracellular Ca2+ increase. Although this treatment blocks proliferation 28 and of Hes1. CCND1 and CCND3 are required during G1, CCNE2 for the G1/S transition and CCNA2 for the S/G2 transition29, Hes1 is a transcription factor involved in the control of the reversibility of cellular quiescence30. Figure 2+ influx through SOC by SKF96365 (10 µM) triggers quiescence of TG1 cells in proliferating medium  , with slow rate for low [Ca2+]cyt and faster rate when the [Ca2+]cyt increases. This sigmoidal behaviour has been mainly attributed to the functions of MICU1 and MICU2. Although their respective gatekeeping functions are still debated, experimental evidences clearly indicate that MICU2 silencing induces an increase of mitochondrial Ca2+ uptake, indicating that MICU2 is an inhibitor of MCU. On the contrary, MICU1 behaves as a Ca2+-sensitive switch for MCU activity; acting as inhibitor of MCU at low [Ca2+]cyt and activator at high [Ca2+]cyt42. We observed that the resting level of cytosolic Ca2+ is lower in proliferating TG1 cells compared to quiescent ones. In proliferating TG1 cells, MICU1 and MICU2 should therefore be inhibitors of MCU and its activity should be low. However, in quiescent TG1 cells we found that MICU2 expression was reduced and as a consequence, its inhibitory effect on MCU released. Although MCU expression is reduced in quiescent cells, MCU activity would be enhanced. Our RNAseq data also pointed to the overexpression, in quiescent cells, of the Na/Ca2+ exchanger SLC8B1, recently identified as one of the components responsible for mitochondrial Ca2+ efflux and which counteracted the accumulation of Ca2+ into mitochondrial matrix by MCU43. Therefore, the transcriptional remodelling of components that control mitochondrial Ca2+ homeostasis may contribute to prevent mitochondrial Ca2+ overload during quiescence.Mitochondria are crucial organelles which not only regulate cellular energy generation and apoptosis but also control intracellular Ca44. Besides these tubular or fragmented shapes, a third shape called donut was reported during hypoxia-reoxygenation stress45. Donut-shaped mitochondria result from the bending and the fusion of the two ends of tubular mitochondria46. We observed that proliferative GSLCs have mainly tubular mitochondria, while quiescent GSLCs mainly show donut-shaped mitochondria. This reshaping of mitochondria is reversible. When GSLCs are transferred from a quiescent to a proliferative medium donut-shaped mitochondria switch back to a tubular shape. Recent studies on the relationship between mitochondrial shapes and stress show that the transition from tubular to donut requires an increase of mitochondrial Ca2+ uptake47 and does not involve fusion mechanism45. Our results are in accordance with the requirement of enhanced mitochondrial Ca2+ uptake and the formation of donut-shaped mitochondria. This remodelling of mitochondrial morphology from tubular to donut may be an additional mechanism explaining the increased mitochondrial Ca2+ uptake capacity observed after SOC stimulation in quiescent GSLCs. An interesting observation that may contribute to the formation of donut-shaped mitochondria during quiescence is the down-regulation of the LETM1 gene (see RNAseq data). LETM1 is a leucine zipper and EF-hand transmembrane protein localized to the inner mitochondrial membrane48 which has been shown to be required to maintain the mitochondrial tubular network independently of the mechanism of mitochondrial fission which involved the DRP1-dependent pathway49.It has been reported that mitochondria are highly dynamic organelles whose morphology is controlled by fission-fusion mechanisms. Under normal conditions mitochondria are essentially tubular but can be fragmented during apoptosis or formed distended swollen structures during necrosis50. On large macro-tumorospheres, we found that a large fraction of mitochondria forms a tubular network in proliferating GSLCs while GSLCs localized to the core of the tumorosphere are characterized by donut-shaped mitochondria. Importantly, the presence of a hypoxic environment has been documented within the core of these macro-tumorospheres14. As suggested by45, the formation of donut-shaped mitochondria might represent a protective mechanism preserving mitochondria against autophagy upon metabolic stress.Mitochondrial dysfunction is one of the hallmarks of cancers and this is associated with cancer cells exhibiting fragmented mitochondria2+, has therefore a great potential for translational research. Mitochondria targeted therapeutic may involve the control of the shape correlated with Ca2+ signalling.In conclusion, our findings on mitochondria in GSLCs point out the regulation of mitochondria dynamics and ER network. The relationship between changes in mitochondrial shape and physiological parameters, particularly mitochondrial Ca51. These cells express stem cells markers, have clonal properties, were able to differentiate and induce tumours when orthotopically grafted in immune-compromised mice52. Their mutational status is however different, while BITC25 is mutated for TP53, TG1 and TG1_C1 are not52. The cells are expanded in NS34 serum-free medium containing EGF and bFGF  as described53 and used up to 10 passages. Under the culture conditions used, TG1, TG_C1 and BTIC25 cell grew as floating neurospheres. To induce quiescence, GSLCs were either cultured 9 days without medium removal as described13 or 5 days in NS34 medium buffered to pH 6.2 or 6.5. Macro-tumorospheres from single GSLCs were obtained as described in14.GSLCs  lines were isolated, established and cultures from surgical resections of primary GBMs from adult patients (age over 18) after informed consent as previously described5cells/ml in 6-well plate and proliferation assessed by direct cell counting method using Neubauer Improved C-C-Chip. Cell viability was evaluated using Trypan blue exclusion assay. The number of cells in S phase was determined with Click-it® Edu Alexa fluor 647 Imaging Kit (Invitrogen) as indicated. At day 5, cells were incubated with 10 µM EdU solution during 2 hours, fixed in 16% paraformaldehyde (PFA) for 30 minutes, transferred on SuperFrost slides  and observed with Leica Sp8 confocal microscope.Cells were seeded at 1∙104 cells per well in soft-agar (0.7% agarose) NS34 medium at pH 7.4 or pH 6.5. This allows to continuously follow sphere formation from individual isolated TG1 cells during 7 days. Pictures were taken with a Nikon Eclipse TS100 Microscope, with 40X/0.55 and 20X/0.40 objectives from day 1 to day 7. Macro-tumorospheres from single GSLCs were obtained as described in14.To assess the ability of TG1 cells to form new clones, spheres were mechanically dissociated into single cell suspension and seeded into 24-wells plates at 10CDKN1A, CCNB1, GOS2, CCND1, CCND3, CCNE2, CCNA2, PCNA, Hes1, STIM1, 2, ORAI1, 2, 3, MICU1, 2 and MCU) relative to TBP (TATA-Box Binding Protein), in proliferating and quiescent TG1 cells were assessed by real-time qRT–PCR. Total RNA was extracted with RNeasy® mini kit (Qiagen) and quantified in a NanoDrop apparatus. RNA (0.4 μg) was reverse transcribed using q-ScriptTM cDNA SuperMix . Amplifications of 8 ng of cDNA were performed in triplicate using Eva-Green® SuperMix (Bio Rad) in a 10 μl reaction mixture containing 500 nM primers . The Qlucore software (htpp://www.glucore.org) used for the analysis. Reads (fastq files) were mapped onto the hg38 assembly of the Homo sapiens genome using Tophat 2.0.1054 and bowtie version 2-2.1.055 to give BAM files. From the BAM files and the hg38 GTF file, Qlucore Omics Explorer creates a count matrix by counting the number of reads overlapping each genomic feature in each sample (each BAM file). The counts are then normalized across samples and across genes, and finally log2-transformed. The processed data have also been deposited in the GEO datasets database under the accession number GSE 93991.To establish RNA signatures of proliferative and quiescent GSLCs, the following strategy was used. Several experiments were performed in order to take into account different types of variabilities . The starting sequence of GA was obtained by PCR from CytGA construct with primers containing a BamHI site at the 5′ end (CCggatccAGCAAGGGCGAGGAGCTGTTC) and XbaI site at the 3′ end (CCtctagaTTAGGGGACAGCTCCACCG). The mitochondrial targeting sequence was fused in frame at the 5′ end with a 86 bp sequence of the human cytochrome c oxidase VIII subunit (COX VIII) isolated from pDsRed2-Mito by digestion with BamHI and XbaI from pDsred2-Mito.The chimeric GFP-aequorin (GA) targeted to the cytosol (CytGA) is a generous gift from Ph. Brûlet6) were electroporated with 2 µg of DNA encoding either the Mitochondrial-targeted EGFP-Aequorin (MitGA) or Cytosolic-targeted EGFP-Aequorin (CytGA) probes, using AMAXA Kit L cell line nucleofactor (Lonza). The localization of the Ca2+ reporter to mitochondria was confirmed using an antibody directed against a mitochondrial protein, TOM20, a 20 kDa translocase of outer mitochondrial membranes. Mitochondrial or cytosolic Ca2+ measurements were performed at 37 °C and the temporal aequorin-generated data were acquired as described14. Ca2+ measurements were performed on isolated TG1 cells immobilized in agar. Ca2+ influx through SOCE was obtained by challenging the SOC channels through depletion, in Ca2+-free medium, of ER store with thapsigargin (1 µM), an inhibitor of the ER Ca2+-ATPase. Following this treatment, bath application of 1 mM Ca2+ restores Ca2+ influx through SOC57.Cells .supplementary information


