More recent studies highlight important functions of cytoskeletal α-smooth muscle actin (α-SMA), α5β1 integrin, and integrin-mediated cell-extracellular matrix (ECM) interactions in Ca2+-dependent regulation of VSMC stiffness and adhesion to the ECM, providing novel insights into the mechanism of calcium action.Vascular smooth muscle cells (VSMCs) are the predominant cell type in the arterial wall. These cells play a critical role in maintaining vascular homeostasis including vasoconstriction and vasodilatation through active contraction and relaxation. Dysregulation of VSMC function alters the response of blood vessels to mechanical stress, contributing to the pathogenesis of vascular diseases, particularly atherosclerosis and hypertension. The stiffness of VSMCs is a major regulator of vascular function. Previous studies suggest that intracellular Ca Vascular smooth muscle cells (VSMCs) are mainly composed of the medial layer of the blood vessels, which are subjected to mechanical stress and pressure of blood flow, and maintain vascular tone and resistance. VSMCs not only have normal cellular functions, maintain their intrinsic properties, support vascular geometry, but also require some important actions to participate in the regulation of the biomechanical and biochemical properties of blood vessels . In addiVSMC carries out biomechanical and biochemical functions that are associated with the transformation of vascular configurations and are characterized by mechanical and physical effects such as blood vessel contraction and relaxation. Due to the various and diverse phenotypes of smooth muscle cells, these diversity effectively allow the blood vessels to gain flexibility and defend various physiological and pathological conditions .via integrin-mediated cell-ECM interaction  in those pathological processes. Currently, many publications contribute valuable data on the elastic changes of VSMC during physiology and disease processes and address some of the new concepts of cell elasticity in vascular disease , 2019. Veraction ,b. Thereeraction .Calcium is a stimulator of many cellular functions, and under physiological conditions, intracellular calcium are present in VSMC to regulate many different biophysical and biochemical processes . The purvia integrin-mediated cell-ECM interactions to alternate cytoskeleton system of the cell. The α5β1 integrin works as the dominant fibronectin (FN) receptor to provide the bio-mechanical linkage between α-SMA and FN in extracellular space  and some internal organelles . In VSMCs, intracellular Ca2+ concentration ([Ca2+]i) is in a range of 100 to 10,000 nM, while Ca2+ concentration in extracellular space is 2 mM (2+]i activates VSMC elastic and adhesive functions by way of two avenues: 1) entry of Ca2+ from extracellular space through specific ion channel, such as L-type Ca2+ channel (LTCC); 2) release of Ca2+ from intracellular organelles (sarco/endoplasmic reticulum)  . [Ca2+]ital axis , 2001. Tignaling . This in2+]i level. Ionomycin, a drug that increases [Ca2+]i levels, was used to treat VSMCs and induce the increases in VSMC elasticity and adhesion activities, whereas they decreased after drug BAPTA-AM treatment to reduce [Ca2+]i. Ionomycin treatment was also observed the area and height of VSMCs to be expanded and increased due to developing cytoskeletal α-SMA production and promoting F-actin assembly. In contrast, BAPTA-AM decreased the area and height of VSMCs due to reducing cytoskeletal α-SMA production and preventing F-actin assembly  is a peptide chain and complicatedly converted by Angiotensin I, and Angiotensin II is found in VSMCs to stimulate the Gq protein and start a series of cascade reactions increasing [Ca]i level . ANG II tiffness .2+]i exceeds the physiological requirement within VSMCs, it also will induce some vascular dysfunctions and diseases, such as hypertension. Hypertension is a common age-related vascular disease, and many factors can induce age-related vascular dysfunctions and diseases. However, a less efficiency of intracellular Ca2+ activity to attenuate the function of ECM-integrin-cytoskeletal axis regulating VSMC elasticity importantly contributes to induce hypertension , collagen I (COL-I), vitronectin (VN), and laminin I (LM), but the characteristics of adhesion to each protein are different with binding and signaling strongly associated with FN and COL-I  to modulate a series of cellular events including VSMC elastic function via some enzymes’ phosphorylation. In VSMCs, G-protein-coupled receptor (GPCR) activates phospholipase C (PLC) and cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3) to form and participate Ca2+ activity. The IP3 diffuses to the sarcoplasmic reticulum (SR) and binds with a receptor on SR membrane to open a ligand-gated Ca2+ channel and increase [Ca2+]i. The DAG activates protein kinase C (PKC). The PKC often directly regulates and controls the opening and closing of L-type Ca2+ channel (LTCC) by phosphorylation and dephosphorylation, while indirectly manipulates VSMC elastic and adhesive functions by triggering [Ca2+]i level  is a Ca2+-binding messenger protein to bind Ca2+ and transduce calcium signals performing its function. [Ca2+]i binding with CaM activates CaM-dependent protein kinase II (CaMKII) to regulate the activation of myosin light chain kinase (MLCK) by phosphorylation and controls the interaction between α-SMA and myosin to affect the VSMC elasticity and adhesion process, while reducing the level of [Ca2+]i by dephosphorylation inhibits the activity of MLCK to promote vascular smooth muscle relaxation  to hydrate the establishment with α-SMA (2+, such as gelsolin, talin, vinculin, and cadherin (2+]i (2+]i level (2+]i (2+ induces to form the complex with FAK, talin, paxillin, and other components, and the complex enhances VSMC adhesion to FN matrix (2+, and VSMCs are regulated by a complex network of phosphorylation and dephosphorylation kinase cascade from Ca2+ generation (2+]i activity for restraining vascular smooth muscle contraction to treat hypertension and some age-related vascular diseases (2+]i (2+]i and attenuate the VSMC elasticity and adhesion (2+]i (2+]i dependent actin-myosin interaction and cause an reduction of VSMC stiffness and adhesion decreasing [Ca2+]i level of VSMC (2+]i and treated VSMCs to show a rapid transient increase in cell stiffness as well as cell adhesion to FN matrix due to transiently increasing in the level of [Ca2+]i, after KCl treatment the increased VSMC stiffness and adhesion to FN matrix were not abolished by MLCK inhibitor (2+-CaM-MLCK pathway in VSMCs is parallel to [Ca2+]i-induced α-SMA and α5β1 integrin expressions for regulating VSMC stiffness and adhesion (Ca]i level . Ca2+ islaxation . Ca2+ alth α-SMA . Many prcadherin . Gelsoliin (2+]i . Cadheri]i level . Vinculiel (2+]i . Active N matrix . The balneration . Blebbisdiseases . The drues (2+]i , whereasadhesion . MLCK anon (2+]i . ML7 is  of VSMC . KCl wasnhibitor . These o2+ pump and Na+/Ca2+ exchanger are two basic mechanisms for Ca2+ entry and exit from cells (2+ level between extracellular space and intracellular cytoplasm, and also keep the fluid and electrolyte balance of VSMCs to regulate cell normal physiological functions and elastic characters. The Na+/Ca2+ exchanger handles vascular tissues and blood pressure in human body through the regulation of VSMC elasticity (2+ pump and Na+/Ca2+ exchanger arise some damages of living organisms, and dehydration and overhydration will derive from the unbalances of electrolytes Na+, Ca2+, K+, Cl−, 2+ (2+ pump and Na+/Ca2+ exchanger occur some pathological damages to produce a lot of crisis and hardness affecting the entry and release of Ca2+ and making [Ca2+]i declination. This declination will reduce Ca2+ signaling function for that phosphorylation and dephosphorylation activate or deactivate the corresponding enzymes to generate a cascade cycle for the modulation of VSMC elastic and adhesive characterizations. The abnormal Ca2+ release and entry promote pathological reactions to induce calcification, and the main form of calcification is calcium phosphate crystalline in VMSCs. Meanwhile, the produced calcium salts harden to lose the corresponding physiological mediation functions (2+ regulation, but calcification cuts off all pathways and makes Ca2+ to fully lose its activity, and further induces VSMCs to thoroughly lose elastic and adhesive functions (ATP-dependent Caom cells . These tasticity . ATP-depand Mg2+ . If fluiand Mg2+ . Additiounctions . Phosphounctions . The calunctions .2+]i directly manipulates the expressions of α-SMA and α5β1 integrin, and the α5β1 integrin regulates [Ca2+]i level in accordance with physiological requirement (2+-CaM-MLCK pathway to influence VSMC stiffness and adhesion via ECM-integrin-cytoskeletal axis without any cascade cycles by phosphorylation and dephosphorylation. However, the physiological mechanism need to be further probed. This investigation factually plays an independent pathway to regulate VSMC stiffness and adhesion via ECM-integrin-cytoskeletal axis and provides a new therapy approach to target α-SMA or α5β1 integrin for influencing VSMC stiffness and adhesion and treat some vascular dysfunctions and diseases. Animal models provide important clues for exploring the pathogenesis of vascular dysfunctions and diseases to study how [Ca2+]i to regulate and mediate VSMC elastic and adhesive functions in living organisms (in vivo rather than in vitro because the normal physiological processes in living organisms provide some novel views to revolve [Ca2+]i role in VSMC elastic and adhesive activities. In general, a more in-depth study of the [Ca2+]i track and role in VSMC elastic and adhesive activities in vivo and in vitro for vascular physiological and pathological processes is the future extension and direction, and the significant advancements will contribute some novel clinic approaches to vascular disease.[Cauirement . This inrganisms . The perYZhu drafted and designed manuscript. YZhu, JQ, LH, SY, and YZhou edited and revised manuscript. YZhu, FZ, and ZZ prepared figures. YZhu, JQ, LH, FZ, ZZ, SY, and YZhou approved final version of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It exists in four main isoforms (PMCA1-4), among which PMCA2 and PMCA3 are considered as fast-acting neuron-specific forms. In the brain, PMCA function declines progressively during aging; thereby impaired calcium homeostasis may contribute to some neurodegenerative diseases. These destructive processes can be propagated by proinflammatory chemokines, including chemokine CCL5, which causes phospholipase C-mediated liberation of Ca2+ from endoplasmic reticulum by IP3-gated channels. Plasma membrane Ca3 receptors expression level was assessed using qRT-PCR and Western blot. To mimic the changes in aged neurons we used stable transfected differentiated PC12 cells with downregulated PMCA2 or PMCA3 and analyzed the effect of CCL5 on calcium transients with Fluo-4 reagent. Chemokine receptors were evaluated using Western blot, and IP2+ by IP3-sensitive receptors, and the time required for Ca2+ clearance was significantly longer. Also, in these lines we detected altered expression level of CCR5 and IP3 receptors. In PMCA-reduced cell lines, CCL5 released more Ca2+ dyshomeostasis, insufficient cell protection may result in progressive degeneration and death of neurons. Although modification of PMCAs composition could provide some protection against calcium overload, reduction of PMCA2 appeared to be more detrimental to the cells than deficiency of PMCA3. Under pathological conditions, including inflammatory CCL5 action and long-lasting Ca All measurements were performed in a calcium-free buffer containing 125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 6 mM glucose, and 25 mM HEPES. In all experiments, after measurement of basal Ca2+ for 150 s, 50 ng/ml of CCL5 (PeproTech) was added. For analysis of CCR/PLC/IP3R pathway, specific inhibitors were added right before measurements—cocktail of CCRs inhibitors: 1 nM BX513 (Abcam) for CCR1, 1 μM SB328437 (Abcam) for CCR3 and 1 nM DAPTA (Abcam) for CCR5 or 4 μM U73122 (Abcam) for PLC, or 100 μM 2-APB (Sigma) for IP3Rs. To determine the role of ER calcium stores in CCL5 response, 1 μM thapsigargin (Santa Cruz Biotechnology) was added 50 s after basal calcium measurement, and next 50 ng/ml of CCL5 at 150 s was applied. All measurements were done in duplicate and the data presented in the figures are the average of at least 5 independent cell cultures.Cells grown to 80% confluence in 25 cmμg of purified RNA and oligo(dT) primers were then used for cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The IP3R genes expression level was quantified in a real-time PCR reaction using 5x HOT FIRE Pol Eva Green qPCR Mix Plus (Solis Biodyne) in the following conditions: 15 min at 95°C followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s using ABI Prism 7000 sequence detection system (Applied Biosciences). Following normalization to the expression of endogenous Gapdh, the fold change of each target gene was calculated using comparative 2−ΔΔCt method as described previously [Total cellular RNA was isolated using Tri Reagent (MRC) according to the manufacturer's protocol. 