FLT3 (FLT3-ITD) is a common mutation for which two inhibitors (midostaurin and gilteritinib) with varied potency and specificity for FLT3 are clinically approved. However, the high rate of relapse or failed initial response of AML patients suggests that the addition of a second targeted therapy may be necessary to improve efficacy. Using an unbiased large-scale CRISPR screen, we genetically identified BCL2 knockout as having synergistic effects with an approved FLT3 inhibitor. Here, we provide supportive studies that validate the therapeutic potential of the combination of FLT3 inhibitors with venetoclax in vitro and in vivo against multiple models of FLT3-ITD-driven AML. Our unbiased approach provides genetic validation for co-targeting FLT3 and BCL2 and repurposes CRISPR screening data, utilizing the genome-wide scope toward mechanistic understanding.Acute myeloid leukemia (AML) is a heterogeneous and complex disease, and treatments for this disease have not been curative for the majority of patients. In younger patients, internal tandem duplication of  FMS-like tyrosine kinase 3 (FLT3) gene are among the most common abnormalities seen in younger adult AML and are present in approximately 30% of patients; it leads to constitutive FLT3 kinase activity and downstream activation of signal transducer and activator of transcription 5 (STAT5) [FLT3-ITD mutations at a high ITD/wild-type ratio has prognostic value both as a risk factor for relapse and poor survival in AML patients [Acute myeloid leukemia (AML) is a molecularly complex disease due to the presence of multiple genetic abnormalities that influence prognosis and therapy outcome . Several STAT5) , 3. The patients . FLT3-ta , 3. Thepatients . Despitepatients , 10. ThiB-cell lymphoma 2 (BCL2) knockout [FLT3-ITD-driven AML beyond the previously studied MV4-11 xenograft model. The high translatability of this combination therapy stands to make a rapid clinical impact for AML patients, especially considering the high relapse rate associated with FLT3 inhibitors. Our approach also illustrates that CRISPR screening is a noteworthy method for identifying rational combination strategies for genetic subsets of AML.We have recently published an unbiased large-scale CRISPR  screen in which we focused on a predicted novel combination strategy of the FLT3 inhibitor midostaurin with the XPO1 (Exportin 1) inhibitor selinexor; however, our data also predicted synergy between midostaurin and genetic knockout   of BCL2 expression with CRISPR ribonucleoprotein in MOLM-13 using two different guides and confirmed reduced protein expression via immunoblot analysis  or BCL2 KD alone (p < 0.001). The combination of BCL2 KD with gilteritinib demonstrated a similar effect, where BCL2 knockdown and gilteritinib in combination significantly decreased viability compared to gilteritinib alone (p < 0.001) or BCL2 KD alone  AML cell lines , while we examined all anti-proliferative effects following a longer duration of treatment (48 h). As shown in Fig. FLT3-ITD responded more robustly to the combination of inhibitors than to either alone  [L), and AMG 176 (MCL1) [L specific) with quizartinib [FLT3-WT using quizartinib, highlighting that there are likely differences in how these two drugs elicit their effects. We sought to further explore how different BCL2 family members act under FLT3 inhibition using our genome-wide CRISPR data.One way AML cells evade apoptosis is by up-regulation of pro-survival members of the BCL2 protein family, including BCL2, MCL1, and B-cell lymphoma-extra large (BCL-XL) . These pL) . Therefo6 (MCL1) . Their rzartinib . Unlike L may also enhance the anti-tumor effect of FLT3 inhibition, though not to the same degree as with BCL2. Interestingly, we were also able to examine what happens when BAX or BAK is knocked out in the presence of FLT3 inhibition and found a pattern of a slight increase in sgRNA levels; this in effect acts like a genetic surrogate for sequestration of these proteins by BCL2 family members and supports the idea that decreasing their efficacy enhances the pro-survival aspect of AML blasts.Data from the midostaurin CRISPR screen show how single guide ribonucleic acid (sgRNA) levels change for each of the BCL2 family members, as well as for BAX and BAK while being treated with midostaurin   of gilteritinib and venetoclax in female immunocompromised triple transgenic NSGS non-obese diabetic/SCID gamma 1Eav/MloySzJ) mice engrafted with MV4-11 cells. We sought to further these studies for clinical translatability with two separate AML mouse models, including a xenotransplantation model and an adoptive transfer of spontaneous murine AML.Following completion of elegant mechanistic studies, Ma et al. concluded with a xenograft mouse study  or 41 days for mice given midostaurin only   mice were transplanted with MOLM-13 luciferase cells and divided into vehicle, venetoclax, gilteritinib, and combination arms , we also expanded our studies to include the in vivo combination of midostaurin and venetoclax , 19. BriFLT3-ITD and TET2 knockout [n = 8–17 mice/group). Such spontaneous models offer an alternative important way to model therapeutic response independent of cell lines conditioned to grow in vitro. Mice were block randomized into six treatment arms upon engraftment: (1) vehicle, (2) midostaurin, (3) venetoclax, (4) midostaurin/venetoclax, (5) gilteritinib, and (6) gilteritinib/venetoclax (Fig. Next, we tested similar drug combinations using an adoptive transfer model, engrafting cells from two donor mice that spontaneously develop AML due to knockout  into NCGetoclax,  midostauBecause NCG mice exclusively express CD45.1, whereas donor mice express only CD45.2, changes in disease burden were analyzed bimonthly to monitor percent CD45.2 in the peripheral blood of recipients. Over an 8-week treatment course, both vehicle and venetoclax-only mice reached nearly 100% CD45.2 in peripheral blood. Treatment with either midostaurin or gilteritinib slowed disease progression leading to an average disease burden of about 70% CD45.2. After 2–4 weeks of treatment, average percent CD45.2 began to decrease in both combination groups and by 8 weeks approached 20% in midostaurin/venetoclax and 40% in gilteritinib/venetoclax (Fig. After 8 weeks of treatment, four mice from midostaurin monotherapy, venetoclax monotherapy, midostaurin/venetoclax, and vehicle groups were killed for interim analysis. We found no evidence of drug-associated toxicologic lesions in any tissues examined for midostaurin or midostaurin/venetoclax therapy (Additional file FLT3-ITD cell lines and primary AML patient samples beyond that of each perturbation alone, an effect which was not observed to the same extent in the case of FLT3-WT. Furthermore, we demonstrated that combination therapy with venetoclax plus midostaurin or gilteritinib can improve survival in xenograft and adoptive transfer murine AML models, examining disease progression, recovered bone marrow cells, and survival. The work presented here supports further clinical development of the combinations of midostaurin and venetoclax as well as gilteritinib and venetoclax and specifically highlights the reproducibility of the synergistic effect across multiple disease-replicating contexts (Additional file Using multiple, orthogonal approaches, we validated the synergistic relationship between the effects of BCL2 and FLT3 inhibition that was predicted by our previous work  and expaAdditional file 1. Additional figures.Additional file 2. Methods.


Mitochondrial Ca2+-targeted fluorescent probes have demonstrated that mitochondria Ca2+ transients are synchronized with Ca2+ fluxes occurring in the sarcoplasmic reticulum (SR). The presence of specialized proteins tethering SR to mitochondria ensures the local Ca2+ flux between these organelles. Furthermore, communication between SR and mitochondria impacts their functionality in a bidirectional manner. Mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniplex is essential for ATP production and controlled reactive oxygen species levels for proper cellular signaling. Conversely, mitochondrial ATP ensures the proper functioning of SR Ca2+-handling proteins, which ensures that mitochondria receive an adequate supply of Ca2+. Recent evidence suggests that altered SR Ca2+ proteins, such as ryanodine receptors and the sarco/endoplasmic reticulum Ca2+ ATPase pump, play an important role in maintaining proper cardiac membrane excitability, which may be initiated and potentiated when mitochondria are dysfunctional. This recognized mitochondrial role offers the opportunity to develop new therapeutic approaches aimed at preventing cardiac arrhythmias in cardiac disease.Ca Arrhythmias can be defined as any disturbance in the normal electrical sequence of the heart. These disturbances may cause the electrical impulse to travel slowly, rapidly, or in an erratic manner. However, few studies have calculated the overall burden of arrhythmias. Incidence has been reported to be about 2.35% in the United Kingdom’s general population . In Mexi2+ homeostasis. Both increased Ca2+ overload conditions and Ryanodine receptor (RyR2) dysfunction (leakiness) promote spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR) and activate the Na+/Ca2+ exchanger (NCX) in its forward mode. In this operating mode, a net inward Na+ current (Iti) is present, which depolarizes the membrane and causes DADs. If these DADs are large enough, they can reach the threshold activation of the Na+ current and generate a full arrhythmogenic AP. Both EADs and DADs are known as trigged activity and play an important role in initiating cardiac arrhythmias. There is compelling evidence that mitochondria play an important role in the generation of Ca2+-triggered arrhythmia, a process that requires an understanding of cellular Ca2+ fluxes.Focal activity and re-entry are proposed as the main mechanisms of cardiac arrhythmia. Re-entry is associated with conduction abnormalities and occurs when a propagating impulse fails to extinguish after normal activation of the heart tissue and re-excites the heart after completion of the refractory period. A reduction in the cardiac impulse’s wavelength, which is a product of the conduction time and refractory period , is a de2+ into the cytosol. RyR2 opening occurs after a small initial amount of Ca2+ passes through L-type Ca2+ channels (LTCCs) in the sarcolemma in response to membrane depolarization during the AP . Ca2+ is extruded into the extracellular space by NCX activation, which counterbalances the entry of Ca2+ through LTCCs. Only a discreet quantity of cytosolic Ca2+ is removed by mitochondria  known as mitochondrial Ca2+ uniplex (mCU) . Mitochondrial Ca2+ extrusion is carried out by the mitochondrial NCX, which possesses slow kinetics compared to the mCU, allowing the accumulation of this ion in the mitochondria. The local SR-Mitochondria Ca2+ flux is facilitated by anchoring proteins that anchor both organelles  production and energetics highlights the importance of coordinated regulation between SR Ca2+ fluxes and mitochondrial homeostasis. In this review, we focus on describing the cross signals that occur between these two organelles and how dysregulation of this intertwined signaling may be involved in altered cardiac rhythms.The SR is the organelle in charge of storing and releasing Cag the AP . The sudo the SR  by the schondria , which oganelles . The dep2+ channel inositol-3-phosphate receptor (IP3R), joined to the mitochondrial voltage-dependent anion channel (VDAC) through protein GRP75  pathway. Similarly, IP3R2 has been described as binding with the FUN14 domain containing 1 (FUNDC1) to modulate SR Ca2+ release , as opposed to the combined pumping force of SERCA in the SR and the sarcolemmal NCX. While it has been calculated that the percentage of cellular Ca2+ taken up by mitochondria is modest  for the electron transport chain (ETC) to use as substrates and generate the mitochondrial membrane potential (ΔΨm) and