We also find that dynamic changes in Ca2+ occur during development of the SAM and this Ca2+ response is required for changes in PIN1 polarity, though not sufficient. In contrast, we find that Ca2+ is not necessary for the response of MTs to mechanical perturbations revealing that Ca2+ specifically acts downstream of mechanics to regulate PIN1 polarity response.In plants mechanical signals pattern morphogenesis through the polar transport of the hormone auxin and through regulation of interphase microtubule (MT) orientation. To date, the mechanisms by which such signals induce changes in cell polarity remain unknown. Through a combination of time-lapse imaging, and chemical and mechanical perturbations, we show that mechanical stimulation of the SAM causes transient changes in cytoplasmic calcium ion concentration (Ca Auxin transport and microtubule orientation respond to mechanical stimulation at the shoot apical meristem. Here Li et al. show that mechanical stimulation causes cytosolic calcium concentration transients, and preventing such changes impairs reorientation of the PIN1 auxin efflux carrier, but not of microtubules. As MTs guide cellulose synthesis3, this is thought to lead to directional reinforcement of the cell wall2. Polar transport of the plant hormone auxin is necessary for floral organ initiation and tissue morphogenesis in SAMs4. In the epidermal cells of the SAM, the plasma membrane-localized transporter of auxin PIN-FORMED 1 (PIN1) is the major auxin efflux carrier that directs auxin flow, and as a consequence controls the formation of leaves and flowers7. The PIN1 protein exhibits dynamic patterns of expression and polarity and its loss of function is sufficient to disrupt floral phyllotaxis8. It has been proposed that the asymmetric distribution of PIN1 in the plasma membrane of inflorescence meristem epidermal cells (PIN1 polarity) is also patterned by mechanical signals, as its membrane localization responds to mechanical changes and correlates with the orientation of cortical MTs6. The current model proposes that PIN1 adjusts its subcellular location to be preferentially in the plasma membrane adjacent to the most stressed side wall6, which directs auxin toward expanding neighbors . This positive feedback controls auxin flow in the epidermis, and as a consequence controls the pattern of formation of leaves and flowers7. The mechanisms by which mechanical signals induce changes in meristem cell polarity remain to be determined.Plant cells respond in a number of ways to mechanical stresses. Cortical microtubules (MTs) align to the direction of maximal anisotropic mechanical stress in shoot apical meristems (SAMs) and in pavement cells2+)10. It is known that Ca2+ signaling plays a role in the development of patterning and morphogenesis in early embryos in ascidians, frogs, and zebrafish. In plants, de la Fuente et al. studied the role of Ca2+ in auxin transport11. Auxin application causes a rapid increase in cytosolic Ca2+ in Zea mays coleoptiles and plant protoplasts14, and Ca2+/calmodulin binding to auxin-induced regulatory proteins has been suggested to control auxin response in maize roots15. These and many other studies indicate that calcium-mediated processes may be involved in modulating auxin response and polar auxin transport17. Yet the link underlying Ca2+ signaling and morphogenesis is not fully understood. Here we show that one prerequisite for the PIN1 response, which takes place over hours, is the much more rapid calcium response, as blocking the calcium response prevents later PIN1 relocalization. This is not a result of irreversible injury to the meristem cells and this is necessary for initiation of PIN1 relocalization and not for the protein vesicle traffic itself. The findings in this study establish a new framework for the function of Ca2+ signals in cellular polarity during organ initiation, tying them directly to morphogenesis.An additional cellular response to mechanical stress is mechanically induced transient change in cytoplasmic calcium ion concentration , known as a plasma membrane Ca2+ channel blocker18, for 2 min every hour for 12 h without rinse after each treatment to maintain the chemical effect. Control samples were treated identically, using water without the inhibitor. After mock treatment, SAMs continued growing   ethane-N,N,N′,N′-tetraacetic acid (BAPTA), an extracellular pH-insensitive calcium chelating agent20, and tested its effect on PIN1 protein polarization dynamics during growth. We found that BAPTA also caused a delay or inhibition of PIN1-GFP polarity convergence on new primordia, although in this case no PIN1-GFP fluorescent signal increase was detected  Fig. . LaCl3-t10) Fig. . Cell diize Fig. . To cont6. We pretreated dissected pPIN1::PIN1-GFP transgenic inflorescence SAMs with LaCl3, then used a glass needle to kill a local group of cells. PIN1 response was monitored 3 h later by laser scanning confocal microscopy, as in Heisler et al. 20106 (n = 5)  , but 5 mM LaCl3 completely prevented PIN1 repolarization. After a 3 h recovery period during which the samples were rinsed in water without LaCl3, PIN1 response to mechanical force change was restored, as PIN1-GFP relocalized around the earlier ablation site, similar to the control meristems  Fig. , b. In t 9) Fig. . Partialems Fig. .Fig. 2Me3 were seen when the SAM was pre-treated with 2 mM BAPTA    24. We found that an individual SAM shows two different patterns of spontaneous Ca2+ concentration dynamics, as inferred from fluorescence intensity. These patterns occur simultaneously and are similar to some aspects of calcium dynamics observed in animals, for example as characterized in larval organs in Drosophila25. Although both patterns consist of a transient increase in Ca2+ concentration, one appears to be a global meristematic behavior, referred to as oscillations hereafter . Supplementary Movie n = 19 peak events). Supplementary Movie n = 4 out of 5 SAMs. The empirical distributions for the intact and excised SAMs, both for inter-peak-times and FWHM, are found to be so similar to each other that the null hypothesis that they are the same could not be rejected by the statistical tests we have applied ; see “Image processing and data analysis” under Analysis of plants expressing R-GECO1 indicates that the oscillations have a typical time between peaks of 22 min ± 4%  of 8 min 35 s, 123 inter-peak times), with event duration of 4 min 51 s ± 3%, estimate as full width at half maximum (FWHM) amplitude   , though 13% of the cells showed prolonged signals lasting longer than 10 s  and lasting for 16 ± 5 s (mean ± SD)      during confocal microscope observation  Fig. . The Ca2TA) Fig. , indicat2+ oscillation signals and Ca2+ wave following mechanical stimulation by a glass pipette using the same sample under the same imaging settings. We found that the spontaneous oscillation signals took 8.8 ± 4.4 times the interval of the time for mechanically triggered waves . Thus the speed of spontaneous Ca2+ signal propagation is much slower than that of deliberately mechanically stimulated waves. We also compared the fluorescence intensity changes between the two responses. The maximum intensity fold change (I − I0)/I0 of the mechanically induced Ca2+ wave is about 2.3 ± 1.1 ; that of oscillation signals is 0.5 ± 0.2    and lasted for 23 ± 8 s (mean ± SD) (Supplementary Table n = 11)   or whether it may only be necessary for the initiation of the PIN1 response, we did meristematic cell ablation with a glass needle, waited for 5 min and then treated with 