1 eviously . Primers  reverse: 5′-GCTTTCGTGGAATACTCGGTC-3′,Ip3r-1 forward: 5′-GTGGAGGTTTCATCTGCAAGC-3′,  reverse: 5′-CCCATGTCTCCATTCTCATAGC-3′,Ip3r-2 forward: 5′-GCTCTTGTCCCTGACATTG-3′,  reverse: 5′-CATCCGTGGGAACCAGTC-3′,Ip3r-3 forward: 5′-AGCGAGAAGCAGAAGAAAG-3′,  reverse: 5′-CTTCCCATTCTCAGCCTTGAC-3′.Gapdh forward: 5′-GCTTACCAG-GGCTGCCTTCT-3′,μg of cell lysate proteins were separated using either 8% or 10% SDS-PAGE and electroblotted onto nitrocellulose membranes using semidry method. The membranes were blocked with 3% BSA in TBS buffer  for 30 min at room temperature prior to exposure to primary antibodies. Membranes were incubated overnight at 4°C with the primary antibodies and for approximately 4 h with the secondary antibodies at room temperature followed by 3 × 15 min wash with TBS-T between each antibody application. The following primary antibodies were used: anti-CCR1 , anti-CCR3 , anti-CCR5 , anti-IP3R-1 , anti-IP3R-2 , monoclonal anti-IP3R-3 , and anti-IP3R-1/2/3 . Monoclonal anti-β-actin antibody (Santa Cruz Biotechnology) was used at 1:1000 dilution as a protein loading control. Secondary antibodies coupled with alkaline phosphatase (Sigma) at 1:5000 dilution were used for staining. Sigma Fast BCIP/NBT was used for visualization according to the manufacturer's instruction. Blots were scanned and band intensities were measured quantitatively using GelDoc™ EQ system with Quantity One 1-D Analysis Software version 4.4.1 (Bio-Rad).Approximately 40-80 4 cells/well on thin-glass bottomed 96-well plate  and differentiated for 48 h. Next, cell cultures were washed once with PBS and fixed for 10 min in room temperature with 2% formaldehyde freshly prepared from paraformaldehyde and buffered to pH = 7.4 in PBS. Subsequently, cells were washed once and further incubated for 1 h with the blocking buffer , following 1 h incubation with primary antibody against CCR5 diluted in the blocking buffer . Next, cells were washed 3 × 5 min with the blocking buffer and incubated for 1 h with secondary goat anti-mouse IgG1 antibody conjugated with Alexa Fluor 488  to fluorescently label immunodetected CCR5. Subsequently, after 3 × 5 min wash with the blocking buffer, plasma membrane marker protein Na+/K+ ATPase was immunostained for 1 h with primary antibody (Abcam) fluorescently labeled with Zenon™ Alexa Fluor 568 Mouse IgG1 Labeling Kit (Thermo Fisher Scientific) and diluted in the blocking buffer (1:50). Finally, cells were washed 3 × 5 min with the blocking buffer, once with PBS, and were fixed again for 10 min in room temperature with 2% formaldehyde in PBS (pH = 7.4). Eventually, cell nuclei were counterstained with 5 μM Hoechst 33342 in PBS, and triple-stained cells were visualized by confocal microscopy. Images were obtained using 780 LSM confocal microscope (Zeiss) with Plan-Apochromat 63x/1.4 Oil DIC M27 objective. ZEISS Microscope Software ZEN2012 was used to calculate mean fluorescence intensities of respective CCR5-positive pixels colocalizing with plasma membrane marker (Na+/K+ ATPase-positive) pixels. Plasma membrane marker-positive pixels were selected based on their fluorescence intensity that ought to be between arbitrary upper (500 units) and lower (40 units) thresholds, set either to eliminate nonspecific background staining or to negate the influence of high-intensity artificial staining (antibody precipitates and others). Analysis of fluorescence intensity of CCR5 staining colocalizing with plasma membrane staining was performed for each cell line for five different fields of view and results were averaged.Cells were seeded at a density of 2 × 10 t-test or ANOVA followed by Tukey or Bonferroni's multiple comparisons tests, wherever applicable. P values were calculated using STATISTICA 12.0 (StatSoft) or GraphPad Prism 7 . P < 0.05 was considered as statistically significant.The data are shown as means ± SD of n separate experiments (n ≥ 3) with the exact n value given under each figure. The comparisons between cell lines were done using Student'sFirst, we evaluated the presence of CCR1, CCR3, and CCR5 in PC12 lines using two approaches. The immunoreactivity of examined receptors was initially assayed in a cell lysate by Western blot . In compSince functionally active chemokine receptors are located at the plasma membranes, we next explored cellular distribution of CCRs by confocal microscopy. Staining of endogenous CCR1 and CCR3 showed low immunosignal intensity, which was concentrated predominantly in the cytosolic area (data not shown). To validate these results, we repeated the assay using another set of antibodies against CCR1 and CCR3; however, we obtained similar results. These data indicated that although CCR1 and CCR3 were present in PC12 cell lines, their participation in CCL5 signaling in our model seems to be negligible. CCR5 was expressed much more abundantly and was mostly distributed in the plasma membranes . Quantif2+ increase in examined PC12 cell lines, chemokine action was assayed in a calcium-free medium to avoid the secondary effect triggered by “calcium-induced calcium release” process, as well as the concomitant participation of membrane calcium channels. Changes in fluorescence were monitored for 250 s at one-second intervals, and CCL5 was always applied after 150 s of measurement. The CCL5 concentration was chosen because it was shown to be effective under inflammatory state, which is supported by literature data. It is also frequently used for in vitro assays including PC12 cells [2+ level in PMCA-reduced PC12 cells was observed . Slower recovery in _2 and _3 lines could result from lowered Ca2+-clearing potency in PMCA-reduced lines, since SERCA affinity for calcium is at low micromolar range [2+-free conditions, the differences in cell response to CCL5 might reflect a balance between intensity of Ca2+ release from ER and Ca2+ extrusion, which varied between cell lines.Treatment with CCL5 at 50 ng/ml increased cytosolic Caar range . In Ca2+μM thapsigargin added after 50 s, and next CCL5 was applied at 150 s. As shown in 2+ reuptake by SERCA, and subsequent addition of chemokine had no effect on calcium transient. To confirm the participation of CCRs in generation of Ca2+ transient, the assay was also performed in the presence of specific CCRs inhibitors. Although CCR5 appeared to be the main functionally active receptor in our cell lines, chemokine action was assessed in the presence of cocktail of CCR1, CCR3, and CCR5 inhibitors added just before measurement  were detected in all lines, but some lower molecular weight bands were also present. They could represent proteolytic fragments of the receptors, as similar observations were also shown in another study [To validate whether the mRNA changes correlated with corresponding receptor protein, Western blot analysis was performed using isoform-specific antibodies and the antibody that recognized all three IPeceptors . Single er study .3R isoforms confirmed that in _2 and _3 lines the protein level of IP3R-1 and 2 was lower than that of control cells, but IP3R-3 increased by ~40% , and certain types of voltage-gated calcium channels [2+ accumulation in the endoplasmic reticulum [2+ concentration to its safe level, but on the other hand, it may potentially increase Ca2+ release after activation of calcium channels in the ER, including activation of IP3 receptors and ryanodine receptors [3R-mediated Ca2+ release from the ER and mitochondrial Ca2+ homeostasis are physiologically coupled, their improper cooperation may significantly affect cell viability [Our previous studies revealed that, in differentiated PC12 cells, downregulation of neurospecific PMCA2 or PMCA3 increased cytosolic Cachannels , 39, 40.eticulum . On the eceptors . Since Iiability .3R-3 proteins, but lowered IP3R-1 and IP3R-2. All these changes are primarily related to elevated cytosolic Ca2+ as a consequence of changed composition of PMCAs. Interestingly, some of them could occur as adaptive processes protecting cells against calcium overload. Since age-related PMCA decrease has been documented [Based on current study, we can add to the list of common features detected after reduction of PMCA2 or PMCA3 increased level of CCR5 and IPcumented , 45–47, 2+ affected chemotaxis, secretion, and gene expression and could lead to inflammatory and degenerative processes in the CNS [2+-induced Ca2+ release (CICR) and involve cooperation with plasma membrane calcium channels [2+ concentration necessary to open these channels [The presence of CCR5 in PC12 cells was shown in several studies , 41, and the CNS , 29. Che the CNS –55. The channels . Crucialchannels . In our 3R pathway. The crucial step appeared to be associated with activation of IP3 receptors. Although IP3R-1 represents a predominant isoform in the central nervous system, other isoforms also exist in some brain areas and may differ in neuronal compartmentalization [3Rs in hippocampus is related to learning and memory abilities, and changes in IP3R isoforms composition during aging may have an impact on increased deficits in these processes. In most cultured cell types IP3R-3 is the principal form, but IP3R-1 and IP3R-2 have also been detected [3R-1 possesses low Ca2+ affinity and medium affinity for IP3, IP3R-2 represents the isoform with the highest affinity for both Ca2+ and IP3, and IP3R-3 is the most sensitive for modulation by Ca2+, but displays the lowest IP3 affinity [3Rs are regulated in a biphasic way by cytosolic Ca2+ and are stimulated at low Ca2+ level, but inhibited by higher than 300 nM Ca2+ [3Rs [3R isoform may produce diversified cell signaling paths that can initiate the adaptive response or can lead to neurodegeneration [Using the selected inhibitors we confirmed that in our cell models CCL5 downstream effects involved CCR5-PLC-IPlization –60. A padetected . All iso nM Ca2+ . Up to na2+ [3Rs . Thus, mneration .3R isoforms were present, with IP3R-3 being the most prominent subtype. An interesting observation made here was the altered composition of IP3Rs in PMCA-reduced lines at both mRNA and protein level. IP3R-1 and IP3R-2 decreased, whereas IP3R-3 amount was higher than that of control cells. Changes in relative mRNA level indicate that the regulation occurs at the level of transcription, suggesting Ca2+-dependent negative feedback loop for IP3R-1 and IP3R-2. Analysis of IP3R protein levels also confirmed the differences in a ratio of IP3R isoforms between control _2 and _3 lines. One can assume that in both PMCA-reduced cells the decreased expression of receptors with higher affinity for IP3 could lower the total apparent sensitivity to IP3 and thereby may provide some protection against calcium overload. The graphical summary of these findings is presented in Our analysis showed that in PC12 cells, all three IP2+ accumulation in the ER; thus potentially more calcium could be released after activation of IP3Rs. Moreover, less efficient PMCA-dependent extrusion system, due to PMCA2 suppression, may prolong calcium signaling, modifying a number of existing pathways. It can also lead to further Ca2+ overload. In fact, in this line we reported decreased cell survival and increased percentage of apoptotic cells [In _2 line, we observed higher Caic cells ; thus thic cells , 63. Takic cells , reducti2 [2 interaction has been proposed [2 by PMCA4 has been demonstrated to protect plasma membrane PIP2 from hydrolysis by PLC. It could potentially limit IP3 production and, subsequently, restrict Ca2+ release from ER. Since for full activity, IP3Rs must bind four IP3 molecules [2 concentration as a substrate for PLC is a necessary requirement. Accordingly, in the _3 line greater protection could result not only from the altered profile of IP3Rs and the presence of highly active PMCA2, but also from higher PMCA4 amount. All these adaptive changes may limit the amount of Ca2+ released from the ER and shorten the time necessary to restore basal Ca2+ level.The presence of an additional protective mechanism may be suggested in the _3 line, where we previously observed increased expression of PMCA4 isoform. One of the well-established mechanisms of PMCA regulation is stimulatory action of PIP2 , 65, 66.proposed . Bindingolecules , a suffi2+-induced signaling that may allow cells to survive but also may promote cell death. The unique role played by “fast” isoforms of plasma membrane calcium pump in neuronal cells suggests that long-lasting calcium dyshomeostasis could markedly increase cell vulnerability to pathological events, including neurodegenerative disorders [2+ extrusion mechanism could accumulate potentially harmful changes in neurons, which increase the risk of developing neurodegenerative diseases.Downregulation of neuron-specific PMCA2 or PMCA3 initiated a set of responses that significantly altered Caisorders , 19, 63.isorders . Here we