subsequent ATP synthesis under energy-demanding states, such as when adrenergic stimulation takes place  increased IKATP and shortened the AP and refractory period, leading to re-entry into myocyte monolayers. The proarrhythmogenic effects of FCCP are attenuated by the IKATP inhibitor glibenclamide . In an isolated rabbit heart, FCCP perfusion caused a higher incidence of APD and Ca2+ transient alternans, increased interventricular heterogeneity, impaired cardiac conduction velocity, and promoted ventricular arrhythmias in optical mapping studies. In this study, APD and Ca2+ alternans were also induced under ischemic conditions in rabbit heart preparation, validating the clinical relevance of blunted mitochondria energetics in cardiac arrhythmogenesis (–/–) mice to I/R injury produced twofold larger infarcts, a high propensity for ventricular tachycardia, and excessive ROS generation when compared to WT mice. Pretreatment with FCCP prior to I/R injury relieved cardiac stress in UCP3–/– hearts and improved ventricular functioning , 6–8 h of tachypacing caused dissipation of the ΔΨm and mitochondrial network, as well as the loss of mitochondrial Ca2+ transients. Interestingly, treatment with SS31, a compound that accelerates the formation of ATP from ADP, protected against mitochondria remodeling, impaired respiration, and Ca2+ homeostasis . This channel is activated by cytoplasmic Ca2+   . The clirhythmia . In line2+ by the SR. This pump is also subjected to oxidative regulation. Cys674 is identified as a critical target for oxidative modification, including oxidation  dysregulation is involved in the initiation of AF and a study demonstrated that the reduction of the atrial arrhythmia burden in patients undergoing isolation of PVs correlated with significantly lower plasma levels of oxidative markers (advanced glycation end-products and thiobarbituric-acid reacting substances) 9 months after the intervention . From th2+ dependent processes including apoptosis and energetics by directly affecting mitochondrial components or their spatial relationship to the SR. For instance, the pharmacological inhibition of mPTP is reduced in hearts from senescent rats compared to younger animals when subjected to ischemia/reperfusion injury H/NAD(P+) ratio in response to electrical pacing. Interestingly, immunolabelling of permeabilized myocytes with anti-RyR and anti-VDAC revealed an age-dependent reduction of the fraction of the RyR2 overlapping with VDAC, whereas quantitative redox proteomics showed augmented oxidation of VDAC proteins. Thus, oxidation of mitochondrial membrane components may contribute to the disruption of the spatial proximity between mitochondria and the SR, which may cause defective Ca2+-dependent mitochondrial energetics  and having a much greater affinity for the mCU, with a Kd of about 0.34 nM. Doses of up to 10 μM had no effect on SERCA, the RyR2, NCX, LTCCs, or myofibrils  injury (360 given before ischemia significantly prevented the occurrence of ventricular fibrillation (VF) and hemodynamic dysfunction. Ru360 administration was associated with protecting mitochondria from depolarization by mPTP opening and thus preserved mitochondrial functioning  injury . In an octioning . Ru360 in curves .360 has been used to research the effects of inhibiting mitochondrial Ca2+ transport on various models of cardiac injury. It has already proved its efficacy in preventing EADs in ventricular myocytes in a non-ischemic HF model   mCU expression using siRNA . TransgeWT group . Combinihe heart . At the ctioning . Notably A (CsA) . N+/H+ eSince increased mitochondrial ROS generation seems to be the initial insult that starts the positive feedback loop, antioxidants could be useful in boosting the myocardium’s ROS defense pool. Since their first trials in animal models, ROS scavengers have had beneficial effects in preventing arrhythmias during I/R. Superoxide dismutase, glutathione, and ascorbic acid significantly reduced reperfusion arrhythmias in isolated rat hearts . N-acetyK1 would predict AP prolongation and the facilitation of EADs. Recently, it has been described that ATF6, an ER transcription factor, is responsible for inducing antioxidative stress programs, such as catalase and ROS scavenging selenoproteins, in a setting with a high level of accumulation of misfolded proteins, such as in I/R  is yet another site where redox signaling is integrated, it is a potential ROS control organelle. Selenium (Se) is essential for the function of at least 25 selenoproteins, including the antioxidant glutathione peroxidase (GPx). In patients undergoing primary coronary artery bypass grafts, preoperative reduced levels of Se, GPx, and malondialdehyde correlated with high incidence of postoperative AF . Moreoves in I/R . Selenops in I/R  and treaS levels . HoweverS levels . Thus, iin vitro models, such as I/R , which restores thiol groups in oxidized proteins to their reduced state, somewhat inhibiting ROS effects. Treatment with DTT reversed Caoduction , suggesthycardia .It was previously thought that antioxidants like quercetin and resveratrol, due to their low bioavailability and non-specificity to any tissue, would make them poor candidates for new therapies . This is2+ leakage, Ca2+ waves, and ROS production, as evidenced by fewer free thiols in the RyR2. These effects were partially prevented using MitoTEMPO or other ROS scavengers , the VDAC, ANT, the phosphate carrier, ATP synthase, and the translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor . A causamponents . Using Texposure . TSPO betivation . These otivation , making tivation . Protecttivation . Hearts recovery . The prorecovery . Notably2+-induced damage  function parameters  is an enzyme normally activated by Ca2+-calmodulin and phosphorylates a wide range of proteins, including RyR2, PLB, and LTCCs. Increased CAMKII activity has been observed in pathological processes, such as cardiac hypertrophy and HF , in which KN-93 administration reduced arrhythmia susceptibility, as well as improved ventricular cardiomyocyte contractile functioning  containing genetic material that coded for SER-CA2a. These patients presented with improvements in symptomatic, functional, biomarker, and LV functional parameters, along with no significant adverse effects in other organs , and more frequent and greater Ca2+ sparks were noted, although the decay times were similar , decreased ATP production, and mPTP opening. Furthermore, increased ROS oxidates the mCU, along with possible phosphorylation from AMPK, secondary to low ATP synthesis, which further increases Ca2+ transport into the matrix, creating a positive feedback loop for more ROS production. ROS can also affect nearby structures, such as SERCA, which decreases its effectiveness in pumping Ca2+ back into the SR. This translates into reduced SR content and higher cytoplasmic Ca2+ concentrations, reducing contractility. Ca2+ can then activate CAMKII, which can also be activated by mitochondrial ROS. CAMKII can then autophosphorylate itself to stay in a permanent active form. RyR2 is activated by CAMKII phosphorylation and ROS, which causes its regulator protein, calstabin, to detach from the RyR2 complex. This increased sensibilization promotes stochastic opening with subsequent Ca2+ leaks and Ca2+ waves, further increasing mitochondrial Ca2+ transport. On the sarcolemma, the oxidation of LTCCs increase the amount of Ca2+ that enters the cardiomyocyte upon activation. A reduction in the ATP concentration caused by mitochondrial dysfunction opens sarcKATP channels, reducing the cardiomyocyte’s bathmotropism and dromotropism. The constantly high Ca2+ concentration also activates NCX, slowly depolarizing the cell and promoting unsolicited depolarizations and APs. Finally, connexin 43 translocation or Panx1 opening due to increased Ca2+ and ROS translocates ATP and adenine into the extracellular matrix, where they activate signaling pathways for TGF-β1 expression, with subsequent fibrosis. This represents two pathways to cardiac tissue fibrosis: cell death-dependent and TGF-β1-dependent. Both have the same outcomes, creating patches of slow conduction and promoting reentry. All these effects make a suitable environment for conduction dysfunction and arrhythmia generation. Up until now, classic antiarrhythmic drugs’ main mechanism known to prevent arrhythmias does not include the mitochondrial-SR interconnection. However, they have effects on either of these organelles  for the Doctorate in Biomedical Sciences. Tecnologico de Monterrey. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: (1) BKca channels (Paxillin, 10 Systemic arterial hypertension is the most common preventable risk factor for cardiovascular disease (CVD)  and is aThe phenolic compounds derive from the secondary metabolism of plants and are chemically characterized by having one or more aromatic rings with one or more hydroxyl groups. The amounts of the phenolic components (150–700 mg/kg) in EVOO depend on many different factors including the cultivars, the degree of maturation, the climate, the production method and the storage . DiffereSome studies have associated the anti-hypertensive effect of EVOO to its acidic component, especially oleic acid ,16; howeca) channels in the vascular smooth muscle cells (VSMCs), but does not involve signaling pathways mediated by nitric oxide (NO)−, prostanoids, or the cyclic nucleotides cAMP and cGMP [ca channels.Previously, we showed that EVOO phenols exert a vasodilatory effect on rat resistance mesenteric artery (MA) , which mand cGMP . The prev/v) was reported as an efficient extraction solvent [The phenolic extract used in this work is derived from crude EVOO. The liquid/liquid partitioning method was used with methanol/water (80:20) . The use solvent  and it i solvent . Oleacein = 16) were used at 18–23 weeks of age. Rats were housed at the University of Calabria under controlled conditions on a 12 h light/dark cycle; commercial chow and tap water were provided ad libitum. All procedures were conducted in accordance with the European Guidelines for the care and use of laboratory animals (Directive 2010/63/EU) and approved by the Italian Institutional Animal Care (n.295/2016-PR). Animals were euthanized with isoflurane and killed by cervical transection; the abdominal cavity was opened immediately, and a section of the mesentery was excised 5 cm distal to the pylorus. The mesenteric tissue was placed in a Sylgard-lined Petri dish containing cold (4 °C) HEPES physiological saline solution (PSS) at pH = 7.4.Male Sprague-Dawley rats , superfused with HEPES-PSS at 37 °C and pressurized using a pressure-servo system . Intraluminal diameter was measured using a video dimension analyzer (Living Systems Instrumentation) and recorded on LabView software.+-rich (60 × 10−3 M) HEPES-PSS, and Acetylcholine (10 μM), to test artery viability and endothelium integrity, respectively. Vessels that did not elicit reproducible responses to either potassium or acetylcholine were excluded from the study. MA were then equilibrated for 30 min in HEPES-PSS at 37 °C and pre-constricted with phenylephrine to produce a 40–60% reduction in lumen diameter [2+ and containing the phosphodiesterase inhibitor, papaverine (10−4 M), to induce maximal vasodilation. To investigate molecular mechanism(s) underlying the EVOO phenol-induced vasodilation, a dose-response curve for phenols (0.1 ÷ 10 μM) was tested in arterial segments pre-incubated for 30 min with one of the following inhibitors, prior to exposure to phenylephrine: (1) Paxillin (10 μM) [ca; (2) Verapamil (10 μM) [3 μM) [αi of G-protein-coupled receptors (GPCR).All arteries were pressurized at an intraluminal pressure of 50 mmHg (as it approximates in vivo conditions) and exposed to Kdiameter . Increas (10 μM) , a selec (10 μM) , a L-typ (10 μM) , an inhi) [3 μM) , an inhi) [3 μM) , an inhi) [3 μM) , a blockThe physiological salt solution HEPES-PSS contained the following (in mmol/L): sodium chloride 141.8, potassium chloride 4.7, magnesium sulfate 1.7, calcium chloride 2.8, potassium phosphate 1.2, HEPES 10.0, EDTA 0.5, and dextrose 5.0. The solution was prepared in deionized water and titrated with sodium hydroxide (10 M) to a physiologic pH of 7.4.