5 mM LaCl3 or 2 mM BAPTA for 15 or 10 min, respectively, without a subsequent sample rinse, as in the earlier experiments. Three hours after stimulation, PIN1 had relocalized in both cases, just as if the SAM had never been treated (n = 4 for LaCl3 and n = 6 for BAPTA)  require earlier changes in cytoplasmic calcium concentration. A previous report, on 31. The major question, then, is how mechanical stress in cell walls translates to changes in the local balance of exocytosis and endocytosis of PIN1 transporting vesicles. What detailed mechanisms might connect calcium response to PIN1 relocalization? The normal asymmetric location of PIN1 in plasma membranes of multiple cell types, including those of the SAM, requires activity of the PINOID gene, coding for a protein kinase that phosphorylates PIN1 on its large central cytoplasmic loop32. PINOID protein has been shown in yeast two-hybrid assays to bind in a calcium-dependent fashion to two different calcium-binding proteins, TOUCH3 , and PINOID BINDING PROTEIN 1 (PBP134). Both TCH3 and PBP1 are members of gene families, TCH3 being related to the many calmodulins and calmodulin-like proteins encoded in the Arabidopsis genome, and PBP1 to multiple calcium-dependent protein kinases. One hypothesis is therefore that calcium transients in the cytoplasm act to facilitate binding of proteins to PIN1, at least one of which can phosphorylate PIN1 so as to motivate trafficking.The mechanism of PIN1 relocalization is not known, but it involves redistribution of PIN1 protein from one membrane region to another by exocytosis and endocytosis that is partly determined by mechanical stress25) in the entire inflorescence meristem. The observed mechanical Ca2+ response could be mediated by a variety of different components. The initial signal could depend upon plasma membrane localized Ca2+ permeable channels, such as those of the CNGC family16, the GLR family38, the MCA family39, the OSCA family41, or by the DEK1 protein42 or TPC143. The intercellular calcium waves may in addition rely upon plasmodesmata for Ca2+ cell-to-cell diffusion37, and Ca2+ pumps, such as the Ca2+-ATPases for cytoplasmic Ca2+ level return to the resting state44.The experiments reported also reveal a new collection of calcium signals in the plant stem cell niche, including mechanically induced slow intercellular calcium waves, spontaneous spikes, and periodic oscillations . The regular oscillations, which occur every 10–30 min, with a fairly regular frequency, remain a mystery in regard to their origin. This study indicates their potential importance for PIN1 protein dynamic polarized movement during growth. It is tempting to imagine that they are related to some regular mechanical oscillation that characterizes the growth of meristems or their cells, such as those that create circumnutation in roots and stems, which is affected by mechanical inputs53. Micronutation of Arabidopsis hypocotyls has a period of 20–60 min54, similar to the calcium oscillation frequency.Spontaneous Ca3 (Sigma-Aldrich) and 100 mM BAPTA  were prepared in water. In PIN1-GFP growth experiments, for LaCl3 treatment, 5 mM solution was applied for 2 min each hour for 12 h to dissected SAMs embedded at the basal end in a 1% agarose plate. During treatment, SAMs were incubated on GM , MES 2-(MN-morpholino)- ethane sulfonic acid (Sigma M2933) brought top pH 7 with 1M KOH, plus 0.8 % Bacto Agar (Difco), 1% MS vitamins (Sigma M3900) agar plus 500 nM 6-benzylaminopurine (BAP). The SAMs were thoroughly washed in water to remove LaCl3 when starting sample recovery. The SAMs were then incubated on GM + BAP plates for 12 h. Microscopy was performed immediately before and 12 h after LaCl3 treatment, and 12 h after recovery. Control samples experienced the same treatment steps except that the solution used was water rather than 5 mM LaCl3. In PIN1-GFP growth experiments, for BAPTA treatment, the procedure was similar to LaCl3 treatment, except that 2 mM BAPTA was used, SAMs were incubated on GM agar plus 500 nM BAP without 3 mM CaCl2 and washed in 3 mM CaCl2 for recovery.Stock solutions of 100 mM LaCl3 solution was applied to the SAM in the same way as in growth tests, but for 15 min before cell ablation. Water served as the control treatment. After ablation, the SAMs (without water rinse) were directly transferred to GM plates to incubate for 3 h. During recovery, SAMs from which the LaCl3 was washed with water were incubated for an additional 3 h on GM plates. Imaging was performed at 0 and 3 h after ablation, and after 3 h of recovery. For BAPTA treatment, 2 mM BAPTA solution was applied to the dissected SAM for 10 min before cell ablation. After ablation, the SAMs were briefly rinsed with water and then transferred to a 1% agarose plate to incubate for 3 h. During recovery, the SAMs were washed with a solution of 3 mM CaCl2 and incubated for an additional 3 h on GM plates. Imaging was performed at 0 and 3 h after ablation, and after 3 h of recovery.In PIN1-GFP ablation experiments, 5 mM LaClIn MBD-GFP experiments, inhibitor treatments were the same as in the PIN1-GFP experiments, except that the incubation time extended to 6 h, after which imaging was done, and recovery experiments were not performed.3 or BAPTA treatment was the same as for other reporters, except the test for blockage of spontaneous Ca2+ reactions (spikes and oscillations) used 0.5 mM LaCl3 and 0.2 mM BAPTA immersion during one-hour imaging. During R-GECO1 recovery experiments, LaCl3− or BAPTA-pretreated SAMs were cell ablated with a pulled glass pipette and incubated for 3 h on a GM plate (for SAMs with LaCl3 treatment) or on a 1% agarose plate (for SAMs with BAPTA treatment). The sample was then placed under the confocal microscope objective and laser scanning was started immediately after adding water.For R-GECO1 and R-GECO1; 29–1-GFP double reporter experiments, LaClpPIN1::PIN1-GFP and pML1::mcherry-MAP4 were carried out on a Leica SP5 upright confocal microscope using a multiphoton laser from Spectra-physics which was controlled by Leica software55. Ablations were performed using 800 nm wavelength with an output power of ~3 W. Each pulse was shot for 1–15 ms, which varied from sample to sample. Ablations were usually accomplished within 1–3 bursts of the laser.For PIN1-GFP and MBD-GFP reporter lines, cellular ablations were performed manually with a pulled glass pipette under a Zeiss SteREO Discovery V8 stereomicroscope. Cellular ablations on the SAMs expressing For observations of R-GECO1 reporter lines, mechanical perturbation  was performed from the side of the SAM using a pulled glass pipette attached to a micromanipulator. Pipettes were fabricated by pulling a 1-mm diameter glass capillary on a micropipette puller . The tip diameters of the micropipettes ranged between 3 and 5 μm, about the size of one meristematic cell. The micropipettes were mounted on a three-axis micromanipulator  allowing precise micropipette movement under the microscope near the SAM  to the part in order to close the open area. The part is mounted on a three-axis micro-manipulator (Narishige Hydraulic Micromanipulator MO-202)  with a time interval of 1 s for Ca2+ wave observation (2D), 1 or 2 s for Ca2+ spike observation (2D), 5 s for Ca2+ spike observation (3D), and 5 or 10 s for Ca2+ oscillation observation (2D). The movie was recorded in 512 × 512 pixels format. The intervals between optical slices in z-stacks ranged from 1 to 2 μm. Confocal z-stack images were processed using ImageJ (imagej.nih.gov/ij/).PIN1-GFP and MBD-GFP fluorescent reporters were imaged using a Zeiss LSM 510 Meta confocal microscope or a Zeiss LSM 780 laser scanning confocal microscope. R-GECO1 and double fluorescent reporters R-GECO1; 29–1-GFP were imaged by using a Zeiss LSM 880 or Zeiss LSM 780 laser scanning confocal microscope. PIN1-GFP and MBD-GFP laser and filter settings were used as described previouslyThe inflorescence meristems of plants carrying GCaMP6f were dissected and mounted as above. The meristems were incubated for ~15 min to minimize calcium response from the dissections. Spikes were captured using a resonance scan mode on Leica TCS-SP5 upright microscope at a scan speed of 8000 Hz. The movie was recorded in 512 × 512 pixels format. For recording GCaMP6f response to mechanical perturbation (wounding), the imaging was performed on a Leica TCS-SP5 upright confocal laser-scanning microscope using a ×25 water-dipping objective (NA-0.95). Five frames were recorded prior to laser ablation and 100 frames were captured immediately after laser-induced