We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca2+ sparks  also became less solid. An increased fraction of small CRUs in HF was linked to augmented ‘silent’ Ca2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca2+ leak and slows Ca2+ release kinetics, leading to weakened contraction in this disease.Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca The muscle cells of the heart coordinate how they contract and relax in order to produce the heartbeat. During heart failure, these cells become less able to contract. As a result the heart becomes inefficient, pumping less blood around the body.For the cardiac muscle cells to contract, the levels of calcium ions in the cells needs to rapidly increase. In failing hearts, these increases in calcium ion levels are smaller, slower and less well coordinated. It was not known what causes these changes, making it difficult to treat heart failure.Calcium ions are released in cardiac muscle cells through protein channels called ryanodine receptors. These receptors form clusters that allow them to synchronize when they open and close. Could the reorganization of ryanodine receptors account for the problems seen in failing hearts? To investigate, Kolstad et al. examined rat hearts using a technique called super-resolution microscopy. This showed that the clusters of ryanodine receptors break apart during heart failure to form smaller clusters. Further experiments showed that calcium ions ‘leak’ from these smaller clusters, reducing the amount of calcium that can be released into cardiac muscle cells during each heartbeat. Released calcium also spreads between the dispersed clusters, resulting in a slower rise of the calcium levels in the cells. Both changes contribute to weakened contractions of cells in failing hearts.Therefore, heart failure can be traced back to very small rearrangements of the ryanodine receptors. This understanding will help researchers as they investigate new ways to treat heart failure. This process of Ca2+-induced Ca2+ release leads to a sharp increase in cytosolic Ca2+ concentration which initiates cardiomyocyte contraction. In ventricular myocytes, Ca2+ release is tightly controlled by the arrangement of LTCCs and RyRs in dyads, with LTCCs present in t-tubules juxtaposed from RyRs across a narrow 12–15 nm dyadic cleft , these functional arrangements of RyR clusters generate Ca2+ sparks, the fundamental units of SR Ca2+ release in cardiomyocytes . SR Ca2+ release is reduced and slowed in this condition, and these changes have been linked to altered dyadic structure  and Structured Illumination Microscopy , the localization of RyRs along z-Lines was clearly apparent, but organization of RyRs within CRUs was not discernable . With dS2+ leak in failing cardiomyocytes. Total Ca2+ leak was assessed in SR microsomes obtained from the left ventricle of Sham and failing hearts.We examined the functional implications of altered nanoscale organization of RyRs, first hypothesizing that RyR dispersion would augment SR Ca2+ uptake by addition of ATP, SERCA function was halted by thapsigargin treatment to reveal RyR-mediated Ca2+ leak  was significantly increased in HF cells, this effect was offset by a tendency toward lower spark frequency . Ca2+ release from the smallest CRUs was never detected, but a progressively greater proportion of events yielded visible sparks as the number of RyRs in these idealized dyad geometries was increased  by creating smaller CRUs which produce Ca2+ release events below the detection limit, and (2) by creating more distributed CRU configurations in which multiple RyR clusters are less likely to co-operatively generate sparks.In summary, these results indicate that nanoscale reorganization of RyRs in HF promotes non-spark-mediated SR Ca2+ release; a hallmark of HF. Representative confocal recordings of Ca2+ sparks and their temporal profiles are shown in 2+ spark kinetics, we again employed our mathematical model with dSTORM-based CRU configurations. During the simulations, the time to opening of each RyR was registered, and the time course of the overall Ca2+ spark determined. Representative simulations show that RyR opening times were delayed in the dispersed, multi-cluster CRUs compared to the solid, single-cluster CRU  across the cell , and remaining ‘fast’ sparks. By this definition, 24% of sparks in HF cells were defined as slow, while only 13% of Sham sparks fit this definition. Representative examples of such sparks and their temporal profiles are shown in 2+ sparks, CRU dispersion during HF slows and desynchronizes the overall Ca2+ transient.Finally, we examined the consequences of increased variability in Cahan Sham . This sl disease . We exam2+ homeostasis in failing cells: increased ‘silent’ RyR leak and slowing of Ca2+ release, which are believed causative for reduced contractility in this condition.In the present study, we have employed dSTORM imaging to reveal key changes in CRU morphology during heart failure. We specifically observed marked dispersion of RyRs, which resulted in a shift towards smaller RyR clusters and CRUs. Remaining larger CRUs became less solid, with more fragmented configurations. Experiments and mathematical modeling linked these changes in RyR arrangement to two central aspects of impaired Caet al.  (0.05 mmol/L). After 10 min of digestion, hearts were cut down, minced, and filtered, and isolated cardiomyocytes were allowed to sediment.Cardiac myocytes from failing and Sham-operated rats were isolated using a standard enzymatic dispersion technique . Excised3 citrate 17.5 mmol/L, 5% goat serum, 3% BSA, 0.02% NaN3, 2 hr). PBS washing was performed in between each step. The cells were then incubated overnight with 1/100 diluted mouse-anti-RyR2 primary antibody  in low blocking buffer, containing 150 mmol/L NaCl, 17.5 mmol/L Na3 citrate, 2% goat serum, 1% BSA, and 0.02% NaN3 at 4°C. This protocol has previously been reported to result in the binding of multiple primary antibodies to each RyR tetramer , and plated on laminin-coated, glass bottom culture dishes . Staining was performed according to a described protocol , with cotetramer . The foltetramer . SpontanData were processed with built-in software  using a Dense Stochastic Sampling Imaging (DSSI) algorithm and multiple Gaussian fits to localize events. Drift correction was performed with a model-based algorithm. Average localization precision was 21.6 nm for the events included in the final reconstructions.http://opencv.willowgarage.com/) modules (https://github.com/TerjePrivate/Ryanodine_Receptor_Dispersion_during_Heart_Failure) (https://github.com/elifesciences-publications/Ryanodine_Receptor_Dispersion_during_Heart_Failure). Images with 10 × 10 nm pixels were convolved with a 2D Gaussian function equal to the calculated resolution of the image (~20 nm), and downscaled by a factor of 3 to produce a final pixel size of 30 × 30 nm. RyR locations were defined using a modified automated thresholding algorithm , and RyR configuration was analyzed. Non-specific labeling was observed to only negligibly increase the number of RyRs/cluster and RyRs/CRU by 3.7% and 3.2%, respectively. Similarly, RyR density was increased by 0.5%, and inter-cluster distance was reduced by 0.2%, supporting that unspecific labelling had a minute influence on the dataset.2+ sparks were recorded from quiescent cardiomyocytes loaded with fluo-4 AM  and superfused with a HEPES Tyrode solution containing (in mmol/L): 140 NaCl, 1.0 CaCl2, 0.5 MgCl2, 5.0 HEPES, 5.5 glucose, 0.4 NaH2PO4, 5.4 KCl, pH 7.4, 37°C. Scans were performed with a 1024 pixel line drawn along the longitudinal axis of the cell with a temporal resolution of 1.5 ms. Ca2+ sparks were analysed with a custom program , as previously (0) of 0.4, to minimise the inclusion of false positives. Linescan images of cells obtained during inhibition of Ca2+ sparks (prolonged exposure to 10 mM caffeine) confirmed the appropriateness of this detection threshold. Ca2+ spark frequency was normalized to cell length and recording time, and spark geometry was assessed by measurements of time to peak (TTP), full width at half maximum (FWHM), and full duration at half maximum (FDHM). Spark-mediated Ca2+ leak was calculated as the product of spark mass (amplitude X FWHM X FDHM) and frequency.Using an LSM 7Live confocal microscope (Zeiss), Caeviously . Sparks 2+ transients were elicited by field-stimulation through a pair of platinum wires (3 ms supra-threshold current pulses at 1 Hz), and recorded as confocal linescans under the same experimental conditions as Ca2+ spark measurements. Global Ca2+ transient characteristics were analyzed by averaging the Ca2+ signal along the linescan, with measurements of transient magnitude , time to half maximal fluorescence (TTF50), and TTP. Local Ca2+ transients were averaged across narrow 2 µm bands of the linescan. Synchrony of Ca2+ release was assessed as previously described .Caescribed , by plot2+ content was assessed by rapidly applying 10 mM caffeine and measuring the amplitude of the elicited Ca2+ transient.SR Ca2+ handling was additionally examined using crude homogenates from rat left ventricle, based on methods described by O’Brien and modified by Li et al.  at 25000 rpm for 3 × 20 s, with a 20 s break between bursts. Homogenates were then aliquoted, frozen in liquid N2, and stored at −80°C until use.Cai et al. . Fresh v2+ uptake and release were measured in 2.2 ml of assay buffer, containing (in mmol/L): 165 KCl, 22 Hepes, 7.5 oxalate, 11 NaN3, 0.0055 TPEN, 4.5 MgCl2, 9 Tris HCl and 0.002 fura-2 salt . Ca2+ fluxes were monitored with an LS50B luminescence spectrometer  after addition of 100 µl of freshly-thawed and vortexed homogenate. Ca2+ uptake by the vesicles was initiated by addition of Na4ATP (2.2 mmol/L), and then blocked by application of thapsigargin (1.5 µmol/L) to assess RyR leak. Releasable SR Ca2+ content was estimated by measuring Ca2+ release induced by application of the RyR opener 4-chloro-m-cresol (4-CMC) (5.5 mmol/L). The fluorescence ratio was calibrated to [Ca2+] using the following equation: [Ca2+]=Kd *((R - Rmin)/(Rmax - R))*(Sf2/Sb2), where R is the 340 nm/380 nm fluorescence ratio, Kd is the dissociation constant of fura-2 and Sf2/Sb2 is the ratio of measured fluorescence intensity at 380 nm when fura-2 is Ca2+ free or saturated, respectively. Rmin is the ratio at very low [Ca2+]i and Rmax is the ratio at saturating [Ca2+]i, obtained by adding 3.3 mmol/L EGTA and 4.8 mmol/L CaCl2 respectively to the cuvette at the end of each recording.CaFrozen tissue from rat left ventricles was homogenized in cold buffer  with the addition of a Complete EDTA free protease inhibitor cocktail tablet  and a PhosSTOP tablet (Roche). SDS was then added to the homogenates to a final concentration of 1%, and protein concentrations were quantified using a micro BCA protein assay kit . Bovine serum albumin (BSA) was used as standard protein.The following primary antibodies were employed for immunoblotting: RyR , BIN1 , junctophilin-2  and GAPDH . Secondary antibodies were anti-rabbit , anti-mouse  or anti-goat  IgG-HRP linked whole antibody. Data were normalized to GAPDH and then to Sham values .Protein homogenates (5 or 15 µg/lane) were size fractionated on 4–15% or 15% Criterion TGX gels  and transferred to 0.45 μM PVDF-membranes . The membranes were blocked in 5% non-fat milk or 5% Casein (Roche Diagnostics) in Tris-buffered saline with 0.1% Tween (TBS-T) for 1 hr at room temperature, and then incubated with primary antibody overnight at 4°C. Secondary antibodies were incubated for 1 hr at room temperature and blots were developed using Enhanced Chemiluminescence . Chemiluminiscense signals were detected by a LAS 4000  and protein levels were quantified using ImageQuant software .2+ spark characteristics. We have made all simulation results, geometries, and code specific to this study available in an online repository (2+ regulation), and because it was built for a similarly constructed  geometry,  across all geometries by modifying the non-junctional SR (nSR) volume as required. Of note, while nSR concentration was modeled as a continuum, global SR calcium concentration was effectively clamped at initial values as expected for the time-scale and spatial-scale simulated.The computational domain in our model , and solved in time using explicit Euler time stepping. Specifically, we used operator splitting and solved each of the reaction and diffusion sub-problems with a fixed ∆t = 0.1µs, except when calculating the RyR release current. Due to the small element volumes and high fluxes this calculation is very stiff, so instead we solve it analytically. If we use x to denote the SR calcium concentration and y to denote the cleft calcium concentration, we can write this sub-problem as:Dyadic CaWhere:Using this scheme it is possible to take arbitrarily long time steps without introducing instabilities.2+, which allowed cooperative opening of adjacent RyR clusters located up to ≈150 nm apart (ie. 4 RyR lengths) if the clusters shared jSR .All raw data acquired and analyzed in this study are publicly available at  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.2+ Release in Failing Cardiac Myocytes" for consideration by eLife. Your article has been reviewed by Harry Dietz as the Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal his identity: David Baddeley (Reviewer #3).Thank you for submitting your article "Ryanodine Receptor Dispersion Disrupts CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2+ imaging and simulations has been used effectively, and the results are convincing. The main finding of the study is that RyR clusters are more dispersed in the failing than in normal cardiomyocytes.This is an interesting and thoughtful manuscript that advances our understanding of the changes underlying the slower and weaker calcium transients in failing cardiac myocytes. The authors describe changes in the arrangement of ryanodine receptors, using super-resolution microscopy and simulations. The combination of structural cluster imaging, functional CaEssential revisions:1) Cluster fragmentation data  need to be shown more clearly and in greater detail. Some of the magnified unthresholded data and the scaled raw image data should be displayed (taking care not to saturate too much). A clear case should be made that the fragmentation is not a thresholding artifact, as it is known that threshold changes could cause apparent fragmentation for a given data set.2) It is critical that the authors provide enough implementation detail for the simulation model so that a reader can use the simulation for stably dealing with the nonlinearities contained in a stochastic RyR switching model. If a custom code was used, it should be provided.It should be clarified if the basic geometry is the same as in the Laver et al. 'induction decay' model, or if not – what is the scope and rationale for the changes made. Also, the authors should explain how they performed the numerical integration  The authors should also provide rationale for the data exclusions and clearly specify which data were used.3) The method is based on the assumption that peripheral couplons are effectively 2D objects parallel to the cell surface and hence the imaging plane. It becomes increasingly hard to justify this assumption when moving away from surface where EM data shows that couplons take on complex 3D geometries, often wrapping around t-tubules. The absence of double-rows would imply that the images taken in this paper are not at the cell surface. While looking at the projected area will still give a reasonable estimate of relative numbers, it is likely that the absolute number of RyRs is underestimated for internal couplons. Regarding the projections to a regular 30nm grid, there is very limited evidence that RyRs form a regular grid in normal conditions within cardiomyocytes. Even if they do form a regular grid, it likely does not align with the imposed sub-sampled pixel grid. The authors should specifically discuss these important aspects of their model. It is also suggested to acknowledge that the estimated numbers are not necessarily very precise and discuss the effect that changing the RyR number would have on the simulation results.4) With regard to the measured parameters, simple t-tests do not suffice. A hierarchical nested model should be used as p<0.05 is not satisfactory when there is large inter-group variability and the number of animals is small. The data needs to be reanalyzed, e.g. using methods described in a recent publication . Essential revisions:1) Cluster fragmentation data  need to be shown more clearly and in greater detail. Some of the magnified unthresholded data and the scaled raw image data should be displayed (taking care not to saturate too much). A clear case should be made that the fragmentation is not a thresholding artifact, as it is known that threshold changes could cause apparent fragmentation for a given data set.Thank you for your helpful comments, which have improved the manuscript. Figure 2 has now been changed to include a new panel which displays a high magnification “high-low/grayscale” image of the region of interest. This has been done to show that the binarized images give a reasonable estimation of RyR clusters based on the raw images for both Sham and HF groups. The added panel has been given a “high-low” look-up table wherein the top and bottom 3% grey value pixels are colored red and blue respectively. This more clearly illustrates that the grayscale images from which the binarization was derived are not heavily saturated.2) It is critical that the authors provide enough implementation detail for the simulation model so that a reader can use the simulation for stably dealing with the nonlinearities contained in a stochastic RyR switching model. If a custom code was used, it should be provided.It should be clarified if the basic geometry is the same as in the Laver et al. 'induction decay' model, or if not – what is the scope and rationale for the changes made. Also, the authors should explain how they performed the numerical integration  The authors should also provide rationale for the data exclusions and clearly specify which data were used.We have now elaborated on each of these points in the Materials and methods section of the manuscript. For the reviewers' reference, the time step was fixed at 0.1 microseconds, which is sufficient for the RyR gating events themselves, and is not limiting for solution of the resulting concentration changes on either side of the jSR membrane, because this sub-problem can be solved analytically.3) The method is based on the assumption that peripheral couplons are effectively 2D objects parallel to the cell surface and hence the imaging plane. It becomes increasingly hard to justify this assumption when moving away from surface where EM data shows that couplons take on complex 3D geometries, often wrapping around t-tubules. The absence of double-rows would imply that the images taken in this paper are not at the cell surface. While looking at the projected area will still give a reasonable estimate of relative numbers, it is likely that the absolute number of RyRs is underestimated for internal couplons. Regarding the projections to a regular 30nm grid, there is very limited evidence that RyRs form a regular grid in normal conditions within cardiomyocytes. Even if they do form a regular grid, it likely does not align with the imposed sub-sampled pixel grid. The authors should specifically discuss these important aspects of their model. It is also suggested to acknowledge that the estimated numbers are not necessarily very precise and discuss the effect that changing the RyR number would have on the simulation results.The reviewers are correct that the RyR clusters imaged in our work are near the surface, but not at the surface. We agree that using grid-based counting methods for determining RyR numbers at internal locations is likely more error prone and may underestimate cluster sizes. The following section has been added to the Discussion section to address these points:“However, it should be noted that the grid-based method for RyR counting assumes that the channels lie parallel to the field of view, with a uniform, grid-like configuration; presumptions which are likely less valid when RyRs are visualized internally than at the cell surface. Due to super-imposition of internal RyRs along the z axis, it is therefore likely that present RyR cluster sizes are somewhat underestimated.”We have made a similar statement in the Discussion section regarding estimates of RyRs / CRU:“Despite possible underestimation of RyR numbers / CRU due to methodological issues noted above, these estimates are in relatively close agreement with an electron microscopy tomography study of mouse ventricular cardiomyocytes ”.2+ spark kinetics. Mean CRU sizes observed experimentally in both Sham and HF cardiomyocytes were within this range . Furthermore, since RyR numbers were likely underestimated experimentally, we do not expect that such counting errors have marked consequences regarding the effects of CRU dispersal on Ca2+ spark parameters. Of note, a lack of sensitivity of spark characteristics to RyR number in average-sized clusters is in agreement with previous work . This finding has been described in the Results section:We have additionally considered the reviewer’s point that underestimation of RyR cluster and CRU sizes by grid-based counting procedures may influence the simulation results. To this end, we have performed additional modeling where RyR number was varied, while maintaining the ratio of jSR to nSR. These new data  showed that for CRUs larger than 20 RyRs there was little effect of varying RyR number on Ca2+ spark kinetics , further confirming a key role of CRU fragmentation in failing cells.”“Of note, although CRUs were observed to contain fewer RyRs in HF than Sham , simply reducing the RyR number to an equivalent degree in the mathematical model did not markedly alter CaWe have also addressed this point in the Discussion section:“Our modeling results also suggest that an altered number of RyRs / CRU, due either to methodological under-estimation or loss of RyRs from CRUs in HF, will not in and of itself promote slowing of sparks kinetics . Indeed, we observed that spark kinetics were relatively insensitive to changes in RyR number for medium-sized CRUs, in agreement with previous work .”4) With regard to the measured parameters, simple t-tests do not suffice. A hierarchical nested model should be used as p<0.05 is not satisfactory when there is large inter-group variability and the number of animals is small. The data needs to be reanalyzed, e.g. using methods described in a recent publication .A hierarchical “nested ANOVA” linear mixed-effects model has been applied to the dataset, which allows RyR configuration to be cumulated and compared within animals. The resulting p-values can be found in a table included in Figure 2—source data 1 and are presented alongside data cumulated within cells. The main differences in RyR configuration between Sham and HF were similar based on these two approaches.