+-rich (60 × 10−3 M) HEPES-PSS has the same composition as the standard solution, HEPES-PSS, except that NaCl was replaced by an equimolar concentration of KCl.KChemicals were purchased from Sigma-Aldrich , Santa-Cruz , Cayman Chemical Co . unless otherwise specified. All drugs tested were administered from stock solutions prepared daily, except for EVOO phenols stock solutions that were frozen in small aliquots.n is both the number of arteries studied and animals used. The vasodilatory effects of EVOO phenols are expressed as percentage of maximal diameter, which was determined in the presence of the relaxing solution. The vasorelaxing efficacy was evaluated as the maximal vasodilatory response (Emax); while the potency was expressed as EC50, calculated as the dilator concentration that caused 50% of the maximum response. Furthermore, we compared the area under the dose-response curve (AUC) at the different experimental conditions. Statistical analysis was performed with paired Student’s t-test. p < 0.05 was considered significant.Data are expressed as means ± Standard Error Media (SEM), where p < 0.001) in a dose-dependent manner, with an Emax of 97.74 ± 0.63% and a EC50 of 0.6 ± 0.04 μM. Vehicle condition for EVOO phenols (Ethanol) was assessed and did not show any effect.We tested the effect of EVOO phenols (0.1–10 μM) on MA pre-constricted with phenylephrine. As shown in ca channnels, Paxillin (10 μM) p < 0.01), and reducing of Emax . Interestingly, Paxillin completely abolished EVOO phenol-mediated vasodilation in the dose range of 0.1 μM ÷ 2 μM.To determine the mechanisms by which EVOO phenols induced vasodilation, a dose-response curve to EVOO phenols was obtained in the presence of the specific inhibitor of BK2+ influx, MA were pre-incubated with the L-type calcium channel blocker, Verapamil (10 μM) 2+ influx, Verapamil induced a rightward shift of the dose-response curve of EVOO phenols, with a significant increase of EC50 , and a significant attenuation of Emax .To establish whether EVOO phenol-mediated vasodilation was associated with extracellular Ca2+ from intracellular stores was also involved in the vasodilation, a set of experiments was performed using two different blockers of intracellular Ca2+ release: Ryanodine (10 μM), a potent inhibitor of RyR, and 2-APB (3 × 103 μM) an inhibitor of IP3R. Ryanodine decreased Emax , and caused a significant increase of EC50 , p > 0.05), but significantly potentiated EC50 , To assess whether the release of Cap < 0.001) and increased EC50 , Furthermore, to confirm the contribution of PLC-initiated in the vasodilatory effect of EVOO phenols, a dose-response curve to EVOO phenols was tested in the presence of the PLC inhibitor U73122 (10 μM), which reduced Emax , significantly decreased Emax , We investigated also whether activation of GPCR is involved in the EVOO phenol-induced vasodilation. Our data showed that inhibition of the subunit GCa channels through an increase of local intracellular Ca2+ level (Ca2+ microdomains) as result of influx across the plasma membrane (extracellular Ca2+) and release from sarcoplasmic reticulum (SR) Ca2+ store. Furthermore, we showed that EVOO phenols activated PLC, which plays a significant role in the regulation of the Ca2+ microdomains.Our study shows that EVOO phenols dilate resistance MA in a dose-dependent manner. The vasodilation is induced by activation of BKCa channels is involved in the EVOO phenol-induced vasodilation of MA. In addition, here we demonstrated that both the release of intracellular Ca2+ from the SR by IP3R and RyR and extracellular Ca2+ influx through the L-type Ca2+ channels are implicated in the EVOO phenols vasodilation. Taken together, our results suggest that EVOO phenols activate BKCa channels potentially through an increase in intracellular localized Ca2+ transients. In agreement with other studies, our data suggest that EVOO phenols may lead to the increase of local Ca2+ microdomains which, in turn, are responsible for the vasodilatory effect on mesenteric resistance arteries. In muscle cells, the release of Ca2+, in the form of Ca2+ sparks, induced by RyR in the sub-plasma membrane of the SR, controls the activity of the nearby BKCa channels in many resistance arteries [2+ influx through the L-type Ca2+ channels has been shown to activate BKCa channels in different types of vessels, including hamster and mouse cremaster arterioles [2+ influx through L-type channels and RyR-based Ca2+ sparks, contribute to activation of BKCa channels [2+ influx through L-type Ca2+ channels can directly activate BKCa channels. Furthermore, it has been demonstrated that, in vascular smooth muscle cells, BKCa and L-type Ca2+ channels can directly interact forming complexes that are accumulated in caveolae by the interaction of caveolin-1, thereby regulating spatiotemporal Ca2+ dynamics including the negative feedback, to control the arterial excitability and contractility [Our results confirm the vasodilation that we showed in our previous study in the same type of artery . A similarteries ,32,33,34terioles ,36, rat terioles  and mousterioles . In strichannels ,36. Studchannels  have shoactility .2+ channels IP3R. Our results showed that EVOO phenols-PLC activation occurred through the subunit Gαi of GPCR since its inhibition by the specific inhibitor pertussin toxin decreased EVOO phenols vasodilation. This can be explained by the fact that Gαi activation inhibits cAMP-PKA signaling which exerts its inhibitory effects by phosphorylation of both PLC and IP3R, thus limiting the Ca2+ mobilization [Furthermore, we demonstrated that EVOO phenols indirectly activated PLC which plays a crucial role in the generation of inositol triphosphate (IP3) a specific agonist of the abundant SR Calization .The involvement of the individual factors PLC, IP3R, RyR and G protein has been reported also in other studies of vasodilation mechanism ,43,44,452+ microdomains-BKCa signal.In conclusion, the present study contributes to understand the molecular mechanisms of the EVOO phenol-induced vasodilation of MA as summarized in


Mag-Fluo-4, loaded into the endoplasmic reticulum (ER) by incubating cells with Mag-Fluo-4 AM, has been used to measure changes in free [Ca2+] within the ER, where the free [Ca2+] is estimated to be between 100 μM and 1 mM. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free [Ca2+] within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for Ca2+ measured in vitro (KDCa ∼22 μM). Using an antibody to quench the fluorescence of indicator that leaked from the ER, we established that the affinity of Mag-Fluo-4 within the ER is much lower (KDCa ∼1 mM) than that measured in vitro. We show that partially de-esterified Mag-Fluo-4 has reduced affinity for Ca2+, suggesting that incomplete de-esterification of Mag-Fluo-4 AM within the ER provides indicators with affinities for Ca2+ that are both appropriate for the ER lumen and capable of reporting a wide range of free [Ca2+].Synthetic Ca Both genetically-encoded Ca2+ indicators (GECI), derived from endogenous Ca2+ sensors , it can also be exploited to allow measurement of [Ca2+] within organelles, like the ER  in the ER lumen is thought to be between 100 μM and 1 mM [2+ indicators [2+] (KDCa < 1 μM). The affinities of Mag-Fura-2 (KDCa = 53 μM) , but Fluo-5 N can be effectively loaded into the ER only with the aid of ER-targeted esterases  in the ER with evident reliability [DCa of 22 μM [2+] thought to occur within the ER. An additional concern is that after Mag-Fluo-4 AM is de-esterified within the ER, some indicator leaks into the medium, possibly through organic anion transporters [2+] ([Ca2+]m) designed to mimic an unstimulated cell (< 200 nM) because such a small fraction (<1%) of the released indicator binds Ca2+, but the leakage becomes more problematic as [Ca2+]m approaches or exceeds the KDCa.Several methods, including targeted expression of esterases , have beand 1 mM , relativdicators , most hasterases . Mag-Flus ∼22 μM , has beendicator . Mag-Fluiability , it is pof 22 μM  should nDCa is ∼1 mM. We provide evidence that the much reduced affinity of the luminal indicator and its broader range of responsiveness (both useful for monitoring ER [Ca2+]) are due to incomplete de-esterification within the ER  within the ER lumen  of 220 nM after addition of 1.5 mM MgATP. We estimate the free [Mg2+] after addition of MgATP to be ∼300 μM, too low to appreciably affect the fluorescence of Mag-Fluo-4  binding because the signals were entirely dependent on addition of MgATP, abolished by inhibition of the ER Ca2+ pump (SERCA) by CPA  were attached to poly-L-lysine-coated 96-well black-walled plates, and fluorescence  was recorded at intervals of 1.44 s using a FlexStation III plate-reader . MgATP (1.5 mM) was added to initiate Ca2+ uptake, and when the ER had loaded to steady state with Ca2+ (∼150 s), ligands were added. The same methods were used for HEK-G-CEPIA1er cells. Where probenecid (2.5 mM) was used, it was present during dye-loading, permeabilization and fluorescence measurements.Mag-Fluo-4 was used to monitor free  and either anti-fluorescein/Oregon Green antibody  or control medium . Free [Ca2+] were estimated using MaxChelator  and supplemented with CPA (10 μM). The cells were distributed into 96-well plates and after 30 s, ionomycin (10 μM) was added to accelerate the equilibration of Ca2+ between the ER lumen and medium. QAb (1:20) was included in all analyses of Mag-Fluo-4. We confirmed that QAb had no effect on the responses recorded from G-CEPIA1er (not shown). Fluorescence was recorded using a FlexStation III  for 15 min at 37 °C. Variations in the durations of the incubations or concentrations of enzyme or Mag-Fluo-4 AM are reported in figure legends. At appropriate times, samples (5 μL) were removed and diluted into CLM-H containing various free [Ca2+] (50 μL) at 22 °C and fluorescence was determined immediately using a FlexStation III (Section Pilot reactions (150 μL) established optimal conditions for de-esterification of Mag-Fluo-4 AM by porcine liver carboxylesterase: 1 μM Mag-Fluo-4 AM and 1–1000 U/mL carboxylesterase in phosphate buffer  were determined. All statistical analyses used log values (pEC50 and pKD), although for greater clarity we also report EC50 and KD values. Student’s t-test or one-way repeated ANOVA with Bonferroni's multiple comparison test was used with P < 0.05 considered significant .For Ca33.13R1 cells with Mag-Fluo-4 trapped within the ER lumen  was 356 ± 52 s and the intercept at the time of CPA addition was 102.2 ± 0.9 % . These results, which concur with our published data [2+] across the entire range of ER Ca2+ contents. This is unexpected for an indicator with a reported KDCa of 22 μM [2+] of between 100 μM and 1 mM m used for calibration. 