ablation at a scan speed of 400 Hz and a scan rate of one optical slice per 0.7 s.pPIN1::PIN1-GFP and pML1::mcherry-MAP4 reporters were imaged on a Leica SP5 TCS microscope. PIN1-GFP was excited using 488 nm argon laser and mcherry was excited using 561 nm white light laser.The meristems carrying The SAM oscillation signal observation in intact plants was performed using an imaging box . Plants were initially grown on soil in small pots until they bolted, then together with the pots, they were transferred into imaging boxes. The flower-dissected SAM was isolated from leaves and roots by placing Parafilm around the stem and sealing with a thin layer of 1% agarose to prevent water leakage. Water immersed SAMs were located under a ×40 objective for signal observation.57 on natural-cubic-smoothing-splines can be found at https://github.com/eldad-a/natural-cubic-smoothing-splines. All other code, including the programming code for quantitative analysis of the meristem calcium oscillations and waves upon mechanical perturbation, are available from the corresponding authors upon request.The corresponding open-source Python code, previously introduced in Refs. Further information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Supplementary Movie 12Source DataReporting Summary


In the legume Medicago truncatula, these nuclear Ca2+ signals are generated by a complex of nuclear membrane-localised ion channels including the DOES NOT MAKE INFECTIONS 1 (DMI1) and the cyclic nucleotide-gated channels (CNGC) 15s. DMI1 and CNCG15s are conserved among land plants, suggesting roles for nuclear Ca2+ signalling that extend beyond symbioses. Here we show that nuclear Ca2+ signalling initiates in the nucleus of Arabidopsis root cells and that these signals are correlated with primary root development, including meristem development and auxin homeostasis. In addition, we demonstrate that altering genetically AtDMI1 is sufficient to modulate the nuclear Ca2+ signatures, and primary root development. This finding supports the postulate that stimulus-specific information can be encoded in the frequency and duration of a Ca2+ signal and thereby regulate cellular function.In plants, nuclear Ca Nuclear Ca2+ signals generated by DMI1 and CNGC15 ion channels in plant roots are needed to establish symbiotic relationships with soil microbes. Here Leitão et al. show that DMI1- dependent nuclear Ca2+ spiking also occurs in Arabidopsis root meristems and are linked to root development Each Ca2+ response can be specified by the spatial release of Ca2+ in specific cell types and cellular compartments, which enables the compartmentalisation of the signal transduced and/or the selective activation of temporally and spatially regulated Ca2+-binding proteins. Within the same cellular compartment, it is postulated that the Ca2+ signature, defined by its amplitude, frequency, and duration, specifies the activation of downstream components2. Although this postulate is well accepted, the genetic demonstration that modulating a Ca2+ signature can change a specific developmental or physiological process is still missing.Calcium  endosymbioses6. The nuclear Ca2+ release is mediated by a complex of nuclear membrane-localised ion channels including DOES NOT MAKE INFECTIONS 1 (DMI1) and the cyclic nucleotide-gated channels (CNGC) 15a, b, c7. The conservation of DMI1 and CNCG15s among all land plants8, including non-symbiotic species such as Arabidopsis thaliana, suggests roles for nuclear Ca2+ signalling that extend beyond symbioses.Ca2+ sensor, discriminating Ca2+ release within the nucleus versus cytoplasmic Ca2+ signals diffusing into the nucleus9 and that Ca2+ release occurs in the nucleus of root meristematic cells. We further demonstrate that the nuclear Ca2+ signal can be modulated genetically by removing or overexpressing the cation channel DMI1, leading to the production of different nuclear Ca2+ signatures and differences in primary root growth. In addition, we provide evidence that nuclear Ca2+ signals contribute to and can be modulated by root auxin homoeostasis. The findings in this study reveal that genetically modulating the activity of ion channels to produce diverse Ca2+ signatures is sufficient to control developmental processes and further highlight the novel function of DMI1 in modulating nuclear Ca2+ signals required for primary root growth.Here we demonstrate, using a dual-localised fluorescent CaMedicago truncatula DMI1 and CNGC15a,b,c identified Arabidopsis DMI1 (At5g49960) and CNGC15 (At2g28260) as orthologues of MtDMI1 and MtCNGC15b,c, respectively8. AtDMI1 was previously characterised as the functional analogue of MtDMI1 in root symbioses10. Similarly, expression of AtCNGC15 in the Mtcngc15b,c double mutant restores the symbiotic defect  that both AtDMI1 and AtCNGC15 are expressed in all tissues analysed including roots  and green fluorescent protein (GFP) under the control of the promoters of AtCNGC15 and AtDMI1, respectively, and showed that these genes are expressed in the primary root apical meristem  or increased (dmi1-2)  displayed longer roots, associated with longer elongating and mature cells 14. Our results demonstrate that AtDMI1 modulates root apical meristem development and growth in a non-symbiotic context. To investigate whether specific nuclear Ca2+ signals occur during root growth, we used a wild-type transgenic line expressing a dual-localised fluorescent Ca2+ sensor  that allows simultaneous recording of cytosolic and nuclear Ca2+ signals9. We monitored the Ca2+ changes at the cellular level in the meristem and elongation zones of 5-day-old growing roots  couples with a Ca2+ channel to mediate Ca2+ release from the ER15. Knocking down of TRIC channels is sufficient to compromise Ca2+ release and notably to extend the duration of Ca2+ release leading to cardiac failure15. Thus overexpressing or removing a counter-ion channel can be sufficient to modulate the Ca2+ release. To assess whether DMI1 can modulate the nuclear Ca2+ release during root growth, we analysed the transgenic lines dmi1-2 and dmi1-1 expressing NRCG-GECO1.2  and wild-type Col were purchased from the Nottingham Arabidopsis Stock Centre. Arabidopsis seeds were surface-sterilised in 1.5% bleach for 15 min, followed by 5 washes in sterile water, and the seeds were then plated in Murashige and Skoog (MS)/2-(N-morpholino)ethanesulfonic acid (MES), 1% sucrose, and 0.8% agar pH 5.8. For bending experiments, the seeds were directly sown on sterile slide cover with MS/MES medium described above with the following modification: 0.8% phytagel. For Ca2+ imaging, as only 2–3 plants could be analysed per day, plants were grown every 3 days until completion of the experiment. After 3–5 days at 4 °C, plates were moved to a growth cabinet .The SALK and SAIL lines  for SALK and SAIL alleles  and assembled as described in Supplementary Table Golden gate cloning followed the principles outlined in Engler et al.Agrobacterium tumefaciens-mediated gene transfer by inflorescence infiltration. Plants were grown for 4–5 weeks (T0 generation) prior to floral dipping, and seeds were collected and germinated under the appropriate selection in the T1 and T2 generations30. dmi1-1 and dmi1-2 plants expressing DII-VENUS or DR5-GFP were obtained by manual hand-pollination of individual lines with Columbia DII-Venus and DR5-GFP described previously19.Arabidopsis stable transgenic lines were generated using M. truncatula roots using Agrobacterium rhizogenes-mediated gene transfer performed as previously described7. The A. rhizogenes strain AR1193 was used. Each construct expresses the fluorescent marker mCherry fused to a nuclear exclusion signal as a plant marker to facilitate the selection of transformed roots by fluorescence microscopy. To monitor AM root length colonisation, plants were grown in Terragreen/Sand  and inoculated with Rhizophagus irregularis  to the ratio 5:5:1 (Terragreen/Sand/Spores). The fungal structures were stained in acidic ink as follows: roots were cleared in 10% KOH 15 min at 95 °C, washed 3 times in water, and subsequently stained in acidic ink  for 4 min at 95 °C. The AM root length colonisation was quantified as previously described7. For nodulation assays, 1-week-old plants were grown in Terragreen/Sand  to a ratio (1:1) and inoculated with Sinorhizobium meliloti 2011 (OD600 = 0.001). Nodules were scored as indicated.The Golden gate constructs pAtUBI10:GFP and pATUBI10:gAtCNGC15:GFP (Supplementary Table E) was first determined through a cDNA dilution series. The qPCR were set up with an