Therefore, BH3 mimetics could potentially be toxic for the pancreas when used to treat cancer. Although ABT‐199 was shown to kill Bcl‐2‐dependent cancer cells without affecting intracellular Ca2+ signalling, its effects on PACs have not yet been determined. Hence, it is essential and timely to assess whether this recently approved anti‐leukaemic drug might potentially have pancreatotoxic effects.Many cancer cells depend on anti‐apoptotic B‐cell lymphoma 2 (Bcl‐2) proteins for their survival. Bcl‐2 antagonism through Bcl‐2 homology 3 (BH3) mimetics has emerged as a novel anti‐cancer therapy. ABT‐199 (Venetoclax), a recently developed BH3 mimetic that selectively inhibits Bcl‐2, was introduced into the clinic for treatment of relapsed chronic lymphocytic leukaemia. Early generations of Bcl‐2 inhibitors evoked sustained Ca2+ measurements and cell death analysis were performed on isolated mouse PACs.Single‐cell Cavia ABT‐199 did not elicit intracellular Ca2+ signalling on its own or potentiate Ca2+ signalling induced by physiological/pathophysiological stimuli in PACs. Although ABT‐199 did not affect cell death in PACs, under conditions that killed ABT‐199‐sensitive cancer cells, cytosolic Ca2+ extrusion was slightly enhanced in the presence of ABT‐199. In contrast, inhibition of Bcl‐xL potentiated pathophysiological Ca2+ responses in PACs, without exacerbating cell death.Inhibition of Bcl‐2 2+ extrusion, ABT‐199 does not substantially alter intracellular Ca2+ homeostasis in normal PACs and should be safe for the pancreas during cancer treatment.Our results demonstrate that apart from having a modest effect on cytosolic Cahttp://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetocThis article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit  Up to five mice were kept per cage with aspen wood bedding material and an enriched environment . All procedures involving animals were performed in accordance with the UK Home Office or the Polish Ministry of Science and Higher Education regulations. The mice were humanely killed by cervical dislocation according to Schedule 1 of Animals (Scientific Procedures) Act 1986. The pancreatic tissue was removed for further experimental procedures. In order to reduce the number of animals needed for the experiments (in line with the 3Rs), cells isolated from one animal were used by two researchers simultaneously.et al.,2+. Immediately after dissection, the pancreatic tissue was washed twice in NaHEPES, treated with collagenase  and then enzymatically digested in the collagenase solution at 37°C for 15 min. After digestion, the pancreas was broken down by pipetting, suspended in NaHEPES, spun , resuspended in NaHEPES and spun again. Finally, isolated cells were suspended in NaHEPES and loaded with a Ca2+‐sensitive dye, Fluo‐4, as described below.PAC isolation was performed as described previously ]. After the incubation, the cells were resuspended in fresh NaHEPES and used for experiments (RT) in a flow chamber perfused with NaHEPES‐based extracellular solution. Experiments were performed using the following equipment: (i) Zeiss LSM 880 confocal microscope (×63 oil objective) at the Jagiellonian University; and (ii) Leica TCS SPE confocal microscope (×63 oil objective) or Leica TCS SP5 II two‐photon confocal microscope (×63 water objective) at Cardiff University. Excitation was set to 488 nm and emission to 500–600 nm. Static images were taken at 512 × 512 pixel resolution, and a series of images were recorded at 256 × 256 pixel resolution; two consecutive frames were averaged, and time resolution was one image per 2 s. Fluorescence signals were plotted as F/FN = 16) or randomized to the experimental groups with five independent pancreata per group (N = 5). Since the NaHEPES‐treated sample served as the control for each individual pancreas isolation  subsequently used in the cell death experiments, N was 16 for this condition. Fifteen minutes before the end of the incubation, Annexin V‐FITC and PI were added to the samples. The cells were visualized with a TCS SP5 II two‐photon confocal microscope (Leica) with a 63× 1.2 NA water objective, and fluorescence/transmitted light images were taken. Annexin‐V‐FITC  specifically stains apoptotic cells, whereas PI  was used for detection of necrotic cells; the cells stained with both fluorescent dyes were classified as secondary necrosis. Fifteen pictures of independent cell clusters were taken at 512 × 512 pixel resolution. The percentage of live, apoptotic, secondary necrotic and necrotic cells were counted in each treatment group by one researcher in a blinded fashion (encoding the group labels).Cell death assay was performed using an Annexin V‐FITC/PI apoptosis detection kit. PACs were isolated as described above, spun down and then suspended in 3 mL of fresh NaHEPES. The cells were divided equally into the experimental groups  and kept until further treatment (4°C). Then 1 mL of the buffer was removed, and the cells were treated with 1 mL of 2× concentrated incubation buffer containing menadione or TLC‐S, and the vehicle ; the isolation quality control group was incubated with NaHEPES only. Also, in some treatment groups, the incubation buffer at this stage was supplemented with the BH3 mimetics: ABT‐199, A‐1155463, ABT‐199 together with A‐1155463, or ABT‐737. The final concentrations were as follows: menadione 60 μM and TLC‐S 200 μM; and the BH3 mimetics: 1 μM ABT‐199, 1 μM A‐1155463, 1 μM ABT‐199 together with 1 μM A‐1155463, or 10 μM ABT‐737. Owing to the limited amount of mouse PACs obtained in a single isolation procedure, all experimental groups but one were equal by design. For the cell death assay, the cells isolated from 16 pancreata were either treated with NaHEPES  whereby viable cells were identified as being Annexin V/7‐AAD negative. The analysis was performed using the FlowJo software.DLBCL cell lines were seeded at 250 000 cells·mLet al.  and treated for 2, 4 or 6 h with ABT‐199 (1 μM) or vehicle. The percentage of apoptotic cells was determined by staining with PI and annexin‐A5‐FITC conjugate (Nexins Research) and analysis on an FACSCalibur flow cytometer with CellQuest Pro version 5.2.1 software (BD Biosciences).Blood samples were collected from patients with CLL according to the principles established by the International Conference on Harmonization Guidelines on Good Clinical Practice. An informed consent was obtained from all patients and approval for the study was obtained from the ethical committee of the Università Cattolica del Sacro Cuore, Fondazione Policlinico A. Gemelli, Rome, Italy (protocol number 14563/15). The collection and analysis of CLL patient samples were performed as reported in Bojarczuk et al. . Briefly2+ stores, PACs were treated with 2 μM thapsigargin (Tg) for 10 min in the absence of extracellular Ca2+. Then the extracellular Ca2+ concentration was increased to 10 mM, which induced Ca2+ influx to the cytosol. Once a [Ca2+]i plateau was achieved, removal of extracellular Ca2+ unmasked the process of Ca2+ extrusion across the plasma membrane. This phase of the response was further analysed and compared between control and ABT‐199 pretreated PACs. First, for every recorded [Ca2+]i trace, the maximum and minimum F/F0 values in the rage of 1000–1400 s of the response were determined (Fmax and Fmin). The normalized fluorescence that corresponds to half the decrease between these two values was calculated as follows: F1/2 = Fmin + (Fmax−Fmin)/2. Next, a linear fit to the extrusion phase was determined. Time values corresponding to Fmax (tmax) and F1/2 (t1/2) were calculated from the linear fit. Finally, t1/2 was calculated as the difference between t(F1/2) and tmax. The t1/2 values obtained for control and ABT‐199‐treated cells were then averaged and presented as dot charts ±SEM. Student's t‐test was applied for statistical comparison, and the significance threshold was set at 0.05.In order to empty the ER Caet al.,N = 5) were performed in each experimental setting (see the text for details). Additionally, in Ca2+ signalling experiments, n values representing the recorded fluorescence of the specific regions of interest (ROI), corresponding to single cells, were provided. Those were not the technical replicates but the independent measurements of the entire cell population in the experiment. Because of the non‐equal numbers cells recorded in the viewing fields, n may vary between treatment groups in the given experimental setting. Quantitative analysis of Ca2+ responses was performed as described previously  × Δt, where F is the recorded fluorescence, F0 is the baseline fluorescence and Δt is the time interval. The values obtained were then averaged and presented as dot charts ±SEM. Student's t‐test was applied for statistical comparison, and the significance threshold was set at 0.05. Quantitative analysis of cell death was performed using SPSS Statistics 24 software (IBM): first, Levene's test was used to assess the equalities of the variances for a variable calculated for the groups, and then ANOVA or a non‐parametric Kruskal–Wallis one‐way ANOVA were applied to compare the differences among group means; for both tests, the significance thresholds were set at 0.05. Finally, Bonferroni's post hoc test (whenever relevant) was performed only if values of the F‐test  achieved the necessary level of statistical significance (P < 0.05).The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis taurolithocholic acid 3‐sulfate (TLC‐S) ; and the BH3 mimetics: the Bcl‐2 inhibitor ABT‐199 , the Bcl‐xL inhibitor A‐1155463  and the non‐selective Bcl‐2, Bcl‐xL and Bcl‐w inhibitor ABT‐737 . NaHEPES buffer was prepared as follows (mM): NaCl 140, KCl 4.7, HEPES 10, MgCl2 1, glucose 10; pH 7.3. The BH3 mimetics were dissolved in DMSO, TLC‐S in NaHEPES and menadione in ethanol.The main reagents for cell isolation and imaging included: Annexin V‐FITC/propidium iodide (PI) apoptosis detection kit, Fluo‐4 AM ; collagenase ; ACh, CCK, inorganic salts, menadione, http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY  was used as a positive control for ER Ca2+‐store loading.In order to compare the effects of acute inhibition of different Bcl‐2‐protein family members on Cad Figure . First, d Figure A. Furthed Figure B. These d Figure C, D. Giv2+ responses occurred in PACs treated with ABT‐737 , 34% produced one or more Ca2+ transients (dark grey) and 20% of cells developed a prolonged cytosolic Ca2+ plateau (black). Interestingly, a similar response pattern was obtained when a combination of two selective inhibitors, ABT‐199 together with A‐1155463, was used , 22% produced one or more Ca2+ transients (dark grey) and 26% developed an increased cytosolic Ca2+ plateau (black). Finally, selective inhibition of a single Bcl‐2‐family member was tested in PACs. Blocking Bcl‐xL with A‐1155463 did not trigger any Ca2+ responses in the majority of cells tested , whereas 24% responded with one or more Ca2+ transients . Circular diagrams summarizing Ca2+ response patterns in PACs to different inhibitors of Bcl‐2 family members are shown in Figure 2+  was shown to induce apoptosis in CLL cells   in PACs  in PACs , induces Cal Figure A, blue al Figure B, red; sl Figure C. Next, d Figure D. Prolond Figure E slightls Figure E, F. TakBH3 mimetics have been developed in order to counteract the Bcl‐2‐dependent evasion of apoptosis, common in cancers. Normal cells, such as PACs, are much less dependent on anti‐apoptotic Bcl‐2 family members than cancer cells. However, even in non‐transformed cells, pathophysiological stimulants that trigger cell death could influence the balance between the pro‐ and anti‐apoptotic Bcl‐2 proteins and thus may ‘prime these cells for death'. Therefore, pathophysiological stress may reveal additional effects of a BH3 mimetic