2+]m in supernatants derived from cells in which the ER had been loaded with Mag-Fluo-4 caused concentration-dependent increases in fluorescence. The KDCa of this leaked Mag-Fluo-4 was ∼38 μM . These values are similar to those we determined for Mag-Fluo-4 pentapotassium salt in vitro  and for cells in the medium (Fpermeabilized cells). Comparison of the fluorescence changes recorded from medium alone or from cells in medium shows that leaked indicator (Fmedium) responds to free [Ca2+] within the 1−300 μM range, while trapped indicator (the difference between Fpermeabilized cells and Fmedium) responds most above 300 μM  of that recovered from control cells. We therefore attempted to quench the fluorescence of leaked Mag-Fluo-4 using an anti-fluorescein/Oregon Green antibody (QAb) that has been shown to quench the fluorescence of Fluo-3 [2+ uptake and medium separated from these cells were pre-incubated with QAb before measuring fluorescence. QAb significantly reduced fluorescence from the leaked indicator (by 70–80 % for [Ca2+]m > 10 μM) (2+]m (permeabilized cells – Fmedium) (2+ uptake (3-evoked Ca2+ release (We were unable to reduce the contribution from leaked indicator by washing cells (not shown), suggesting rapid re-equilibration after removal of Mag-Fluo-4 from the medium. Probenecid (2.5 mM), which blocks organic anion transporters and is often used to prevent loss of indicator from cells ,33, was f Fluo-3 ,35. Perm> 10 μM) D, withouM) (2+]m E. QAb reFmedium) E and F. + uptake G or IP3- release H. The fo release G.2+]m. QAb also provides the tool needed to allow the KDCa of indicator trapped within the ER to be determined.We conclude that quenching of leaked Mag-Fluo-4 by QAb allows the trapped indicator to be used with permeabilized cells in medium with high m. Ionomycin (10 μM) was then added to accelerate equilibration of luminal [Ca2+] with [Ca2+]m, and, for Mag-Fluo-4 . The results indicate KDCa values of 1.15 mM for Mag-Fluo-4 and 1.55 mM for G-CEPIA1er, and h of 0.42 and 1.54, respectively . The values for G-CEPIA1er are comparable to published values  m. As expected, Mag-Fluo-4 AM was not fluorescent at any [Ca2+]m  was similar to the KDCa of Mag-Fluo-4 pentapotassium salt (16.2 μM) (DCa for Mag-Fluo-4 (22 μM) [2+]m and fluorescence (reported by h) changed with enzyme concentration (h was 0.97 ± 0.06 at the lowest enzyme concentration (1 U/mL), it decreased with increasing enzyme concentrations (10–100 U/mL) to a minimal value of 0.32 ± 0.01 (with 30 U/mL), and h then increased towards a maximal value of 1.40 ± 0.03 with further increases in enzyme concentration. The h value obtained with the greatest enzyme concentration (1000 U/mL) was similar to that observed for Mag-Fluo-4 pentapotassium salt (h = 1.20 ± 0.01)  C and the (22 μM) . The slontration D: h was  ± 0.01) D. IncubaDCa or h B–D, confesterase E-H.DCa of Mag-Fluo-4 measured in vitro (∼20 μM) (2+-sensitive form indistinguishable from Mag-Fluo-4 (2+ with reduced affinity. Incubation of Mag-Fluo-4 AM with the lowest concentration of carboxylesterase used (1 U/mL) produced a homogenous population of indicator (h = 0.97 ± 0.06) that bound Ca2+ with extremely low affinity (KDCa ∼ 93 mM). This species is not unmodified Mag-Fluo-AM since fluorescence of the latter is entirely insensitive to Ca2+  or more prolonged incubations provides a heterogenous population of Ca2+-binding sites (h < 1) with average affinities that increase with enzyme concentration (KDCa =34 mM - 24 μM) or incubation time. We have not resolved whether mixtures of only the two forms of indicator, namely the very-low affinity (KDCa =93 mM), partially de-esterified form and Mag-Fluo-4 itself (KDCa ∼20 μM), are sufficient to explain our results.Our results confirm both the K(∼20 μM) C and thag-Fluo-4 . Howeverluo-4 AM A. We sug2+ indicators, namely 5F-BAPTA m used in these studies were too low ethane-N,N,N′,N′-tetraacetic acid), wherein four carboxylate groups coordinate Ca2+ . Firstly, partial de-esterification of Mag-Fluo-4 AM provides an average KDCa appropriate for an organelle with a steady-state free [Ca2+] between 100 μM and 1 mM [DCa  provides sensitivity across a wide range of free [Ca2+]  substantially ameliorates the problems without affecting biological responses (Three features of Mag-Fluo-4 AM behaviour fortuitously contribute to its utility as a reliable reporter of ER free [Caand 1 mM . Secondle [Ca2+] . Finallye [Ca2+] A by meche [Ca2+] C. This lesponses G and H oesponses G. More i2+].We conclude that partial de-esterification of Mag-Fluo-4 AM within the ER alongside extrusion of the fully de-esterified form allow the indicator to reliably report changes in ER free [CaA.M.R. performed all experiments. A.M.R and C.W.T. designed the study, analysed and interpreted results, and wrote the manuscript.The authors declare they have no competing interests.


Entamoeba histolytica. Motility, adhesion, cytolysis, and phagocytosis/trogocytosis are important steps in invasion and pathogenesis of E. histolytica, and Ca2+ signaling is thought to be associated with these processes leading to tissue invasion. There are a large number of Ca2+-binding proteins (CaBPs) in E. histolytica, and a number of these proteins appear to be associated with different steps in pathogenesis. The genome encodes 27 EF-hand–containing CaBPs in addition to a number of other Ca2+-binding domain/motif-containing proteins, which suggest intricate calcium signaling network in this parasite. Unlike other eukaryotes, a typical calmodulin-like protein has not been seen in E. histolytica. Though none of the CaBPs display sequence similarity with a typical calmodulin, extensive structural similarity has been seen in spite of lack of significant functional overlap with that of typical calmodulins. One of the unique features observed in E. histolytica is the identification of CaBPs  that have the ability to directly bind actin and modulate actin dynamics. Direct interaction of CaBPs with actin has not been seen in any other system. Pseudopod formation and phagocytosis are some of the processes that require actin dynamics, and some of the amoebic CaBPs  participate in this process. None of these E. histolytica CaBPs have any homolog in organisms other than different species of Entamoeba, suggesting a novel Ca2+ signaling pathway that has evolved in this genus.Calcium signaling plays a key role in many essential processes in almost all eukaryotic systems. It is believed that it may also be an important signaling system of the protist parasite  Entamoeba histolytica causes human amebiasis, a major public health problem in developing countries. Though great strides have been made in understanding the pathobiological mechanisms of the disease in the last few decades, details about the molecular pathways that are involved in tissue invasion and damage during both intestinal and extraintestinal diseases are not clear. Because only a fraction of infected individuals (about 10%) display invasive disease, an understanding of the signaling system that triggers invasion by the parasite is needed for the development of better therapeutic molecules. Clear linkage between the genotype of the parasite with invasive disease or with extraintestinal invasion has not been seen, though a number of virulence factors have been identified in recent years [E. histolytica to invade [The protist parasite ls about 0% displant years . Gut bacnt years . It is io invade ,4. Howev2+ is one of the versatile, ubiquitous second messengers that mediate pathways by altering the shape, charge, and electrostatic interaction of downstream effector molecules [2+ and release it as and when needed [2+-mobilizing signals that regulate the level of Ca2+ in different cellular compartments by activating various ion channels and transporting systems. Once Ca2+ is released, a repertoire of CaBPs, Ca2+ buffers, and Ca2+-regulated enzymes subtly translate these Ca2+ signals into a cellular response. After initiation of a response by activating the appropriate pathway, Ca2+ is rapidly removed from the cytoplasm by various pumps and exchangers [E. histolytica encodes most of the molecules needed for release and sequestering of Ca2+ in response to a signal. Only a handful of molecules have been reported. A figure summarizing our current understanding and the molecules involved is shown in 2+-ATPases, out of which 3 belong to plasma membrane Ca2+-ATPase (PMCA) and 2 to sarcoendoplasmic reticulum ATPase (SERCA), and these are present in vacuoles and in the cytoplasmic network, respectively [2+-ATPases from E. histolytica (Eh), namely EhSPCA  and EhCCX (Ca2+/cation exchanger), have been identified. These are present on the membrane of some cytoplasmic vesicles [Caolecules . In orden needed . This tochangers . It is nectively ,8. More vesicles ,10. Intevesicles .E. histolytica has ionophore-releasable Ca2+, comprising around 70% of the total Ca2+ pool that can be divided into 2 parts. One is stimulated by the second messenger inositol 1,4,5-triphosphate P3) releasing internal Ca2+ from endoplasmic reticulum-like structures [2+ stores, it is not clear whether there is a link between them in this organism. E. histolytica also encodes a calpain-like protein and many nucleotidases that require Ca2+, such as Ca2+-dependent ATPase/ADPase, Ca2+-dependent thiamine pyrophosphatase, and acid phosphatase. The calpain-like protein is thought to be associated with apoptosis of the parasite because its level is increased during programmed cell death of trophozoites. It was also found in the cytoplasm and near the nucleus [E. histolytica [2+ buffers, thereby participating in the regulation of Ca2+ concentration in different cellular compartments.ructures . The secructures . Though  nucleus ,14, wher nucleus –17. It itolytica . Some ofN-acetyl-d-galactosamine  lectin, a 260-kDa heterodimeric cell-surface protein consisting of a 170-kDa heavy chain (hgl) bound to a 35/31-kDa light chain (Igl) through disulfide linkage [The initial step during the process of invasion is adherence to the target cells after the contact has been made. A number of molecules that are involved in this process have been identified. The most prominent among them is galactose-and  linkage . The lig linkage . CRD rec linkage . Light s linkage . The gly linkage .2+ also participates in the binding of ligands by Gal/GalNAc lectin [2+ binding site, and in one study, it was shown that Ca2+ binding is required for interaction with the ligand [2+ binding site was identified, and a mutant that lost the ability to bind Ca2+ was generated. Though carbohydrate-binding function was retained by the mutant protein, the ability to agglutinate red blood cells (RBCs) was lost, suggesting that some properties of Gal/GalNAc lectin are modulated by Ca2+ ions [2+-binding chaperone protein calreticulin (CRT) is an E. histolytica cell-surface protein that binds complement component 1q (C1q) [2+.Cac lectin . CRD hase ligand . In a moa2+ ions . The Ca21q (C1q) . It part1q (C1q) . Moreove1q (C1q) . The patE. histolytica to target cells is required for subsequent cell lysis and tissue invasion [E. histolytica or through stimulation of apoptotic pathway initiated after contact with the parasitic cells [E. dispar [E. histolytica, amebapores, were also implicated in cytolysis carried out by amebic cells [E. histolytica with target cells is a dramatic rise of Ca2+ levels in the latter after contact. Blocking target cell Ca2+ channels inhibited cell death. This is thought to be initiated by Gal/GalNAc lectin because the purified protein itself enhances Ca2+ levels in target cells [2+ release on contact with E. histolytica is not clear.It has been shown very clearly that adherence of invasion –30. Deatic cells . Amebic ic cells . Among t. dispar . Porin-lic cells –36. One et cells ,37. Howe2+ signaling is also involved in the ability of E. histolytica to initiate target cell killing. Blocking the rise of intracellular Ca2+ in the parasite prevents the initiation of the process of cytolysis [2+ in amebic virulence was seen when a Ca2+-binding transcription factor upstream regulatory element 3 binding protein (URE3BP) was found to regulate gene expression of virulence-associated genes. UREBP binds the promoter element of Gal/GalNAc lectin gene hgl5 [2+-binding EF-hand motifs and negatively regulates transcription in presence of Ca2+; that is, it binds the promoter DNA motif only in the absence of Ca2+ [2+-binding–defective mutant displayed a dominant positive phenotype, and cells expressing the mutant protein were more virulent [2+-dependent binding partner known as EhC2A [2+-dependent phospholipase activity that may have a role in virulence [A number of studies suggest that Caytolysis ,29,38,39ene hgl5 ,41. It h of Ca2+ ,42. A Cavirulent . URE3BP virulent . URE3BP as EhC2A . Apart firulence .E. histolytica. It displays a high rate of pinocytosis and phagocytosis that results in plasma membrane renewal every 30 min [2+ in phagocytosis was also