initial denaturation step (2 min at 95 °C), followed by 40 cycles of amplification and quantification . A melt curve was also generated to verify the specificity of the amplification reaction . Calculation of the normalised expression and fold change ratio was performed using the mathematical model described previously31. Primers used for qPCR are listed in Supplementary Table Total RNA was extracted using the RNeasy® Plant Mini Kit (QIAGEN) according to the instructions of the manufacturer. A subsequent step of on-column DNase digestion was included. Concentration and purity were determined by spectrophotometry (A260/280 and A260/230 ratios) and integrity was confirmed by gel electrophoresis (1% (w/v) agarose). cDNA was obtained from 500 to 2000 ng of RNA using the SuperScript™ III Reverse Transcriptase (Invitrogen™) according to the instructions of the manufacturer. qPCR was performed using a CFX96 Touch™ Real-Time PCR Detection System (BIO-RAD) with SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma-Aldrich). For each primer set, amplification efficiency .Seedlings were grown vertically on MS–MES plates as follows: 6 seedlings of control genotype and 6 seedlings of test genotype, per plate, and photographed at days 6 and 12 for the primary root length phenotyping; 4 seedlings of control genotype and 4 seedlings of test genotype, per plate, and photographed at days 3 and 5 for the growth rate followed by Ca2O, washed with 20 mM phosphate buffer, and incubated overnight at 37 °C in a α-amylase suspended in 20 mM phosphate buffer. Samples were then washed with dH2O, incubated in 1% periodic acid at room temperature for 40 min, washed with dH2O, incubated in freshly prepared Schiff’s reagent  for 30 min or until roots had acquired a pink colour, washed with dH2O, and incubated overnight at room temperature in chloral hydrate:glycerol:water (8 g:1 mL:2 mL). Samples were then mounted in chloral hydrate and imaged using a Zeiss LSM 780 confocal microscope (Carl Zeiss). Cell counting and cell length measuring were performed using ImageJ 1.48v 32. This macro was used to count and measure the cells along the cortex file. The first rapidly elongating cortex cell was identified using the Cell-o-Tape plugin and confirmed by visual inspection.Seedlings were grown vertically on MS–MES plates  and analysed at days 6 or 12. Seedlings were fixed in 50% methanol 10% acetic acid, for at least 24 h at 4 °C. Samples were then washed with dH2HPO4, 0.5 M NaH2PO4, 0.1 M K3Fe(CN)6, 0.1 M K4Fe(CN)6). The rinse solution was removed, and stain solution added (rinse solution complemented with 2 mM x-Gluc (5‐bromo‐4‐chloro‐3‐indolyl‐beta‐D‐glucuronide). Samples were then gently vacuum-infiltrated for 30 min and then incubated at 37 °C, in the dark, for 30 min or 24 h. Samples were then washed in water and cleared in chloral hydrate  for at least 45 min, before mounting in chloral hydrate.Seedlings were fixed in cold 90% acetone for at least 30 min at 4 °C. Acetone was removed, and the material was washed twice with rinse solution (0.5 M NaEmbedding primary roots GUS stained for sectioning was performed using Technovit 7100 according to the manufacturer’s instruction (Electron Microscopy Science). Sectioning was performed using a Leica UC7 (Ultra-microtome). Images were obtained with a DM6000 microscope (Leica).2+ imaging was performed using a Nikon ECLIPSE FN1 as described in Kelner et al.13. Seedlings were collected 5 days after germination and carefully placed in a small chamber made on a coverslip using high-vacuum grease . The chamber was filled with a small volume of MS (50–100 µL) and closed with a smaller coverslip, covering the entire root. The seedling was then incubated at room temperature for at least 45 min before imaging. Images were collected every 2 or 3 s, for an average period of 1.5 h, focussed on the tip of the root upwards as indicated.Ca2+ imaging of gravistimulated roots, the plants were grown vertically on top of MS medium (0.8% phytagel) over cover slides. Two hours before imaging, a coverslip was gently put on top of the root and the plate, remaining vertical, was rotated 90 degrees. After 2 h of gravistimulation, the sample was imaged as described above, for 1 h. For all the Ca2+ imaging experiments, the number of biological replicates is n/3 as the number of plants recorded per day is 3.For Cahttp://bradbusse.net/sciencedownloads.html), and application of a lookup table. Image data were obtained from processed images using Time Series Analyser V3_2 (https://imagej.nih.gov/ij/plugins/time-series.html). Normalised data sets (ΔF/F) were calculated as (F − F0)/F0, where F0 represent the average of the first 20 frames of baseline values. To analyse spike shape, an algorithm was designed to quantify the rise and fall times of the spikes observed during Ca2+ imaging. The script developed to analyse the fall and rise times of the Ca2+ spike is included as Supplementary Data https://gitlab.com/rosscarter33/calcium_splike_analysis. Data were detrended by subtracting a polynomial fit to the raw fluorescence data. The detrended signal and the gradient of the detrended signal were used to identify the peak  and the beginning and end of the pulse, these being defined as the first point in time before and after the peak, respectively, where both the detrended signal and the gradient fall below a threshold. These values are then used to calculate the rise and fall times.For image processing, the following steps were conducted using ImageJ 1.48v: background subtraction, registration using MultiStackReg v1.45 .Statistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, La Jolla, CA, USA, Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Data 1Reporting SummarySource Data


This article comments on:et al. 2018. 2+ signals and efflux from cells via anion channels. Journal of Experimental Botany 69, 3477–3489Novel roles of ascorbate in plants: induction of cytosolic Ca.Makavitskaya M, Svistunenko D, Navaselsky I,  For the latter, NOX-produced superoxide anions need to be converted to hydroxyl radicals.Reactive oxygen species (ROS) and intracellular Caet al. (2018)Makavitskaya 2+ in the roots.have now demonstrated that salt stress promotes ascorbate efflux, which, via reduction of the apoplastic copper and iron ions, assists in the generation of hydroxyl radicals, thus inducing a rise in intracellular Ca2O2)  . At this2+-binding sites (EF-hand motifs) in their N-terminus (2+ binding to the residues located in the N-terminus. Activation of Ca2+ influx conductance by HO• (less commonly by H2O2), as for the first time demonstrated by 2+ provides a feed-forward mechanism for ROS and Ca2+ crosstalk  is involved in plant growth and development, phytohormone signaling, hypersensitive responses to pathogens and responses to abiotic stress . The electron-transport chain from NADPH to O2 includes FAD and b-type hemes, located in the RBOH C-terminus and between transmembrane domains, respectively. The latter is converted to peroxide (H2O2) spontaneously or catalysed by superoxide dismutase (SOD). In the presence of reduced transient valency metals, copper (Cu+) or iron (Fe2+), H2O2 is reduced to the hydroxyl radical (HO•) by means of the Fenton reaction. Apoplastic Fe and Cu form part of low molecular weight complexes with organic acids or of metalloproteins, cell wall-bound peroxidases  and diamine oxidase . Ascorbate in the apoplast is mainly exported from the cytosol.Plant NADPH-oxidase (RBOH) is an integral plasma membrane protein which mediates a single-electron transmembrane transfer from NADPH to molecular oxygen, generating the superoxide-anion  in the N-terminus or via Ca2+-dependent phosphorylation of different residues either by CDPK or the CBL-CIPK couple  as compared to iron .Different ROS species have different capabilities in the activation of Ca to iron . This is2O2 scavenger) in the cytosol is well established, but this process employs a sophisticated four-enzyme system, which may be less abundant in the apoplast (2+) form, export of ascorbate to the apoplast and its role in the reduction of Fe3+ to Fe2+ have been shown to be essential for iron uptake  and Fe (1 mM) and suppressed by the HO• scavenger thiourea, specific Cu and Fe chelators and low external Ca2+. Collectively, these results imply that an ascorbate-induced increase in cytosolic Ca2+ is caused by Ca2+ influx via HO•-activated plasma membrane conductance, where HO• is generated by means of the Fenton reaction. Despite a higher affinity for copper as compared to iron, naturally occurring apoplastic Fe