that do not surface under physiological conditions.2+ signals in PACs under a pathophysiological stimulus, the cells were incubated (2 h) with different BH3 mimetics , whereas A‐1155463 (blocker of Bcl‐xL) potentiated TLC‐S‐elicited Ca2+ release (P < 0.05). Interestingly, these two opposite effects cancelled out when ABT‐199 and A‐1155463 were applied simultaneously. Under the inhibition of a wide range of Bcl‐2 family proteins by ABT‐737, TLC‐S‐induced Ca2+ responses were potentiated (P < 0.05).Substantial elevations in cytosolic CaS Figure A. These 2+ elevations in PACs , as well as ABT‐737, both increased the pathophysiological Ca2+ elevations induced by menadione (P < 0.05). Taken together, pharmacological inhibition of Bcl‐xL via its hydrophobic cleft appears to increase pathophysiological Ca2+ responses, whereas inhibition of Bcl‐2 does not have a major impact on Ca2+ homeostasis both in physiology and pathology. Since ABT‐199 does not exacerbate pathophysiological Ca2+ signals, it may have a more advantageous safety profile compared to inhibitors that target Bcl‐xL.A similar experimental approach was used to test the effects of BH3 mimetics on 60 μM menadione‐elicited Cas Figure B. ABT‐192+ responses in PACs. Since Ca2+ release is associated with the induction of necrosis and apoptosis, application of Bcl‐2 protein inhibitors may modulate PAC death. To determine this, PACs were incubated for 2 h with either TLC‐S (200 μM) or menadione (60 μM) in the presence of BH3 mimetics or appropriate vehicles  incubation with ABT‐199 (1 μM) did not potentiate the increased cell death that spontaneously occurred in PACs maintained s Figure D. Second2+ extrusion in PACs by modulating 2+ ATPase (PMCA)plasma membrane Ca activity  for ABT‐199‐treated PACs compared to control cells (87.7 ± 5.7 s), which translates into a shorter time required for the fluorescence of the Ca2+ indicator to decrease by half the difference between the [Ca2+]i plateau and the baseline , the anti‐apoptotic Bcl‐2‐family members regulate several proteins involved in Ca2+ signalling (Vervliet et al.,3R (Rong et al.,et al.,3R results in sensitization of the receptor and triggers pro‐survival Ca2+ oscillations (White et al.,et al.,3R via its BH4 domain thereby inhibiting excessive pro‐apoptotic Ca2+ release (Chen et al.,et al.,et al.,3Rs, the anti‐apoptotic Bcl‐2 proteins also regulate intracellular Ca2+ handling via RyRs (Vervliet et al.,et al.,et al.,et al.,2+ homeostasis.It is very well established that the Bcl‐2 family proteins play a role in the regulation of intracellular Ca2+ responses induced in normal mouse PACs and in the rat pancreatic cancer cell line AR42J (Gerasimenko et al.,et al.,2+ stores, causing the ER stress‐mediated cell death (Hermanson et al.,et al.,Inhibition of the anti‐apoptotic Bcl‐2 members by the early generation BH3 mimetics, BH3I‐2′ and HA14‐1, was associated with global and sustained Ca2+ release in PACs (Figure 2+ responses elicited by endogenous agonists at physiologically relevant concentrations (Figures 2+ homeostasis, occasionally inducing infrequent Ca2+ transients in PACs (Figure via simultaneous application of ABT‐199 and A‐1155463 (Figure 2+ rises in PACs or a prolonged Ca2+ plateau. This suggests that the toxic effects of BH3 mimetics on Ca2+ associated with the inhibition of a wide range of the anti‐apoptotic Bcl‐2 proteins could be avoided by selectively targeting only one family member, specifically Bcl‐2 itself (Figure 2+ signalling in cell lines, including Bcl‐2‐dependent DLBCL cancer cell models (Vervloessem et al.,This study demonstrates that selective inhibition of Bcl‐2 by ABT‐199 neither triggers substantial intracellular Ca Figures . Targete2+ responses elicited by physiological or supramaximal concentrations of ACh (Figure 3R‐mediated Ca2+ release from the ER and further amplified by RyRs (Wakui et al.,3Rs and RyRs by Bcl‐2 is independent of its hydrophobic cleft (Vervliet et al.,et al.,ABT‐199 did not alter Ca2+ responses induced by TLC‐S or menadione, selective inhibition of Bcl‐2 by ABT‐199 did not (Figure 2+ responses were even reduced in the presence of ABT‐199 (Figure 2+ extrusion (Figure et al.,et al.,2+ extrusion by alleviating Bcl‐2's inhibitory effect on PMCA.Although inhibition of Bcl‐xL by A‐1155463 or pan‐inhibition of Bcl‐2/Bcl‐xL/Bcl‐w by ABT‐737 potentiated pathophysiological Ca2+ signals by ABT‐199 was reversed by A‐1155463, when PACs were pre‐incubated with both inhibitors before application of TLC‐S, indicating that the effects on Ca2+ associated with Bcl‐xL inhibition dominate over those triggered by inhibition of Bcl‐2 (Figure 2+ signalling in response to pathophysiological stimulants. The mechanisms remain unclear, although it may relate to interference by these drugs of Bcl‐xL‐dependent regulation of Ca2+‐release systems like IP3Rs (Yang et al.,The moderate suppression of pathological Ca2+ responses induced by TLC‐S and menadione (Figure 2+ homeostasis in PACs are insufficient to increase the sensitivity of normal cells to cell death inducers. This finding could be particularly relevant to the situation when a BH3 mimetic is used in combination with other chemotherapeutic agents. Such combined therapies are currently undergoing clinical trials (Ferdek and Jakubowska, Despite the aforementioned effects on pathophysiological Cae Figure , neithere Figure A,D. Thes2+ extrusion (Figure 2+ signals on its own (Figure 2+ homeostasis or sensitize healthy PACs to inducers of cell death, it should be safe for the pancreas when used in therapy for leukaemia.In conclusion, we report that selective inhibition of Bcl‐2 by ABT‐199, apart from having a modest effect on cytosolic Can Figure , does non Figure  and pathn Figure . As a con Figure . TherefoG.B. conceived the study with further input of M.J., P.F. and T.V.; G.B., T.V. and P.F. coordinated the work. M.J., P.F., M.K., C.M. and T.V. performed the experiments. M.J. and P.F. performed the data analysis and made the figures. The manuscript was drafted by T.V., M.J., G.B. and P.F. All authors were involved in the interpretation of the data and critically revised and approved the manuscript for submission.The authors declare no conflicts of interest.Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.This


Striga spp. Because organic synthesis of natural SLs is laborious, there is a large need for easy-to-synthesize and efficient analogs. Here, we investigated the effect of a structural modification of the D-ring, a conserved structural element in SLs. We synthesized and investigated the activity of two analogs, MP13 and MP26, which differ from previously published AR8 and AR36 only in the absence of methylation at C-3′. The de-methylated MP13 and MP26 were much more efficient in regulating plant development and inducing Striga seed germination, compared with AR8. Hydrolysis assays performed with purified Striga SL receptor and docking of AR8 and MP13 to the corresponding active site confirmed and explained the higher activity. Field trials performed in a naturally Striga-infested African farmer’s field unraveled MP13 as a promising candidate for combating Striga by inducing germination in host’s absence. Our findings demonstrate that methylation of the C-3′ in D-ring in SL analogs has a negative impact on their activity and identify MP13 and, particularly, MP26 as potent SL analogs with simple structures, which can be employed to control Striga, a major threat to global food security.Strigolactones (SLs) regulate plant development and induce seed germination in obligate root parasitic weeds, e.g.  For exar hyphae . Howeverarasites .R configuration to a second moiety that consists of a tricyclic lactone (ABC-ring) in canonical SLs, such as strigol and orobanchol. Non-canonical SLs, such as carlactonoic acid, contain less conserved second moiety instead of the ABC-ring  and CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8. These three enzymes are plastid-localized and conduct the steps converting all-trans-β-carotene into the key intermediate carlactone (CL)  MORE AUXILLARY GROWTH 1 (MAX1) homologs and other enzymes convert it into SLs (Striga hermonthica HYPOSENSITIVE TO LIGHT (ShHTLs), which have evolved through extensive gene duplication of KAI2/HTL in Orobanchaceae and new functionalization that enables sensing SLs  . CL is tinto SLs . Plant Sinto SLs . Followiinto SLs . KARRIKIinto SLs . It is asing SLs . ShHTL7  process . This prmination .Striga spp. (Witchweeds), and Phelipanche and Orobanche spp. (Broomrapes), are of great economic importance, as they infest a wide range of cereal and non-cereal crops, causing enormous yield losses . Seeds of P. aegyptiaca (collected from tomato field) were provided by Prof. Mohamed Ewis Abdelaziz, Cairo University, Egypt. Seeds of rice cv IAC 165 were obtained from Africa Rice, Tanzania (courtesy of Jonne Rodenburg). The seeds of d10 and d3 mutants were kindly provided by Dr Junko Kyozuka, Tohoku University, Japan. Arabidopsis (Columbia) seeds were multiplied locally. Rice was grown at 28°C and 70% relative humidity with fluorescent white light (130–180 μM m−2 s−1) 12-h day/night period. For root and hypocotyl studies, Arabidopsis plants were grown 16 h at 22°C/8 h at 16°C day/night, 60% relative humidity, and 60–70 μmol m−2 s−1 white light. Striga preconditioning and germination were carried out at 30°C under moist conditions in the dark.Seeds of rac-GR24). Cyclohexanone was purchased from Sigma-Aldrich. The formulated emulsifier was prepared as previously described -furanone  (2SO4), and evaporated. The residue was column chromatographed on silica gel using a mixture of hexane and ethyl acetate as eluent to give (E)-2-methyl-3-[oxy]acrylaldehyde (MP13). Yield was 57%. The physicochemical properties of MP13 are: 1H NMR : δ 9.34 , 7.17 , 6.98 , 6.19 , 2.04 , 1.70 . HRMS-Esi: m/z [M-Na]− Calcd for C9H10Na1O4:205.04768, found: 205.06819. 13C-NMR  δ: 192.19, 170.54, 162.58, 141.40, 135.18, 122.52, 100.29, 10.38, 6.20. Melting point was 88°C  and 7.7 N NaOH aq 390 μl  were heated at 70°C until the reaction mixture became homogeneous. Then the reaction mixture was dried with evaporation under reduced pressure. To the solution of the resultant residue in DMSO (5.0 ml), 5-bromo-3-methyl-2(5.1 mmol)  was addewas 88°C .in vacuo with an evaporator to give residue, which was then purified with silica gel column with ethyl acetate-hexane as eluent to give MP26 in 55% yield. The physicochemical properties of MP26 are: 1H NMR : δ 7.25 , 6.91 , 6.73 , 6.06 , 5.81, 3.74 , 2.00 , 1.74 . 