seen when chelation of Ca2+ in the cytoplasm led to inhibition of the process [E. histolytica [2+ plays a critical role from the initiation stage to the formation of phagosomes.Phagocytosis is intimately associated with the biology of y 30 min . It phagy 30 min ,48. Wheny 30 min . Moreove process ,51. In ttolytica . It is c2+ and C2 domains and takes place when C2 binds membranes in the presence of Ca2+ [2+ is not required at this step. Once EhCaBP1 is at the phagocytic initiation site, it binds and recruits the atypical protein kinase EhAK1 in presence of Ca2+ [2+ [E. histolytica because no conserved gene has been seen in this system. EhCaBP3 is thought to be the closest homolog because it displays the highest degree of sequence similarity (about 49%) with calmodulins [2+ [2+-binding–defective mutants of all these proteins helped to delineate the participation of Ca2+ in different steps. Generally, these mutants display a dominant negative phenotype with respect to phagocytosis. Interestingly, overexpression of Ca2+-binding–defective EhCaBP1 did not interfere with the formation of phagocytic cups or the process of recruitment, but the process of transition from cups to phagosomes was blocked, thereby indicating that EhCaBP1 recruitment is independent of Ca2+ [2+ has both direct and indirect roles in the phagocytosis of E. histolytica.Phagocytosis is initiated by the recruitment of a C2-domain–containing protein kinase (EhC2PK) at the particle attachment site . This re of Ca2+ ,53. We b of Ca2+ ,55. EhAK of Ca2+ . Arp2/3 Ca2+ [2+ . A typicmodulins . Both Ehlins [2+ ,59. Myoslins [2+ . Imaginglins [2+ . The rollins [2+ ,59. Over of Ca2+ . TherefoE. histolytica [2+ also plays an important role in trogocytosis.Trogocytosis has recently been shown to be a novel mechanism of target cell killing and virulence of tolytica ,61. The tolytica . SubsequE. histolytica. The most prominent among these are 2 novel granule proteins grainin 1 and 2, which not only show a considerable structural similarity to EF-hand-motif–containing CaBPs but also bind Ca2+ [2+-binding EF-hand domains [2+-dependent manner [E. histolytica beta-tubulin.A number of as yet functionally uncharacterized CaBPs have been described in ind Ca2+ –65. Thesind Ca2+ . EhCaBP2 domains  Fig 2)..E. histo domains , suggest domains . Unlike t manner –68. Howet manner ,70. It w2+ is also required for a number of processes, including pathogenesis in E. histolytica. The surprising part is the extensive participation of Ca2+ and CaBPs in a few systems such as phagocytosis not observed in any other eukaryotic systems (see 2+ signaling is a rapid response, and mobilization of these EhCaBPs may not cause time delays after binding of a particle to the cell surface. Fast imaging of mobilization of different proteins during phagocytosis may help us to understand the nature of this rapid assembly process. The involvement of Ca2+ in many other amebic processes has not been investigated. Given the large number of CaBPs encoded by the E. histolytica genome, it will not be surprising to find Ca2+ signaling regulating a large number of pathways affecting the overall biology of E. histolytica.Calcium is known to be involved in many cellular processes in almost all eukaryotic systems. Therefore, it is not surprising that Catems see . Moreove


The pathogenicity of each hepatitis E virus (HEV) genotypes/subtypes may be different. This study aimed to investigate the infectivity and pathogenicity of different HEV genotypes/subtypes from different mammalian sources especially human in rabbits, and to assess whether rabbits are an appropriate animal model to study different HEV genotypes/subtypes. Thirty-seven rabbits were randomly divided into nine groups and inoculated with eight different HEV strains, including human-derived HEV3b (hHEV-3b), hHEV-4a, hHEV-4d and hHEV-4h, swine-derived HEV4d (sHEV-4d) and sHEV-4h, rabbit-derived HEV3 (HEV-3ra) and camel-derived HEV8. HEV RNA, antigen, anti-HEV and alanine aminotransferase (ALT) in serum or/and feces were monitored weekly. One rabbit from each group was euthanized at seven weeks post inoculation and the liver specimens were taken for histopathological analysis and immunofluorescence staining of HEV ORF2 proteins. hHEV-4d, sHEV-4d and HEV-3ra infections were successfully established in rabbits and typical acute hepatitis symptoms were observed, including viraemia/antigenemia, fecal virus/antigen shedding, elevated ALT level and liver histopathological changes. One rabbit infected with HEV-3ra showed chronic infection. hHEV-4d and sHEV-4d are less infectious and pathogenic than HEV-3ra in rabbits. hHEV-3b and HEV8 only caused inapparent infection in rabbits as 60% (3/5) and 20% (1/5) of the rabbits seroconverted to anti-HEV, respectively. No obvious signs of HEV infection in rabbits inoculated with hHEV-4a, hHEV-4h and sHEV-4h. The infectivity and pathogenicity of different HEV genotypes/subtypes in rabbits is different, which may be related to the species specificity of HEV. Rabbit can be used as an animal model for the study of HEV-3ra and more importantly human HEV-4d. Hepatitis E virus (HEV), mainly transmitted via fecal-oral route, is a single-stranded positive-sense RNA virus with a genome of about 7.2 kb in length and three open reading frames (ORF) flanked by two untranslated regions at the 5′ and 3′ end ,2. HEV iHepeviridae, which includes five species (Orthohepevirus A–D and Piscihepevirus). Species Orthohepevirus A is closely related to infections in humans and consists of at least 8 genotypes (HEV1-8) ..Hepeviri(HEV1-8)  and was (HEV1-8) . Later s(HEV1-8) ,14. HEV5(HEV1-8) ,16. HEV7(HEV1-8)  and subs(HEV1-8) . In 2016(HEV1-8) , and coutentions ,22. TherThe infectivity and pathogenicity of each HEV genotypes/subtypes may be different. Therefore, it is essential to develop an appropriate animal model to evaluate the infectivity and pathogenicity of different HEV genotypes. Commonly used animal models for HEV research include non-human primates, pigs, humanized mice and rabbits . The afog at 4°C for 30 min. The clarified suspension was filtered through 0.45 and 0.22 μm filters and titrated subsequently. All eight HEV-positive inocula were adjusted to 1 × 106 copies/mL. The negative inoculum was prepared from fecal sample of a non-infected rabbit.In this study, a total of eight different HEV strains of genotypes/subtypes obtained from different mammalian sources were used and the details of the eight strains are listed in Thirty-seven 3-month-old Japanese white rabbits weighing about 2–3 kg were randomly selected. Serum and fecal samples were collected weekly for two consecutive weeks to test alanine aminotransferase (ALT) for establishing the baselines. All samples were negative for anti-HEV antibodies by an enzyme-linked immunosorbent assay (ELISA) and HEV RNA by a previously described reverse transcription-nested polymerase chain reaction (RT-nPCR) .n = 5, each group) were inoculated intravenously with 1 mL of human-derived HEV-3b (hHEV-3b), human-derived HEV-4d (hHEV-4d), swine-derived HEV-4d (sHEV-4d), swine-derived HEV-4h (sHEV-4h), Bactrian camel-derived HEV8 and rabbit-derived HEV-3 (HEV-3ra) positive inoculum, respectively. The hHEV-4a and hHEV-4h strains were made from limited clinical samples and were not enough for the inoculation of five rabbits; therefore, Group G and H consist of two rabbits each. Group I rabbits (n = 3) were inoculated intravenously with 1 mL of negative inoculum as a negative control  was euthanized and liver specimens were taken and fixed in neutral 4% paraformaldehyde for 3 days for histopathological analysis and immunofluorescence staining of HEV ORF2 proteins.The serum samples of rabbits were tested for serum ALT concentrations using standard methods on a Hitachi Automatic Clinical Analyzer 7180. Rabbits were considered to have liver injury when the serum ALT concentration exceeded the pre-challenge level by more than two-fold.Anti-HEV antibody and HEV antigen were detected by commercial ELISA kits  according to the manufacturer’s instructions. Signal-to-cut-off (S/CO) values were calculated and values >1 were considered positive.Total RNA was extracted from 100 μL of serum or fecal suspensions prepared as above using TRIzol reagent  following a standard instruction. The RNA was then amplified by RT-nPCR for partial ORF2 genome on an automatic PCR system  and HEV viral load was determined using a commercial one-step real-time quantitative PCR assay , as previously described .The liver tissue were fixed in 10% neutral buffered formalin and embedded in paraffin and cut into 5 μm serial sections. Slides were stained with haematoxylin and eosin (H&E). The samples were photographed and analysed under a microscope  equipped with a digital camera with the assistance of Motic Images Plus 2.0 software.For immunofluorescence, tissue and organ sections were deparaffinized, hydrated, and water-bath heated for antigen retrieval. HEV proteins were visualized by using HEV ORF2-specific antibodies . Nuclear staining was achieved with DAPI . Staining sections were photographed and analysed by fluorescence microscopy .Nucleotide sequences of the eight strains, and other sixty-six ICTV proposed reference sequences obtained from GenBank, were aligned and analysed with MEGA X . PhylogeThe phylogenetic tree was constructed by the neighbour-joining method based on a partial nucleotide sequence about 350 bp of the ORF2 region and involved seventy-four nucleotide sequences including the eight strains used in the current study and the ICTV proposed reference strains . The phyRabbits were inoculated intravenously with the eight HEV strains of different genotypes/subtypes from different mammalian sources . The rab5–107 copies/g) was higher than that in Group B and C  and 20% (1/5) of rabbits were seroconverted to anti-HEV antibodies, respectively, which suggested an inapparent infection. In Group B, each rabbit was seroconverted for anti-HEV antibodies at 10 and 14 wpi, respectively, while the remaining rabbits stayed seronegative during the study. Only one rabbit in Group C showed seroconversion for anti-HEV antibodies at 7 wpi. All rabbits in Group F were seroconverted for anti-HEV antibodies. The rabbits in Group D, G, H and I did not show seroconversion during the entire study  and the negative control group (Group I) did not increase significantly during the study  was euthanized at 7 wpi. H&E staining showed the disordered structure of liver tissues and infiltration of inflammatory cells in liver sections in Group B, C and F. Mild cholestasis was also observed in Group B. No obvious pathological changes were seen in Groups A, D, E and negative control Group I .Figure The current study investigated the infectivity and pathogenicity of eight different HEV genotypes/strains in rabbits, including human-derived HEV-3b, HEV-4a, HEV-4d and HEV-4h, swine-derived HEV-4d and HEV-4h, rabbit-derived HEV3 and camel-derived HEV8, providing valuable information of the susceptibility and future application of the rabbit model in HEV study especially information about human HEV4 strains. These isolated strains are representative strains of the HEV genotypes/subtypes circulating in China. Although HEV3 is prevalent in many developed countries, it is not the dominant genotype in China ,34. TherAmong the existing animal models for HEV infection, nonhuman primates, pigs, rabbits and human liver chimeric mice are the most frequently used ones . Each moHEV4 is the dominant genotype in China and is prevalent in many other Asia countries including Japan, South Korea and Singapore . RecentlIn summary, the present study demonstrated that different HEV genotypes/subtypes showed different infectivity and pathogenicity in rabbits, which may be related to the species specificity of HEV. Rabbit can be used as an animal model for the study of rabbit-derived HEV3 and more importantly, human HEV4. Further study using more HEV isolates of different genotypes/subtypes is warranted.