and Cu centers displayed comparable roles as Fenton catalysts in Arabidopsis roots. Their absence in cell wall-free preparations (root protoplasts) precluded a rise in ascorbate-induced cytosolic Ca2+. Up to this point, HO• generation in the discussed work was achieved at least partially by the artificial, external application of Fenton reagents. It was previously demonstrated that acute salt stress provokes very significant generation of HO• by Arabidopsis roots (•–), released by Arabidopsis NaCl-stressed roots. Another important finding of this study was a direct recording of ascorbate efflux by means of the patch-clamp technique. Whereas import of the oxidized form of ascorbate for its recycling in the cytosol is relatively well understood (•– formation and whole-cell current (patch-clamp measurements) were suppressed by A9C with the same potency  and massive (up to 20% of cellular ascorbate) ascorbate release from plant cell cultures, induced by 1–10 mM H2O2  and/or be a consequence of electrocoupling with the ROS-induced K+ efflux .Another open question is how ascorbate export is stimulated under stress (i.e. how the activity of transporters is potentiated). One interesting possibility may be feed-forward regulation by ROS . The ESR mM H2O2 . The exa2+ self-amplifying loop is gaining shape and has been shown to be involved in diverse plant responses. Ascorbate efflux could potentially intensify the operation of such a loop, assisting the formation of powerful hydroxyl radicals. However, whether such efflux is a common component of the self-amplifying mechanism and how it is co-ordinated in time and space with the operation of RBOH and/or ROS-activated plasma membrane channels will need to be addressed in future studies.The ROS-Ca


Acute myeloid leukemia (AML) is a clonal disease caused by genetic abberations occurring predominantly in the elderly. Next generation sequencing (NGS) analysis has led to a deeper genetic understanding of the pathogenesis and the role of recently discovered genetic precursor lesions ) in the evolution of AML. These advances are reflected by the inclusion of certain mutations in the updated World Health Organization (WHO) 2016 classification and current treatment guidelines by the European Leukemia Net (ELN) and National Comprehensive Cancer Network (NCCN) and results of mutational testing are already influencing the choice and timing of (targeted) treatment. Genetic profiling and stratification of patients into molecularly defined subgroups are expected to gain ever more weight in daily clinical practice. Our aim is to provide a concise summary of current evidence regarding the relevance of NGS for the diagnosis, risk stratification, treatment planning and response assessment in AML, including minimal residual disease (MRD) guided approaches. We also summarize recently approved drugs targeting genetically defined patient populations with risk adapted- and individualized treatment strategies. Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by uncontrolled proliferation and accumulation of immature myeloid precursor cells in the bone marrow leading to impaired hematopoiesis and bone marrow failure . It is tAML is a clonal disease caused by genetic mutations in normal myeloid hematopoetic progenitor cells, leading to altered self-renewal, differentiation and proliferation . In the clinicaltrials.gov for ongoing trials with targeted agents.Here, we systematically review current literature on the use of NGS for the diagnosis, risk stratification, treatment initiation and/or modulation, as well as for response assessment in AML. We conducted a literature search using the PubMed database and screened titles and abstracts with relevant data for the use of NGS in AML (search terms used: AML or acute myeloid leukemia and NGS or next generation sequencing). For targeted therapies we used the search term AML or acute myeloid leukemia and filtered the results for clinical trials within the last 10 years. Papers were included if NGS was used for either diagnostic, prognostic or therapeutic purposes or for response assessment. Medical therapy was included if a molecularly targeted therapy was used alone or in combination with other substances. We searched The aim of the treatment part of this review is not to give an overview of AML treatment per se, but to specifically summarize which substances are thought to specifically target certain mutations (or groups of mutations), and thus might be used as a result of mutational profiling by NGS .https://emea.illumina.com/science/technology/next-generation-sequencing/deep-sequencing.html?langsel=/at/; accessed on 4 January 2019).Next-generation sequencing (NGS), also known as high-throughput sequencing, is the term used to describe a number of different modern massively parallel sequencing technologies. For clarification of termini, ‘(ultra) deep sequencing’ refers to NGS approaches enabling the detection of rare clonal cells comprising 1% or less of the original sample (https://www.genome.gov/27565109/the-cost-of-sequencing-a-human-genome/).NGS used to be an extremely expensive and time-consuming procedure. The costs for sequencing a whole human genome have dramatically sunk in the last two decades: from a phenomenal 100 million USD in 2001, to approximately 1000 USD in 2015; similarly, the costs for sequencing per raw megabase sunk from approximately 10.000 USD in 2001 to less than 10 cents in 2011  platforms use sequencing by synthesis methodologies. The iSeq100, miniSeq, miSeq and the nextSeq System are Illuminas benchtop sequencers. They also have the high-throughput platforms HiSeq2500, HiSeqX Ten and the latest NovaSeq.Thermo Fisher Scientific  offers the Ion Proton System, Ion PGM System, Ion S5 System, Ion S5 XL System, Ion GeneStudio S5 System and the HID GeneStudio S5 System. Using semiconductors, these platforms work via measuring the pH changes resulting from hydrogen ions released during the addition of nucleic acids. Pacific Bioscience  offers the PacBio System platform, using single molecule realtime technology. Oxford Nanopore Technology  have electronics-based DNA/RNA sequencing platforms. They use nanopore sequencing, which measures changes in the electrical current when molecules pass the nanopore. They offer a portable pocketsize MinION sequencing device and two benchtop sequencers: GridION and PromethION, which can run MinION flowcells. Their smallest device so far is the SmidgION for portable DNA analysis, which can even be used with a smartphone.There are huge differences in costs between whole genome sequencing (WGS), whole exome sequencing (WES) and targeted sequencing (where chosen regions of interest are sequenced). Depending on sample number, read size, number of megabases in the panel, the quality control methods applied and the sequencing platform used, the costs for targeted sequencing range between ~200–1000 € or more per run, and the running time of the sequencing analyses may range anywhere from 2 h to a few days.® Myeloid Sequencing Panel (Illumina); and (iv) Human Myeloid Neoplasms Panel . A comparison of the covered genes is provided in Currently, commercially available myleoid NGS gene panels include: (i) SureSeq myPanel™ NGS Custom AML ; (ii) Leuko-Vantage Myeloid Neoplasm Mutation Panel ; (iii) AmpliSeqThe diagnosis of AML requires the presence of at least 20 percent blast forms of myeloid origin in a bone marrow aspirate or peripheral blood smear . IrrespeIn the 2016 update of the WHO classification for AML with recurrent genetic abnormalities, AML with NPM1 and biallelic CEBPA mutations became full entities with a favorable prognosis. In addition, AML with RUNX1 mutations was added as a provisional entity, as patients with RUNX1 mutations often present with immature morphology and/or secondary AML with an inferior prognosis .n = 50) or WES (n = 150) . ID. ID99]. ClinicalTrials.gov, Identifier: NCT01915498) including 239 patients with R/R IDH2 mutated AML, enasidenib was tested at increasing doses (50–650 mg/day) [n = 34; 19.3%). IDH2 mutation type  did not have any influence on treatment outcomes in this trial [In a phase I clinical trial ( mg/day) . The med mg/day) . The oveis trial .https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm569482.htm; accessed on 4 January 2019). EMA granted enasidenib orphan drug designation on 28 April 2016, and agreed to a pediatric investigation plan on 4 October 2017 .Based on the results of the above trial, FDA approved enasidenib on 1 August 2017 for the treatment of adult patients with R/R AML with an