13C-NMR  δ: 170.70, 167.66, 148.56, 144.82, 141.55, 135.22, 117.16, 115.06, 100.00, 51.39, 10.62, 9.31. Melting point was 146–149°C (Methyl (triphenylphosphoranylidene) acetate (2.1 mmol) was mixed with MP13 (1.13 mmol) and toluene (20 ml/mmol) in a round-bottom flask and stirred for 23 h at room temperature. Then, the reaction mixture was concentrated 46–149°C .d10) were sterilized with 2.5% sodium hypochlorite for 15 min. After thorough washing with sterile MilliQ water, the seeds were kept in water for 2 days at 30°C in the dark for imbibition and pre-germination. The pre-germinated seeds were then transferred to moist filter paper in 90-mm petri plates and incubated at 30°C overnight. The plates with germinated seeds were then shifted in a growth cabinet with fluorescent white light (130–180 μM m−2 s−1) for 1 week. Seven days-old seedlings were moved to 50-ml falcon tubes (one seedling per tube) containing modified half-strength Hoagland nutrient solution in greenhouse. After 1 week, rice plants were treated with 2.5 μM of each SL analog. Mock and GR24 (2.5 μM) were included as control treatments. Six applications of each SL analog were done two times in a week. After 3 weeks of application, number of tillers per plant, plant height, and fresh biomass were measured at final harvest.Rice seeds . The sterilized Arabidopsis seeds were grown in petri plates that were first kept at 4°C in darkness for 3 days. For hypocotyl study, the petri plates were exposed to continuous white light for 24 h, then transferred to continuous monochromatic red light  conditions for another 4 days. For lateral root density, the plates were vertically grown at 22°C in Percival incubator under long day conditions  for 8 days. After scanning, the hypocotyl length and number of lateral roots were recorded using publicly available ImageJ software.Half-strength MS prepared with 0.5 glS. hermonthica and P. aegyptiaca seeds was tested by following the procedure described before . GR24 and sterile MilliQ water were included as positive and negative controls, respectively. The treated seeds were allowed to germinate in the dark for 24 h at 30°C (for Striga) and for 1 week at 25°C (for Phelipanche). The germinated and non-germinated seeds were counted under a binocular microscope, and germination rate (in %) was calculated.Germination activity of SL analogs on d before . The pre−1, pH, 6.8) by adopting the previously described procedure with minor modifications  was spiked in 975-μl test solution. The time course of degradation was monitored by ultra-performance liquid chromatography (UPLC) analysis using a Zorbax Eclipse XDB-C18 column , eluted first by 5% acetonitrile in water for 0.5 min, then by a gradient flow from 5 to 100% acetonitrile within 17.5 min, and by 100% acetonitrile for 5 min. The column was operated at 40°C with a flow rate of 0.35 ml min−1, and the temperature of auto-sampler was set at 21°C. Compounds eluted from the column were detected with a photodiode array detector, and the relative quantity of non-degraded amount was calculated by integrating corresponding peak areas and comparison with the internal standard Indanol. Stability was monitored at 3-day intervals up to 3 weeks.The SL analogs GR24, AR8, MP13, and MP26 were first tested for their stability by high-performance liquid chromatography (HPLC) in an aqueous solution  was added in a black Eppendorf tube weekly up to 6 weeks and incubated at 30°C. On the seventh week, about 50 μl of each analog was applied on a glass fiber disc containing preconditioned Striga seed and put again in the dark in sealed petri plates at 30°C for 24 h. Germinated and non-germinated seeds were counted from each disc.The stability of formulated SL analogs was further tested in sand culture. About 1.5 ml of silver sand was added in each of 6 wells of 24-well plates. Then, 800 μl of formulated SL analogs (1.0 μM) was added in each of the six wells. The plates were sealed firmly with para-film, wrapped in aluminum foil, and placed at 30°C from day 1 to day 7. After 7 days, glass fiber discs containing preconditioned Oryza sativa D14 (OsD14) was carried out according to the procedure described earlier (In vitro Yoshimulactone Green (YLG) hydrolysis assays  were co-incubated with 1.0 μM of YLG  for 60 min at room temperature. Fluorescent intensity was measured by spectraMax i5 (Molecular Devices) at excitation by 480 nm and detection by 520 nm. The change in fluorescence observed over the course of 1 h of YLG in buffer without protein was subtracted from the data collected in presence of protein. IC50 values were calculated by GraphPad (Prism 7).Protein expression and purification of ShHTL7 and  earlier . In vitrs assays  were carAR8 and MP13 were docked to ShHTL7 (pdb:5z8p) and OsD14 (pdb:5dj5) based on the structure bound to GR24 (pdb:5dj5) as template using COOT  program.ca 4,000) Striga seeds were thoroughly mixed in 2 L of sand and soil mixture (1:1) and added in 3-L perforated plastic pot containing 0.5 L of clean soil in the bottom. Striga seeds were first preconditioned by giving light irrigation and putting pots at 35°C in greenhouse for 10 days. Each pot was sprayed with 20-ml (1.0 μM) formulated SL analogs up to 10 days at 2-day intervals. A light irrigation was given next day of each application to move the compound to Striga seeds and they were allowed to germinate for suicidal death without host for another 1 week. Then, three rice seedlings (5 days old) were planted in the middle of each pot. The rice plants were grown under normal growth condition  for 8 weeks. Number of emerged Striga plants in each pot were counted.About 10 mg of   to test suicidal activity of formulated MP13, taking formulated water treatment as control treatment. After onset of few rains, the field was thoroughly prepared by subsequent ploughing and planking. The experiment was laid out in a randomized complete block design with four plots or replications. Each plot consisted of two 3-m-long mounds and spaced with one mound between rows. Formulated MP13 and normal water (control) were applied twice in each plot (25 ml m−2) following rains (~10 mm) to get a final concentration of 0.5 μM. Local pearl millet crop was sown at least 1 week after the second application. Data on the number of emerged Striga plants at three counting dates were collected in both fields on a plot basis. The three Striga counts were then used to calculate the average number of emerged Striga plants throughout the season.A naturally Striga infestation and plant biology. Half maximum effective concentration (EC50) was calculated using IC50 toolkit.Collected data were analyzed statistically using statistical software package R (version 3.2.2). One-way analysis of variance (ANOVA) and least significant difference (LSD) multiple range test were used for analyzing the effect of different SL analogs on The two SL analogs MP13 and MP26 differ from the previously described AR8 and AR36  by the ad10 mutant grown hydroponically. On an average, the high-tillering d10 mutant showed six tillers per plant. This number decreased to 4.5 (23% inhibition) and 2.5 tillers per plant (57% inhibition), upon treatment with MP13 and MP26, respectively. Treatment with GR24 reduced the number of tillers to 1.4 tillers per plant (76% inhibition) while AR8 showed the weakest effect (5.6 tillers per plant)  ,B. Applir plant) . In anott growth . SeedlinStriga seeds to examine their germination-inducing activity (Striga germination rate (37–70%), compared to the parent analog AR8 (9%). However, the activity of MP13 and MP26 was slightly lower than that of the standard SL analog GR24 (75%). To further validate these results, related half maximal effective concentration (EC50) of formulated and non-formulated SL analogs was determined  and 1.9 × 10−9 M (non-formulated). MP26 was the second most effective analog, with EC50 values of 9.8 × 10−8 (formulated) and 3.5 × 10−7 (non-formulated), followed by MP13, with EC50 values of 1.1 × 10−7 (formulated) and 6.4 × 10−7 M (non-formulated). AR8 exhibited the lowest activity and the highest EC50 value for formulated (9.9 × 10−7 M) and non-formulated (6.9 × 10−4 M) application.Firstly, new SL analogs were applied on preconditioned activity . Applicatermined . Both foP. aegyptiaca seeds and determined their germination-inducing activity in this root parasitic plant. Application of non-formulated MP13 and MP26 at 1.0 μM concentration led to germination rate of around 68%, which is much higher than that of AR8 (32%) and similar  to that of GR24 (75%) , followed by MP26 . Surprisingly, non-formulated MP13 showed better performance with less EC50 value (3.2 × 10−8), compared with formulated application (2.5 × 10−7 M). The previously published analog AR8 showed again highest EC50 values in both formulated (3.2 × 10−7 M) and non-formulated (9.7 × 10−7 M) application  . We alsolication .The stability of MP13 and MP26 was compared to the parent SL analogs AR8 and GR24 . InitialStriga germination after 1 day of incubation in sand.The stability of MP13 and MP26 was also studied in sand culture at 30°C for 7 days . IncubatStriga seed germinating activity (germination rate) as a readout  in aqueous solution, using  readout . The actStriga SL receptor in ShHTL7 (Striga hermonthica Hyposensitive to Light 7) (50: 1.64 ± 0.52 μM), followed by MP13 (IC50: 3.87 ± 0.90 μM), MP26 (IC50: 5.30 ± 0.71 μM), and finally AR8 (IC50 of 6.90 ± 0.97 μM). Similarly, highest OsD14 binding activity was observed for GR24 (IC50: 1.24 ± 0.09 μM), followed by MP26 (IC50: 2.07 ± 0.80 μM), MP13 (IC50: 3.45 ± 1.2 μM), and finally AR8 (IC50 of 4.87 ± 0.78 μM). Docking of AR8 and MP13 to ShHTL7 active site further showed that the extra methyl group in AR8 could cause steric obstruction with the ShHTL7 Ile193 side chain, making it less active   and riceLight 7) ,B. Resuls active . The de-s active ,B. Simils active ,B.Striga Incidence Under Greenhouse and Naturally Infested Farmer Field Conditions.Testing of the Suicidal Germination Activity of Formulated SL Analogs on Striga suicidal germination-inducing activity of MP13 and MP26 in artificially infested pots using rice as host crop (Striga emergence on weekly basis (Striga emergence in the AR8 treated pots (11 plants per pot) at 10 weeks after sowing (WAS), compared to other SL analogs tested. Nevertheless, AR8 treatment resulted in around 18% decrease in Striga emergence, compared to the mock control. At the same time point, pots treated with MP13 and MP26 showed an average of six and four Striga plants per pot, respectively, which corresponds to 52 and 69% reduction, compared to the mock treatment. Treatment with GR24 led to the most pronounced decrease (82%) in Striga emergence. Statistically, the effect of MP13 and MP26 was comparable to that of GR24 during the whole growth period. The severity of Striga infestation (number of emerging Striga plants) inversely correlated with the growth of the host (height). Plants grown in mock were the shortest one, followed by those grown in AR8-, MP13-, MP26-, and finally GR24-treated pots . For this purpose, we applied MP13 two times after onset of rainfall with final conc. of 0.5 μM. After few days of last application, a local pearl millet cultivar was sown as a host crop. At the end of the season, we counted 91 Striga plants m−2, compared to 154 plants m−2 in the mock field. In other words, application of formulated MP13 led to around 41% reduction in Striga emergence  from plant sources and the difficulties in producing them by organic synthesis  showed high activity with P. aegyptiaca, which was statistically similar to that of GR24.We are very interested in developing simple and low-cost SL analogs to combat root parasitic weeds, in particular Striga germination-inducing activity than AR8. We hypothesized that the observed increase in Striga germination activity by modified MPs is due to their high affinity to Striga receptor. We confirmed this hypothesis by assessing the competitive inhibition of ShHT7 and OsD14-mediated YLG hydrolysis assays. In these experiments, the lower IC50 value (and hence stronger ShHTL7 and OsD14 interaction) of MP13 and MP26 compared with AR8 explained the increased sensitivity of Striga and rice to MP13 and MP26. The higher activity of MP26 for Striga germination despite a 2-fold lower IC50 compared to MP13 might be due to the increased stability of MP26. While better tillering inhibition by MP26 than MP13 and AR8 might be due to its higher binding activity to OsD14 (Stability is a major factor in determining the activity of SL analogs. Therefore, we determined the stability of MP13 and MP26 in formulated and non-formulated forms, and under different conditions, including incubation in sand at 30°C. In general, our results show that GR24 is the most stable analog tested in this study, followed by MP26, MP13, and finally AR8. Stability test showed that MP13 and MP26 are less stable than GR24 in sand. This result indicates that our new analogs might have less impact on the environment, compared to