Lycii radicis cortex (LRC) extract inhibited ovariectomized (OVX)-induced bone loss in mice. This study investigated the anti-osteoporotic effects of bioactive constituent(s) from the LRC extract. The effective compound(s) were screened, and a single compound, scopolin, which acts as a phytoalexin, was chosen as a candidate component. Scopolin treatment enhanced alkaline phosphatase activity and increased mineralized nodule formation in MC3T3-E1 pre-osteoblastic cells. However, osteoclast differentiation in primary-cultured monocytes was reduced by treatment with scopolin. Consistently, scopolin treatment increased osteoblast differentiation in the co-culture of monocytes (osteoclasts) and MC3T3-E1 (osteoblast) cells. Scopolin treatment prevented bone mineral density loss in OVX-induced osteoporotic mice. These results suggest that scopolin could be a therapeutic bioactive constituent for the treatment and prevention of osteoporosis.Bone remodeling is a renewal process regulated by bone synthesis (osteoblasts) and bone destruction (osteoclasts). A previous study demonstrated that  Bone remodeling during vertebrate development is governed by the coordinated action of bone formation (osteoblasts) and resorption (osteoclasts) ,2. Bone Lycii radicis cortex (LRC), the root bark of Lycium chinense, is broadly regarded as a traditional medicine in eastern Asia [During the past decade, traditional herbal plants have been used for medicinal purposes because they are more suitable for long-term treatment with fewer side effects than pharmacological medications. Herbal medicines are widely used to improve bone health and as alternative therapeutic options for osteoporosis . Many reern Asia . Physiolern Asia ,16. Treaern Asia . Studiesern Asia ,18. Our ern Asia . DespiteHere, we explored the therapeutic possibilities of a single anti-osteoporotic compound (scopolin) isolated from LRC extract. We evaluated the bone formation-enhancing effects of scopolin on osteoblastic MC3T3-E1 cells. In addition, we examined bone formation in a monocyte/MC3T3-E1co-culture system to characterize the differentiation of osteoblast and osteoclast cells. Finally, we investigated the protective effect of scopolin on OVX-induced bone loss in an osteoporotic mouse model.2O, and then partitioned successively into CH2Cl2 , EtOAc , n-BuOH , and aqueous  fractions. Fraction D was chromatographed on a Diaion HP-20 gel (1000 g) column and eluted with a H2O-MeOH solvent gradient system  to give four fractions (D1-D4). Fraction D2 (14.5 g) was subjected to RP-18 gel (200 g) column chromatography and eluted with H2O-MeOH (100:0 to 0:100) to produce seven subfractions (D2-1 to D2-7). Subfraction D2-5 (524 mg) was subjected to preparative HPLC and eluted with MeOH-H2O/0.1% formic acid (10:90) to give compound 1 (2.1 mg) . Voucher specimens (GL0180), authenticated by Dr. Chun Whan Choi, were deposited at the herbarium of the Bio-center, Gyeonggi Business & Science & Accelerator, Suwon, South Korea. The ethanol extract of LRC (254 g) was evaporated, suspended in H(2.1 mg) . The strtrometry  and compBuOH C, 1.1 g, and2, and the medium was changed every 3 days.Mouse preosteoblastic MC3T3-E1 cells were obtained from the RIKEN Cell Bank  and cultured in α-modified minimal essential (α-MEM) medium  supplemented with streptomycin , penicillin , and 10% fetal bovine serum (FBS) . Preosteoblast differentiation was stimulated by adding β-glycerophosphate (10 mM)  and an osteogenic medium containing ascorbic acid (50 μg/mL)  after allowing 24 h for cell adherence (day 0). The cells were incubated in a humidified atmosphere at 37 °C containing 5% COThe cells were cultured in a 96-well plate overnight and co-treated with different concentrations of scopolin  in the induction medium for 48 h. Cell viability was determined with a D-Plus™ CCK cell viability assay kit . The cells were incubated with water-soluble tetrazolium salt (WST) solution (5 mg/mL in PBS) for 2 h, and their absorbance values were analyzed using a microplate reader  at 450 nm and 655 nm (reference wavelength).2+, 0.5% Triton X-100, 1 mmol/L of Tris-HCl (pH 8.8), and 5 mmol/L of p-nitro-phenylphosphate. The absorbance value was measured at 405 nm . ALP staining in the differentiated osteoblasts was performed by fixing them with 4% paraformaldehyde (PFA), followed by staining with SIGMAFAST™ BCIP®/NBT . The ALP-positive osteoblast cells were examined under a microscope .Osteoblast differentiation was measured using ALP activity in total cell lysates in a buffer containing 10 mmol/L of MgThe MC3T3-E1 cells were incubated in a 48-well plate and treated with three different concentrations of scopolin  for 21 days. Matrix mineralized nodule formation was measured using Alizarin Red S  staining. After 21 days of incubation with Scopolin in the osteoblast induction medium, the induction medium was removed and the mineralized osteoblastic cells were washed three times with PBS. The cells were fixed with 70% ethanol for 10 min, and nodule formation was evaluated by staining with 40 mM alizarin red S (Sigma-Aldrich). Positive alizarin red S staining was identified using microscopes .4 cells/well) and the isolated monocyte cells (4 × 104 cells/well) were co-incubated in media containing β-glycerophosphate (10 mM) and ascorbic acid (50 µg/mL).For the differentiation of osteoclasts, monocytes were separated from mouse bone marrow cells in induction medium containing 1 mM ascorbate-2-phosphate . The identification of monocyte cells was confirmed via a monocyte marker , using a FACS Aria III cell sorter  with the FACS Diva software ). For induction of differentiation to osteoclasts, the monocytes were incubated in the buffer containing 50 ng/mL of receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)  and 30 ng/mL macrophage colony-stimulating factor-1 (M-CSF)  . The difAfter induction of osteoclast differentiation, the cells were fixed with 4% PFA for 10 min and washed three times with PBS. The differentiated osteoclasts were analyzed using TRAP staining and activity assay with an Acid-Phosphatase Kit . TRAP-positive cells, including three or more multinucleated nuclei, were calculated under microscopes . Osteoclastogenesis-induced monocytes were subjected to TRAP activity assay using TRACP & ALP Assay Kit  as described by the manufacturer’s protocol. The absorbance of TRAP activity was analyzed at 405 nm  and expressed as a percentage of the untreated control.Alpl, 5′-CCA ACT CTT TTG TGC CAG AGA-3′ and 5′- TGA CAT TCT TGG CTA CAT TGG TG-3′; for mouse Runx2, 5′-TAA AGT GAC AGT GGA CGG TCC C-3′ and 5′- TGC GCC CTA AAT CAC TGA GG-3′; for mouse Bglap, 5′-TAG TGA ACA GAC TCC GGC GCT A-3′ and 5′-TGT AGG CGG TCT TCA AGC CAT-3′; for mouse Sp7, 5′-ATG GCG TCC TCT CTG CTT G-3′ and 5′-TGA AAG GTC AGC GTA TGG CTT-3′; for mouse Tnfsf11, 5′-TGC CTA CAG CAT GGG CTT T-3′ and 5′-AGA GAT GAA CGT GGA GTT ACT-3′; for mouse Nfatc1, 5′-GGA GAG TCC GAG AAT CGA GAT-3′ and 5′-TTG CAG CTA GGA AGT ACG TCT-3′; and for mouse Gapdh, 5′-AGG TCG GTG TGA ACG GAT TTG -3′ and 5′- TGT AGA CCA TGT AGT TGA GGT CA-3′. Gene expression was quantified by normalizing to Gapdh expression using the comparative threshold (Ct) method, as described by the manufacturer (Bio-Rad software). The values were expressed as fold change compared to the control. The relative gene expression was calculated as 2−ΔCt (ΔCt = Ct target gene − Ct Gapdh). The fold change was calculated as 2−ΔΔCt (ΔΔCt = ΔCt control − Ct treatment).Total RNA was extracted from the cultured cells using TRIzol reagent  and complementary DNA (cDNA) was produced using a RevertAid™ H Minus First Strand cDNA Synthesis Kit , according to the manufacturer’s recommendations. PCR amplifications were performed using a SYBR Green I qPCR kit (Takara Bio Inc.) at 40 cycles with 150 ng of cDNA using Bio-Rad CFX . The specific primers were as follows: for mouse n = 24) and sham-operated , were purchased from the Shizuoka Laboratory Center, Inc. . After surgery, the mice were acclimated for 1 week before scopolin treatment and were divided into five groups comprising six mice each: 1) Sham, 2) OVX control, 3) OVX treated with 10 mg.kg−1day−1 of SrCl2 as a positive control, 4) OVX treated with 20 mg.kg−1day−1 of scopolin, and 5) OVX treated with 40 mg.kg−1day−1 of scopolin. The mice were maintained on a diet of Formula-M07  and tap water (15 mL/day). All the mice were kept in clear individual plastic cages under illumination (12-h light/dark cycle), temperature (23 ± 2 °C), and humidity (55% ± 5%). The mice were orally treated with the liquid form of scopolin alone (20 and 40 mg.kg−1day−1) by gavage for 12 weeks. The animal research protocol was approved by the Animal Care and Use Committee of the Ajou University School of Medicine, and all experiments were conducted in accordance with the institutional guidelines established by the Committee (AMC-133).Seven-week-old female ddY mice , including ovariectomized , and placed carefully on a specimen tray in the same position for measurements of right femur BMD using a PIXI-mus bone densitometer with the on-board PIXI-mus software .2O/O2 inhalation anesthesia and injected with Tc-99m hydroxymethylene diphosphonate (HDP)  via the tail vein. Skeletal technetium (Tc-99m) HDP uptake on planar HDP bone scans of mice receiving intravenous treatments was plotted as a function of skeletal blood flow and osteogenic activity. In vivo planar images were obtained using an Inveon SPECT scanner  equipped with a low-energy all-purpose collimator. The images were acquired until 100,000 total counts were measured per body.Transverse micro-CT images of the samples were scanned using a micro-CT scanner . Reconstruction was performed using the Inveon Research Workplace and COBRA_Exxim . Two-dimensional (2D) axial and three-dimensional (3D) images were reconstructed for qualitative and quantitative analyses. These analyses provided information regarding the main histomorphometric parameters, including percentage bone volume , trabecular number , thickness , and