IDH2 mutation as detected by an FDA-approved test, at a starting dose of 100 mg once daily. FDA concurrently approved the RealTime IDH2 Assay (Abbot Molecular) as companion diagnostic method .Of importance, FDA issued a warning and health care alert on 29 November 2018, that signs and symptoms of the life-threatening side effect differentiation syndrome are not being recognized, despite this information being present in the prescribing information. FDA stressed the importance of early recognition and aggressive management of this side effect to lessen the likelihood of death , 179 patients with R/R AML with IDH1 mutations, confirmed using the Abbott RealTime IDH1 Assay, where treated with 500 mg of oral ivosidenib daily [IDH1 mutations occur in about 6–10% of patients with AML [https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm614128.htm; accessed on 4 January 2019). Ivosidenib received a positive aviso for orphan drug designation for the treatment of AML from EMA on 12 December 2016 .Based on these results, ivosidenib received FDA approval on 20 July 2018, for the treatment of adult patients with R/R AML with a susceptible IDH mutation as detected by the Abbott RealTime IDH1 Assay, which was approved on the same day. , an anti-apoptotic protein commonly expressed in hematologic malignancies, has been shown to be involved in tumor survival and chemoresistance . VenetocClinicalTrials.gov Identifier: NCT01994837) [Venetoclax was first approved for the treatment of chronic lymphocytic leukemia after showing impressive results in relapsed and/or high risk patients . In AML,1994837) . Thirty-1994837) .In order to predict possible in vivo response to venetoclax, Kontro et al. performed whole exome sequencing and gene expression profiling of bone marrow and peripheral blood samples obtained from patients with de novo or relapsed AML in comparison to bone marrow samples from healthy controls and CLL patients . They diwww.clinicaltrials.gov, NCT02203773) [n = 29 in the 400 mg cohort and n = 37 in the 800 mg cohort) or dectiabine (n = 31 in the 400 mg cohort and n = 37 in the 800 mg cohort) as backbone. Venetoclax was coadministered daily with 20 mg/m2 of decitabine on days 1 to 5 or 75 mg/m2 of azacitidine on days 1 to 7, each 28-day cycle. Sixty, 74, and 11 patients received venetoclax at 400, 800, and 1.200 mg, respectively. The most common grade 3 or worse adverse events included febrile neutropenia (43%), thrombocytopenia (23%), and neutropenia (16%). At the 400 mg dose of venetoclax, the rate of complete remission/complete remission with incomplete blood cell count recovery was 73% (76% with azacitidine and 71% with decitabine). Even more encouraging, almost half of the responding patients achieved minimal residual disease negativity (assessed by a multiparameter flow cytometry) [Very recently, durable responses with venetoclax in combination with azacitidine or decitabine in elderly (>65 years) patients with de novo AML have been reported in a phase IB study (2203773) . Here, ptometry) . Furthertometry) ,57,58,60https://www.genengnews.com/topics/drug-discovery/sunesis-withdraws-european-maa-for-aml-drug-vosaroxin/; https://www.esmo.org/Oncology-News/Withdrawal-of-the-Marketing-Authorisation-Application-for-Vosaroxin; accessed on 4 January 2019).For the sake of completeness we will briefly mention vosaroxin, which induces apoptosis of AML cells in a TP53-independent manner, highlighting its potential usefulness in TP53-mutated AML . Despitehttps://www.ema.europa.eu/en/medicines/human/EPAR/jakavi; https://www.drugs.com/history/jakafi.html; accessed 6 January 2019). A phase I/II study tested the drug in 26 patients with R/R AML at increasing doses (50–200 mg BID) [Ruxolitinib is an inhibitor of Janus kinase 1 and 2 (JAK1 and 2) approved for the treatment of primary myelofibrosis and other chronic myeloid neoplasms ,125 by t mg BID) . Infecti mg BID) .www.clinicaltrials.gov; NCT00674479), ruxolitinib showed modest antileukemic activity with achievement of CR in 3 of 18 patients with post-myeloproliferative neoplasm AML [In another phase II study including 38 patients with R/R AML . Other JAK2 inhibitors currently in clinical trials include fedratinib (phase III), pacritinib, momelotinib and SB1518 (phase I and II). Allthough these inhibitors are mainly tested in chronic myeloid neoplasms, trials evaluating there use in (secundary) AML are underway.So far ruxolitinib has shown modest response rates in AML. However, due to its good tolerability, further research in (JAK mutated) AML in combination with other substances is ongoing. Currently, four clinical trials evaluating ruxolitinib in AML are actively recruiting  negativity as a new response category  . Two metComplete remission requires the presence of < 5% myeloblasts in the bone marrow, absence of circulating blasts and hematologic recovery. Allmost all (94%) long term survivors of AML have achieved CR after primary treatment of AML (either with intensive chemotherapy or lower intensitiy treatment), indicating that CR is a prerequisite for long term cure . Howeverp < 0.001), as well as lower rates of relapse-free survival  and overall survival . Mutations in NRAS, PTPN11, KIT and KRAS were independtly associated with worse survival in multivariate analysis .As compared to RT-PCR, NGS panels have the advantage to cover a broader range of genes. In a recent large series including 482 patients with AML treated with intensive induction chemotherapy, Jongen-Lavrencic et al. performed targeted NGS with a 54 gene panel at diagnosis and after induction chemotherapy (in CR) . They idp < 0.001). Interestingly, an increase in NPM1 transcripts upon sequential monitoring was a very reliable predictor of relapse (69 of 70 patients).A similar observation came from another study in standard risk AML patients with NPM1 mutations . Here, 3As descriped above MRD negativity (assessed by flow cytometry or PCR) has been clearly associated with better clinical outcomes and its role in predicting survival is certainly increasing. MRD guided treatment approaches have been reported recently.p < 0.0001; disease-free survival: 61.7% vs. 19.6%, p = 0.001), whereas outcomes with chemotherapy were superior for MRD-negative patients  [Zhu et al. prospectively evaluated an MRD-directed (using RT-PCR for RUNX1) consolidation approach for 116 patients with t AML who were in first CR after induction chemotherapy . In thisl 94.7%) .p < 0.001) and a shorter overall survival . MRD positivity was an independent negative prognostic value in multivariate analysis . Disease free survival and overall survival were significantly improved by alloHSCT in patients who were MRD positive , a benefit not observed in MRD negative patients .Similarly, Balsat et al. reported outcomes for ELN high-risk NPM1-mutant patients . Here, pp < 0.0001). The authors concluded that pre-emptive therapy with azacitidine can prevent or substantially delay haematological relapse in MRD-positive patients with MDS or AML [Very recently, results of the RELAZA2 trial, an open-label, multicentre, phase II trial have been reported . In thisS or AML .These studies imply that a tailored postremission approach using MRD is feasible, but further information from prospective trials is needed. Also, the sensitivity of NGS based MRD assessment is expected to be improved with methods like droplet digital PCR in the future. As such, MRD assessment by NGS adds further information on relapse risk and survival. However, efforts to standardize MRD assessment in AML and guidelines on MRD based treatment modulation have to be developed in the future.Next generation sequencing is an exciting tool that is significantly and continuously increasing our understanding of AML pathogenesis and treatment induced clonal evoluation during the course of the disease. Mutations detected by pretreatment molecular analysis can have major implications on prognostic stratification and treatment decisions, which already has led to changes in disease classification by WHO. Response assessment by MRD is superior to morphologic assessment and may help the clinician to guide decisions on treatment choice, initation time point, and/or intensifcation. For a broad use in daily clinical practice, standards for NGS based treatment decisions and monitoring have to be further defined in the future including but not limited to: (i) at what timepoints during the course of treatment should NGS analysis be performed? (ii) which target genes should be included in a NGS panel and which sequencing coverage should be used.