GR24. Nevertheless, toxicity tests and precise evaluation of the effect on soil organisms need to be provided before applying these compounds in field. It is important to note that although MP13 showed early degradation in HPLC, it still showed better to OsD14 ,B. Our cto OsD14 ,B.Striga germination-inducing activity in pots under greenhouse conditions. This test showed quite promising results. The application of formulated MP13 and MP26 significantly alleviated Striga emergence. The effect of these compounds was much higher than that of AR8, comparable with GR24 (Striga infestation, compared with the corresponding control (−1. This amount is significantly less than the amounts used to apply the SL analog T-010 as suicidal germination agent (To get information on the application potential of MP13 and MP26, we first tested their ith GR24 –C. The i control ,E. This  control . It shouon agent . It can Striga in infested regions of sub-Saharan Africa.Our findings show that the presence of methyl group at the C-3′ in D-ring has a negative impact on SL analogs’ activity and provides structural explanation for this effect. The developed MP13 and MP26 are promising SL analogs that can be applied to regulate crop architecture. MP13 has large potential for application in combating the root parasitic weed in silico analysis. MJ, SA, TA, SA-B, and others wrote the manuscript and respective parts. All authors read, edited, and approved the final manuscript.SA-B and MJ conceived and designed the experiments. BK, IH, JW, FA, RZ, K-PJ, DY, IT, TO, and TA performed the experiments. UH and SA did the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Context: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities.Objective: The effects of fucoidan on Ca2+ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined.Materials and methods: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca2+ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5 mg/mL or 1.5 mg/mL was applied for 3 min to determine its effects on Ca2+ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5 mg/mL for 3 h.Results: The Ca2+ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca2+ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca2+ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca2+ channels, PR1/PR2.Discussion and conclusions: Our data indicate that fucoidan suppresses the intracellular Ca2+ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca2+ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future. Fucoidan could protect rat cholinergic basal forebrain neurons against β-amyloid-induced death in vitro  disorders , compound 48/80 and acetylcholine. Histamine and compound 48/80 are the agonists only for stimulating G-protein-coupled receptors to induce the Ca2+ responses  including both ionotropic type (iGluRs) and metabotropic type (mGluRs)  and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the presence of extracellular Ca2+ and mGluRs agonists such as quisqualate and -1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) in the absence of extracellular Ca2+ were used. Glutamate, an agonist for both iGluRs and mGluRs, was also used in the presence or absence of extracellular Ca2+. In contrast to our expectation, fucoidan showed a very selective inhibition of only the NMDA receptor (NMDAR) (one of iGluRs) but not other types of receptors examined.In the present study, we investigated the effects of fucoidan mainly on glutamate receptors in cortical and hippocampal neurons. In an attempt to induce the CaFucus vesiculosus Linn. (Fucaceae)), poly-d-lysine, cytosine β-d-arabinofuranoside (cytosine arabinoside), ACPD, Bay K8644, AMPA and NMDA were obtained from Sigma-Aldrich .Fura-2 acetoxymethyl ester (Fura-2/AM) was purchased from Dojindo Laboratories . Horse serum was from Life Technology . B27 supplement was purchased from Invitrogen . Quisqualic acid  was from Wako Pure Chemical Industries, Ltd. . Fucoidan (purified from d-lysine coated glass-bottomed dishes at a density of about 2 × 105 cells per dish. The cell suspension (around 200 µL) in the dishes was incubated in a humidified incubator with 5% CO2 for 20 min at 37 °C to allow cell adhesion, and then, additional neuron medium (2 mL) was supplemented into the dishes. On day 1, 2 and 6, 500 µL of medium was replaced with 700 µL of fresh neuron medium. The neuron medium was a mixture of Neurobasal A  containing: 10% horse serum; 2 mM l-glutamine ; 10 µg/mL gentamycin ; and 2% B27 supplement. From day 2, 1 µM of cytosine arabinoside was added into neuron medium. The cells cultured for 10–14 days were used for following experiments.The animal procedures were approved by the institutional review committee and were kept in accordance with the Guide for the Care and Use of Laboratory Animals at Hamamatsu University School of Medicine. Wistar rats in postnatal day 1 purchased from a local animal centre  were anaesthetized with ether, and then the cortex or hippocampus was dissected from their brain. The tissue was cut into small pieces with a pair of scissors, and then the pieces were dispersed into a cell suspension in the neuron medium using plastic pipette. The density and viability of the isolated cells was determined using a hematocytometer and trypan blue staining, respectively. These cells were seeded into 35-mm poly-2+]i) was measured using procedures similar to those we used for HeLa cells  for 20 min at 37 °C in the dark and then rinsed twice with artificial cerebro-spinal fluid (aCSF) for calcium imaging. The aCSF contained (in mM): NaCl, 140; KCl, 5; CaCl2, 2; MgCl2, 1.2; glucose, 10; and HEPES (pH 7.2) 10. Fura-2/AM loaded cells were placed on the stage of an inverted microscope  and were illuminated at wavelengths of 340 and 380 nm every 3 s alternately. An intensified charge-coupled device (CCD) camera  was used to capture the calcium images at 510 nm with a 40× objective lens . In the control, the agonists, such as glutamine, NMDA, and so on, were applied for 3 min after recording the baseline for 3 min. In the test, fucoidan was added into the aCSF during the same baseline period (3 min), and then without removal of fucoidan, the agonists were further added into aCSF. The changes in [Ca2+]i in individual cells were determined by the 340/380 nm fluorescence ratio with an image-analysis software . A calcium-free aCSF was also used, in which case, CaCl2 was removed and ethyleneglycol tetraacetic acid (EGTA) was added at 1 mM (pH 7.4).The intracellular calcium concentration  . RT-PCR was performed using Real Time PCR EasyTM-SYBR Green I Kit  according to the manufacturer's instructions. The conditions consisted of an initial denaturation step at 95 °C for 2 min, followed by 40 cycles of 30 s denaturation steps at 95 °C, 30 s annealing step at 53.5 °C to 56.5 °C, and 1 min extension step at 72 °C. A final extension step of 5 min at 72 °C was also performed. PCR amplification products were analysed by electrophoresis on a 1% agarose gel. All PCR results were derived with cycle number producing a signal in the linear portion of the amplification curve. The primers were listed as follows  was used to measure the mRNA expressions of iGluRs and mGluRs. Cortical and hippocampal neurons were serum-starved for 24 h and treated with 0.5 mg/mL fucoidan for 3 h. Total RNA was resolved in 10 μL of diethylpyrocarbonate-treated water, and 1 μg of each RNA samples was reverse-transcribed into cDNA by RT Easy follows . The β-a2+ responses, 10–20 cells were analysed in each observation field. All observations were repeated using 3–5 culture dishes. For statistical analyses, software implemented in Microsoft Excel 2010 was used. The integrated amplitude of the Ca2+ response was calculated by using an extended baseline for subtraction. The results were expressed as mean ± SEM.For the quantification of Ca2+ responses were examined by time-lapse imaging with Fura-2/AM (2.5 µM). The cells were preincubated with 0.5 and 1.5 mg/mL fucoidan and then stimulated with glutamate (50 µM). In the presence of extracellular Ca2+, glutamate exposure largely increased the [Ca2+]i in cortical neurons  and G-protein-coupled receptors (mGluRs)  in the presence of extracellular 0.4 mM Mg2+ and 2 mM Ca2+ , a nonspecific agonist activating AMPA receptor (AMPAR) and ACPD receptor , treatment of glutamate has no significant influence on [Ca2+]i in cortical neurons within the concentration range of 50–1000 µM (data not shown). Interestingly, glutamate (50 µM) could induce the Ca2+ responses in hippocampal neurons in the absence of extracellular Ca2+ , but it did in the hippocampal neurons   .l-type Ca2+ channels. In the presence of this agonist, Ca2+ channels tend to open for longer periods causing an increase in [Ca2+]i. Administration of Bay K8644 (20 µM) induced rapid Ca2+ responses in cortical and hippocampal neurons (2+ responses were inhibited greatly by fucoidan at a concentration of 0.5 mg/mL (2+ response (data not shown).Bay K8644 is an opener of all  neurons . The Ca2.5 mg/mL . Moreove2+ responses in neurons. This is inconsistent to our previous study which indicated that fucoidan mainly worked on G-protein-coupled receptors in HeLa cells. We wanted to confirm more about the effects of fucoidan on different G-protein-coupled receptors, for example, adrenergic receptors, expressed in hippocampal neurons. Adrenergic receptors are members of the G-protein-coupled receptor superfamily, and their activation causes an increase in inositol 1,4,5-trisphosphate (IP3) and [Ca2+]i. In hippocampal neurons, adrenaline (100 µM) treatment could apparently induce the Ca2+ responses in the both presence and absence of extracellular Ca2+ , we measured the mRNA expressions of iGluRs , PR1/PR2 and mGluRs  using RT-PCR. Results showed that after 3 h of exposure to fucoidan (0.5 mg/mL), no apparent change of the mRNA expressions of GluR2 and GluR6 was observed in cortical and hippocampal neurons   was applied, which fucoidan failed to inhibit. This indicates that the bulk of the glutamate-induced Ca2+ transients in cortical neurons was mediated by simultaneous activation of the NMDARs and Ca2+ permeable AMPA/KARs, and also that about 50% reduction of Ca2+ responses by fucoidan was ascribed to the inhibition of iGluRs of NMDARs but not of AMPARs in primary cultured neurons. The mGluRs are present on the cell membrane of hippocampal neurons, and they are insensitive to fucoidan. At the same time, Bay K8644 induced the Ca2+ responses in the presence of extracellular Ca2+, and the Ca2+ responses are suppressed by fucoidan by more than 90%. Similarly to NMDARs, l-type Ca2+ channels themselves are sensitive to fucoidan.In the current study, our data demonstrated that fucoidan attenuated the intracellular Cators see . Glutama2+ permeability of receptors. In fact, an asparagine residue occupies the place in NMDAR which is equivalent to the glutamate-arginine editing site in AMPA/kinate receptors , a transmembrane domain, and an intracellular C-terminal domain , which is a heterogeneous mixture of more than 15 different fucans with varied proportions of individual monosaccharide (Nishino et al. So far, the molecular weight of fucoidan is known not as a single value but rather a broad distribution around an average. In the present study, we used commercially available fucoidan purified from 2+ responses of neurons by selectively inhibiting NMDARs in cortical neurons and l-type Ca2+ channels in hippocampal neurons. However, further studies are needed to determine how fucoidan regulates the specific receptors. Since fucoidan alone influences the mRNA expression in the neurons, fucoidan is proven to bind these cells probably at specific receptor sites. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.The present study shows that fucoidan suppresses the intracellular Ca