separation . Axial images were displayed using the Inveon Research Workplace  for the region of interest (ROI) measurement and analyses. Axial reformats were performed to allow slice-by-slice manual tracing of the contours of the trabecular bone. A region 300 μm below the growth plate was analyzed for a cross-sectional study of the trabecular bone. For the single-photon emission computerized tomography (SPECT) scan, the mice were anesthetized with isoflurane/Ng for 15 min at 4 °C and assayed for the measurement of OPG and RANKL using a Luminex multiplex assay , according to the manufacturer’s instructions. The ratio of OPG to RANKL (OPG/RANKL) was calculated to explain the process of bone formation coupled with bone resorption.The animals were euthanized, and fresh blood samples were collected from the left ventricle in heparinized syringes. Serum was immediately separated via centrifugation at 1200× t-test. A one-way analysis of variance was conducted for the comparisons of multiple groups, followed by Tukey’s honest significant difference post hoc test. P-values less than 0.05 (p < 0.05) were considered statistically significant.At least three biological and technical replicates were conducted during all experiments. The statistical package for the social sciences  was used for statistical analyses. The results of bar graphs are expressed as the mean ± standard error of the mean. Differences between groups were assessed using Student’s Our previous study suggested that natural herbal medicine (LRC extract) prevented OVX-induced BMD loss in an osteoporotic mouse model by promoting the differentiation of osteoblast lineage cells . In the We investigated the specific anti-osteoporotic effects of scopolin on osteoblastic differentiation in the pre-osteoblastic MC3T3-E1 cell line. The cells were treated with three different concentrations of scopolin  for either 3 or 5 days . After iAlpl , Runx2 (runt-related transcription factor 2), Bglap , and Sp7 (osterix), as previously suggested [Alpl, Runx2, Bglap, and Sp7 was significantly increased by scopolin treatment compared to the control group .Next, we examined the mRNA expression of bone remodeling markers, including uggested ,27. MC3Tol group A–D. ThesTnfsf11), and nuclear factor of activated T cells cytoplasmic 1 (Nfatc1) were measured to further confirm the effects of scopolin on the mRNA levels of osteoclast markers. Scopolin treatment significantly reduced the expression of osteoclast differentiation-induced genes  restored BMD in the right femur bone with improved trabecular bone structural properties, including BV/TV, Tb.Th, Tb.N, and Tb.Sp  and bone mineral content (BMC) . BMD wasnd Tb.Sp A–C. The ol group A. In addol group B,C. For ol group D.2 increased OPG and decreased RANKL serum levels compared to non-treated OVX mice. Scopolin treatment also significantly increased serum OPG levels and decreased serum RANKL levels , and RANKL, using enzyme-linked immunosorbent assay (ELISA). SrClL levels E. Thus, L levels E.Alpl, Runx2, Bglap, and sp7. In contrast, decreased osteoclast differentiation in primary cultured mouse monocytes from bone marrow was observed after scopolin treatment. Consistently, treatment of a monocyte and MC3T3-E1 co-culture system with scopolin resulted in enhanced osteoblast differentiation, and scopolin treatment prevented OVX-induced bone loss and osteoclastogenesis in an OVX animal model.In this study, we isolated and identified a single bioactive compound, scopolin, from the fractionation of LRC. Scopolin treatment enhanced osteoblast differentiation and mineralization in MC3T3-E1 cells by upregulating the mRNA expression of Alpl, Runx2, Bglap, and sp7, which play crucial roles in the mineralization of newly formed bone.A previous study demonstrated that LRC might be an alternative herbal drug for effective osteoporosis treatment in vivo and in vitro and suggested 13 major compounds identified in LRC extract . In addiA previous study demonstrated that scopolin exerted suppressive effects on the differentiation of pro-osteoclastic RAW 264.7 cells by inhibiting RANKL-induced osteoclast differentiation . Similar2 as a positive anti-osteoporotic agent because a previous report suggested the protective effects of SrCl2 against bone loss [We also found that scopolin treatment inhibited OVX-induced bone loss in mice. We selected SrClone loss . Scopolione loss . RANKL ione loss ,38. OVX one loss . Howeverone loss . Thus, sWe investigated the effect of scopolin treatment on OVX-induced bone loss in an osteoporotic animal model. Our data suggested that scopolin treatment enhanced osteoblastic differentiation by upregulating osteoblastic-inducing genes. Consequently, the activation of osteoblastic signaling induced by scopolin treatment prevented OVX-induced bone loss in mice. Our observations suggest that scopolin might be a potential alternative herbal medicine for the prevention of bone loss.


We previously demonstrated that canstatin, a C-terminal fragment of type IV collagen α2 chain, regulated Ca2+ handling in rat heart. In this study, we aimed to clarify the effects of canstatin on I/R-induced ventricular arrhythmia in rats. Male Wistar rats were subjected to I/R injury by ligating the left anterior descending artery followed by reperfusion. Ventricular arrhythmia (ventricular tachycardia and ventricular fibrillation) was recorded by electrocardiogram. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and ROS production in neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen glucose deprivation/reperfusion (OGD/R) were measured by lucigenin assay and 2′,7′-dichlorodihydrofluorescein diacetate staining, respectively. The H2O2-induced intracellular Ca2+ ([Ca2+]i) rise in NRCMs was measured by a fluorescent Ca2+ indicator. Canstatin (20 µg/kg) inhibited I/R-induced ventricular arrhythmia in rats. Canstatin (250 ng/mL) inhibited OGD/R-induced NOX activation and ROS production and suppressed the H2O2-induced [Ca2+]i rise in NRCMs. We for the first time demonstrated that canstatin exerts a preventive effect against I/R-induced ventricular arrhythmia, perhaps in part through the suppression of ROS production and the subsequent [Ca2+]i rise.Ventricular arrhythmia induced by ischemia/reperfusion (I/R) injury is a clinical problem in reperfusion therapies for acute myocardial infarction. Ca Myocardial infarction (MI) is caused by a coronary thrombosis mainly due to the disruption of atherosclerotic plaque . Reperfu2+ overload via increasing intracellular Ca2+ ([Ca2+]i) through the regulation of Ca2+ handling proteins, such as L-type Ca2+ channel (LTCC), Na+-Ca2+ exchanger (NCX), ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+-ATPase (SERCA), in cardiomyocytes i rise in neonatal rat cardiomyocytes (NRCMs) i rise in cardiomyocytes leads to ventricular arrhythmias [2+]i increase induced by H2O2 (100 µM) in NRCMs. H2O2 induced a [Ca2+]i rise (0.107 ± 0.009), which was significantly inhibited by canstatin    A,B. Temp, n = 7) A,B.2O2-induced [Ca2+]i rise in NRCMs.In the present study, we for the first time demonstrated that canstatin suppressed I/R-induced ventricular arrhythmia in rats. Furthermore, canstatin inhibited OGD/R-induced NOX activation and ROS production, and suppressed the H2+ overload, which leads to ventricular arrhythmia [2+]i rise.ST elevation is a characteristic waveform observed during ischemia in a rat model of I/R-induced ventricular arrhythmia . In thisrhythmia . In thisvβ3 and αvβ5 integrins i rise through the activation of Ca2+ handling proteins, such as LTCC, NCX and RyR [2O2-induced [Ca2+]i rise in NRCMs . PBS  was used as a vehicle for recombinant canstatin.The reagent sources were as follows: recombinant mouse canstatin (produced by eviously ), verapaAll animal experiments were approved by the President of Kitasato University through the judgement of Institutional Animal Care and Use Committee of Kitasato University , 19-126 (29 August 2019)). Male Wistar rats  were cared in accordance with the guideline for animal care and treatment of the Kitasato University. For the production of an in vivo model of I/R-induced ventricular arrhythmia and the isolation of NRCMs, 10-week-old and 1–3-day-old rats were used, respectively.n = 13) were anesthetized with urethane . Then, the rats were artificially ventilated  following an endotracheal intubation, and intravenous cannulation to the saphenous vein was performed for the administration of drugs. After a left thoracotomy was performed, 3 Spring Clip Electrodes  connected to a 3-Lead Shielded Bio Amp Cable  were fixed on the right axillary (cathode), left pectoral (anode) and right inguinal (earth) regions. ECG recording was performed by using a Bio Amp  and PowerLab system  in lead II configuration. A 6-0 nylon suture was passed under LAD. The suture was threaded through a polyethylene tube to create a snare. Recombinant canstatin , PBS (n = 5) or verapamil  was intravenously administered for 5 min. Verapamil was used as a positive control. Then, LAD ligation was performed by tightening the snare (ischemia). Ten minutes after the ligation, it was released (reperfusion). VT and VF were recorded for 10 min after reperfusion  containing 10% feral bovine serum , 1% antibiotic-antimycotic mixed solution  and 100 µM bromodeoxyuridine (BrdU). The cell suspension was pre-plated for 90 min twice to remove the attached non-cardiomyocytes. The non-attached cardiomyocytes were collected, seeded and cultured on culture dishes (for lucigenin assay) or coverslips coated with 1% gelatin (for DCF-DA staining or measurement of [Ca2+]i) in high-glucose DMEM containing 10% FBS, 1% antibiotic-antimycotic mixed solution and 100 µM BrdU.NRCMs were isolated from neonatal Wistar rats as described previously . The hea2, 94% N2 and 5% CO2 at 37 °C) in the multi-gas incubator  for 10 min (OGD). Then, the cells were cultured with the high-glucose DMEM under normoxic conditions (95% air and 5% CO2 at 37 °C) for 5 min (R). Recombinant canstatin, PBS, gp91 ds-tat or tempol were treated throughout the experiment. Control cells were cultured in high-glucose-containing DMEM under normoxic conditions. After the OGD/R stimulation, the cells were used for lucigenin assay or DCF-DA staining.To mimic I/R injury, OGD/R was performed in NRCMs as described previously  Figure . After N2PO4 and NaHPO4, 1 mM EGTA and 150 mM sucrose at pH 7.0) containing lucigenin (10 µM), NADPH (1 mM) and cell lysate (20 µg) was poured into assay wells (96-well plates). Then, the chemiluminescence was continuously measured for 30 min at 37 °C by a TriStar LB941 luminometer . The chemiluminescence of relative light units per second (RLU/s) was obtained every 10 s, and the results were calculated as area under the curve.To assess the activity of NOX in NRCMs, a lucigenin assay was performed as described previously . Total cTo evaluate intracellular ROS production in NRCMs, DCF-DA staining was performed as described previously . The cel2+]i in NRCMs was measured by using Fura-2 acetoxymethyl ester (AM)  as described previously i rise in cardiomyocytes. It is expected that canstatin contributes to the development of a novel therapeutic strategy to suppress I/R-induced ventricular arrhythmia in AMI patients.Our data suggest that canstatin is preventive against I/R-induced ventricular arrhythmia, perhaps in part through the suppression of ROS production and the subsequent [Ca