The therapeutic approach for acute myeloid leukemia (AML) remains challenging, since over the last four decades a stagnation in standard cytotoxic treatment has been observed. But within recent years, remarkable advances in the understanding of the molecular heterogeneity and complexity of this disease have led to the identification of novel therapeutic targets. In the last two years, seven new targeted agents  have received US Food and Drug Administration (FDA) approval for the treatment of AML. These drugs did not just prove to have a clinical benefit as single agents but have especially improved AML patient outcomes if they are combined with conventional therapy. In this review, we will focus on currently approved and promising upcoming agents and we will discuss controversial aspects and limitations of targeted treatment strategies. NPM1, FLT3, CEBPA and IDH1/IDH2 have updated prognosis and guide AML therapy  and elderly de novo FLT3-ITD positive AML patients   but had no impact on OS , disrupters of telomeric silencing 1-like (DOT1L) and lysine specific demethylase (LSD1), as well as a KMT2A-menin- inhibitors. Furthermore, immunogenic drugs like CD33/CD3 or CD123/CD3-bispecific T-cell engaging (BiTE) antibody construct and chimeric antigen receptor (CAR)-T-cells might be promising approaches for future AML treatment. The discovery that epigenetic readers of the BRD and BET protein family seem to be crucial for leukemic blasts by transcription of oncogenic c-MYC led to rapid development of BET inhibitors currently explored in clinical trials. Data for AML are limited so far but among 36 r/r AML patients the BET inhibitor OTX015 showed modest response with an ORR of 13% . Furthern = 3) [LSD1, a histone H3K4me1/2 demethylase, is critical for hematopoietic differentiation and is overexpressed in multiple cancers. Recent studies suggest that inhibition of LSD1 may reactivate the all-trans retinoic acid receptor pathway in AML . Currentn = 3) . Pinemosn = 3) . Based on = 3) . A curren = 3) . In addin = 3) . Anothern = 3) . In addin = 3) . The facFLT3 and IDH1/2 mutated AML. For fit AML patients IC remains the treatment back bone and for FLT3 mutated patients accounting for about 25% the addition of midostaurin is now standard of care. The benefit of additional enzyme inhibition in combination with IC in first line treatment for IDH1/2 mutated AML needs to be further evaluated. In addition, it has to be taken into account that only a small fraction of AML patients harbour these mutations (8% IDH1 and 12% IDH2). Moreover, new agents targeting both mutant IDH enzymes like AG-881 (NCT02492737) may be more relevant for future clinical use. Further questions to be addressed are combinations, scheduling and timing of targeted maintenance therapy. Still challenging are r/r AML, especially if they occur during or immediately after targeted therapy. For FLT3 mutated AML, second generation TKIs like gilteritinib, crenolanib and quizartinib have shown benefit for r/r FLT3 mutated AML facilitating HSCT in CR. While mutational driven targeted therapies are now available for about 45–50% of AML patients  half of the AML patients do not yet have the opportunity to be treated with targeted therapies. While the benefit of midostaurin for FLT3 WT AML patients is currently being investigated, one additional option for these patients might be the addition of leukemia specific pathway inhibition by venetoclax or glasdegib to low intensive therapy, in particular in older AML patients. The impact of these drugs combined with IC on outcome of younger AML patients will be seen over the next years. The combination of mutational driven and pathway driven agents may give us even more therapeutic options and the upcoming new agents provide additional hope. However, the progress being accomplished through these new drugs comes with an economic burden. This has to be critically rendered and kept in an acceptable setting to make novel treatment strategies accessible for as many patients as possible.Despite the compelling progress in AML treatment and the development of new targeted therapies, most adult patients will still die from the disease. Today, the most curative treatment approach remains HSCT but is associated with a high therapy related mortality and most AML patients are not eligible for this intensive treatment route. This emphasizes the need for more targeted and less toxic treatment. The identification of molecular aberrations guided the development of mutational driven therapies for In conclusion, targeted therapy seems to be the future for AML management, in particular for unfit patients and we may hope that someday we do not have rely on IC and allogeneic HSCT alone anymore. The FDA approval of midostaurin, gilteritinib, enasidenib, ivosidenib, glasdegib, venetoclax and gemtuzumab ozogamicin within one year seems to be just the beginning and new pathway inhibiting compounds as well as immunogenic based treatment strategies are already under further clinical investigation. It will be interesting to pursue how the combination of certain targeted agents will further improve the outcome of AML patients.


Cold-induced urticaria is a kind of physical urticaria characterized by the appearance of wheals after exposure to cold. The atypical form is a rare sub-type characterized by appearance of hives even in areas not directly exposed to the cold and by a negative cold stimulation test. Its diagnosis is often challenging because of the lack of specific tests and it is usually based on the patient’s clinical history. Hypotension due to generalized exposure to the cold is described both in the typical and the atypical forms.We describe a 9-year-old boy who, at the beginning of the summer after the first swim in the sea, developed generalized urticaria, dyspnea, conjunctival hyperemia, blurred vision and loss of strength. The child was treated with intramuscular steroid and intravenous antihistamine, and the symptoms quickly resolved. Insect bite, contact with fish and drug ingestion were denied, and no unusual food had been eaten before the swim. A tentative diagnosis was made of either aquagenic urticaria or cold urticaria, but the specific tests were negative. Although the cause was unknown, prophylactic treatment with antihistamines was prescribed but in spite of this, wheals developed all over the body, after every swim in the sea. The child then came to our attention and relying on clinical history a diagnosis of atypical cold urticaria was made: development of hives even in areas not directly exposed to cold and a negative response to the cold stimulation test, are the characteristic features of this rare form of cold urticaria.Atypical cold urticaria should be suspected in all cases of anaphylaxis related to cold exposure (i.e. contact with water) with a negative cold stimulation test. Cold-induced urticaria (CU) is a physical urticaria characterized by the appearance of wheals after exposure to cold. It represents one third of all cases of physical urticaria . The mosA 9-year-old boy complained of malaise, just a few minutes after his first summer swim in the sea; soon after, he presented generalized urticaria, dyspnea, conjunctival hyperemia, blurred vision and faintness. When first aid arrived, since anaphylactic shock was suspected, intramuscular steroids, intravenous antihistamine and nebulized salbutamol were administered, with rapid improvement on the part of the patient. The only thing of note in the child’s medical history was allergy to dust mite, and no other allergies were reported. There was no evidence of any insect bite or drug ingestion; an hour before the swim, the child had eaten his usual breakfast, with hot chocolate. Apparently, there was not contact with fish during the swim. The child had never complained of similar symptoms before and had never had urticaria after contact with water, be it seawater or tap water. No familiarity for allergic disease or chronic urticaria was reported. The child was referred to the local Allergy Department and in order to identify the offender, skin tests and specific IgE assays were performed. In detail, they tested allergy to milk, due the history of milk intake before the appearance of symptoms, and to insect venom and fish, because of the possibility of contact with insects and fish during the bath; all the tests were negative. Although the patient developed no symptoms on contact with tap water, an aquagenic urticaria was suspected, but the specific test was negative. Finally, a cold urticaria was suspected but the cold stimulation test (CST) was negative too. Given the severity of the reaction, prophylactic antihistamine therapy was commenced, but in spite of this, throughout the summer the patient continued to develop wheals all over his body after every swim in the sea Fig. , even inUrticaria is a common finding in pediatrics. It is characterized by the presence of wheals and\or angioedema. Wheals are skin lesions characterized by a central swelling and peripheral erythema; the single skin lesion lasts 1–24 h, but the duration of the whole disease can be longer . AngioedIf urticaria lasts more than 6 weeks, it is defined as chronic urticaria. The prevalence of chronic urticaria in children is about 0.1–0.3% . In abouAs previous reported CU accounts for about 30% of physical urticaria in childhood, even if its prevalence may vary according latitude . It is mThe systemic form of ACU is rare, but it should be promptly recognized because it is usually characterized by more severe reactions then TCU. Unlikely, its diagnosis is often challenging because of the lack of specific diagnostic tests and because it can be precipitated by many different sources, kinds and degrees of cold. Moreover, the clinical association with the cold is usually less obvious than in TCU: in fact, in ACU, the appearance of wheals is caused by a decrease in body temperature rather than by direct contact with a cold object . It is aIt is by now recognized that in TCU, generalized exposure to the cold  can cause severe systemic symptoms, including hypotension ; systemip = 0.01). No statistically significant difference regarding symptom severity were found between ACU and TCU patients, but severe systemic reactions  were more common in the TCU group than in the ACU one  and among children than adults  [A recent retrospective study  comparedThere is no specific diagnostic test for ACU, although some Authors suggest a specific test in which the patient, wearing light clothes, is to be placed in a cold room (at a temperature of 4 °C) for 30 min . This teJust like the typical form of CU, ACU usually responds well to antihistamine therapy, both in presence of acute symptoms and for prophylaxis . AccordiThe other key aspect of treatment is prevention: patients should avoid abrupt exposure to intense cold and should take prophylactic antihistamine therapy if a high-risk activity is programmed (such as sailing in winter); in high-risk patients self-injectable epinephrine should also be prescribed , 13. Evep = 0.02) [Even if life-threating symptoms, as the ones described in our patient, are possible, most patients with CU have mild and intermittent symptoms. The mean duration of symptoms of CU is 4.8 to 7.9 years ; disease = 0.02) .ACU can show with severe systemic symptoms until anaphylaxis, so it must be suspected in all cases of anaphylaxis related to any kind of generalized cold exposure and in all cases of CU related to cold exposure, especially in those children who seem to be more likely to have generalized symptoms. The case described is emblematic of the potential severity of this condition and shows the challenges involved in recognizing this rare form of urticaria. However, diagnosis can be easily made without the need for allergy tests just by remembering the typical features of ACU: 1) correlation to various cold stimuli, 2) appearance of wheals even in areas not directly exposed to the cold and 3) a negative response to the cold stimulation CST.