Charge acceleration during an intense light field application to solids attracts much attention as elementary processes in high-harmonic generation and photoelectron emission. For manipulating such attosecond dynamics of charge, carrier-envelope-phase (CEP: relative phase between carrier oscillation of light field and its envelope function) control has been employed in insulators, nanometal and graphene. In superconducting materials, collective control of charge motion is expected because of its strongly coherent nature of quasi-particles. Here we report that, in a layered organic superconductor, a non-linear petahertz current driven by a single-cycle 6 femtosecond near infrared field shows up as second harmonic generation (SHG), which is in contrast to the common belief that even harmonics are forbidden in the centrosymmetric system. The SHG represents a CEP sensitive nature and an enhancement near the superconducting temperature. The result and its quantum many-body analysis indicate that a polarized current is induced by non-linear acceleration of charge, which is amplified by superconducting fluctuations. This will lead to petahertz functions of superconductors and of strongly correlated systems. 2Cu[N(CN)2]Br. They find unusual temperature dependence and CEP-sensitive nature of the SHG which are explained in terms of nonlinear current.Here the authors show second harmonic generation (SHG) from a centrosymmetric organic superconductor κ-(BEDT-TTF) Considering non-linear light–matter interactions during an ultrashort pulse, we can expect petahertz control of the polarized current in superconducting materials.An electromagnetic oscillation of light cannot directly drive a polarized current because of its symmetric nature on the time axis . However, recent developments of ultrashort laser technologies enable us to control the direction of charge motion by carrier-envelope phase (CEP) control of a strong light fieldD(t) is induced by an electric field), a net current given by E0 (t): envelope function of a single-cycle pulse, E(t) of the moment), resulting in current-induced second harmonic generation (SHG)16. Thus, the SHG  is induced even in the centrosymmetric system, if a non-linear current is driven by light.In the non-linear regime 2]Br21 with a transition temperature TSC = 11.6 K  have been discussed in analogy with a pseudogap in high-TSC superconducting cuprates in the temperature-t/Udimer phase diagram21, where t/Udimer is the ratio of an inter-dimer transfer integral t to the effective on-site Coulomb energy for a dimer Udimer, as shown in Fig. 25, coherent excitation of the Higgs mode26 and light-induced superconductivity28. On the other hand, petahertz light functions, driven by the non-linear current during a light pulse is also expected to be characteristic, reflecting the ultrafast time scale of superconducting fluctuations in κ-(BEDT–TTF)2Cu[N(CN)2]Br29.Our target material is a layered organic superconductor κ-(BEDT–TTF)6 K Fig. . Superco2Cu[N(CN)2]Br . A CEP dependence of the SHG shows that it is induced by the non-linear current. Superconducting fluctuations above TSC amplify the SHG.In this article, we report SHG for a strong light field (16 MV/cm = 1.6 V/nm) in a centrosymmetric organic superconductor κ-(BEDT–TTF)EFund | |c, ESH | | c) and green  lines] and 2.2 eV  for the fundamental photon energy of 0.75 eV. Here, EFund, ESH, and ETH indicate the electric fields of the fundamental light, SHG and THG, respectively. Note that the SHG is originally forbidden in this centrosymmetric system (orthorhombic with Pnma symmetry31) in a conventional perturbation theory. However, the SHG is observed, and it is ca. 1/50 times as intensive as the THG.Figure c-axis (ESH | | c) for both excitation polarizations , although the THG shows the usual polarization which is the same as that of the fundamental pulse (lower panel). Such unusual polarization dependence of the SHG is discussed below. We performed SHG and THG measurements with an incident angle of smaller than three-degree to minimize surface SHG32. In fact, the results above  and  are confirmed by rotating the sample by 90° not to depend on the s-polarized or p-polarized configuration under such a restricted condition, although the s-polarized or p-polarized configuration is indistinguishable for the incident angle of 0°. Moreover, the dependences on the CEP and the temperature (which are shown below) are incompatible with SHG induced by static geometries such as a surface or a point defect. Thus, the observed SHG is not attributed to surface SHG. The possibility of symmetry breaking induced by photo-carrier generation near the surface is also excluded by the fact that the lifetime of carriers (ca. 500 fs)29 , open red circles ] and THG (ITH: blue) , indicating that the SHG grows up toward TSC from high temperature, although ITH does not depend on the temperature. The fluctuation of ISH for  is larger than that for  because of stray lights from the intense/ broad THG. The spectral shape is almost independent of the temperature in the range between 6 and 60 K as shown in Fig. TSC. Such temperature dependence of the SHG, which cannot be understood by a conventional perturbation theory, clearly shows that the observed SHG is related to the superconducting fluctuations21.Figure h/] where the transfer integral is about 0.1 eV in organic super conductors, a possible mechanism for the unconventional SHG is the non-linear current discussed above. Furthermore, the temperature dependence . During a period of the CEP, the SHG shows two maxima at around ΔφCEP = 1/2π and 3/2π. Considering that the directions of the light-induced current cannot be distinguished by the SHG measurement, i.e., that currents with a same amplitude and opposite directions give same SHG intensities, the above ΔφCEP dependence is quite reasonable. This CEP dependence of the SHG is completely consistent with the fact that the unconventional SHG is attributed to the non-linear light-induced current.We can demonstrate relevance of this scenario by measuring the CEP dependence of the SHG, because the non-linear current is sensitive to the CEP as mentioned above Fig. . Figure j in a two-dimensional three-quarter-filled Hubbard model for a 98 × 98-site system in the framework of the time-dependent Hartree–Fock approximation. The details are described in Supplementary note ωJ, J denotes the absolute value of the Fourier transform of j9) for ESH | | c, ETH | | c with electric field amplitudes (F [V/Å]) of 0.16, 0.06, and 0.006  shows the polarization that is parallel to the fundamental polarization for both Efund | |c and Efund | |a. The polarization dependence cannot be understood simply by a point group analysis of χ(2) tensor for the orthorhombic structure [class mm2(C2v) ]   owing to an absorption loss is not the reason of the narrow bandwidth  in Fig. h/(0.1 eV)) in organic superconductors, indicating that the coherence time is governed by the electronic scattering  is comparable to the electronic scattering time of ∼40 fs (=2Cu[N(CN)2]Br. The SHG is enhanced toward TSC. A narrow bandwidth of 130 meV shows that the coherence of the SHG survives for 30 fs after the 6 fs light-field application. The CEP dependence indicates that the SHG is induced by the non-linear petahertz current.In summary, this article demonstrates SHG in the centrosymmetric organic superconductor κ-(BEDT–TTF)2Cu[N(CN)2]Br were grown under dried nitrogen gas atmosphere by electrochemical oxidation of BEDT–TTF (80 mg) dissolved in 100 ml of 1, 1, 2-trichloroethane (10% v/v ethanol) in the presence of supporting electrolytes NaN(CN)2, CuBr, and 18-crown-6 ether29. A thick plate-like crystal  was selected for optical measurements. The a–c crystal plane corresponds to the two-dimensional conductive layer.Single crystals of κ-(BEDT–TTF)34.A broadband infrared spectrum covering 1.2–2.3 μm of the 6 fs pulse is obtained by focusing a carrier-envelope phase (CEP) stabilized idler pulse (1.7 μm) from an optical parametric amplifier (Quantronix HE-TOPAS pumped by Spectra-Physics Spitfire-Ace) onto a hollow fiber set within a Kr-filled chamber (Femtolasers). Pulse compression is performed using both active mirror  and chirped mirror (Femtolasers and Sigma-Koki) techniques2) pulse are controlled by a pair of wire-grid CaF2 polarizers34. The SHG and the THG are detected by a photomultiplier tube (Hamamatsu R13456) after passing through a spectrometer . The CEP of the fundamental pulse is controlled by a pair of glass plates with the incidence angle of θ  . The intensity and polarization of the fundamental  Fig. .Supplementary Information