Upward lightning studies took place in Rapid City, South Dakota, USA and S. Paulo, Brazil during the summer thunderstorm seasons from 2011 to 2016. One of the main objectives of these campaigns was to evaluate and characterize the triggering of upward positive leaders from tall objects due to preceding nearby flash activity. 110 upward flashes were observed with a combination of high- and standard-speed video and digital still cameras, electric field meters, fast electric-field antenna systems, and for two seasons, a Lightning Mapping Array. These data were analyzed, along with correlated lightning location system data, to determine the triggering flash type responsible for the initiation of upward leaders from towers. In this paper, we describe the various processes during flash activity that can trigger upward leaders from tall objects in the USA and in Brazil. We conclude that the most effective triggering component is the propagation of the in-cloud negative leader during the continuing current that follows a positive return stroke. Upward lightning is the development of a self-propagating lightning leader from a tall object that travels upward toward the overlaying electrified storm cloud. Upward lightning can initiate without any preceding lightning activity, herein classified as self-initiated upward lightning. For self-initiated upward lightning to occur, storm electrification and the resulting presence of a cloud charge region is required to generate an electric field necessary for the initiation and growth of an upward leader. The elevated vertical shape of the tall object enhances the electric field locally resulting in conditions favorable for initiation of an upward leader. However there is no proceeding nearby lightning activity prior to the initiation of the upward leader. An upward leader from a tall object can also initiate and develop in response to an electric field change created by a nearby preceding lightning flash . Since the upward leader is essentially triggered by the nearby lightning activity, we refer to this type of upward lightning as lightning-triggered upward lightning.Past research efforts have sought to characterize the type of triggering flashes and electric field environment prior to and during the initiation of upward leaders from tall objects as well as characterize weather conditions favorable for upward flashes. The following is a literature review specifically relating to lightning-triggered upward lightning.Wang et al.1 presented electric field changes for 14 upward flashes between 2005–2006 from a wind turbine and its protection tower in Japan. It was found that 4 upward flashes exhibited only a unidirectional negative field change (physics sign convention) that correlated with the initiation and development of an upward positive leader (UPL) without any preceding flash activity. They called this type of electric field change Type 1. The remaining 10 upward flashes showed an initial positive field change in response to nearby flash activity before the field change reversed polarity to negative in response to the development of an UPL. They defined this second type of electric field change behavior as Type 2.et al.2 documented direct evidence that an upward lightning flash can trigger other upward lightning of opposite polarity from nearby tall structures.Later, Lu Wang and Takagi3 reported on 53 winter upward flashes to a wind turbine and its protection tower in Japan between 2005 and 2010. Here, 28 upward leaders were triggered by preceding lightning activity and 25 developed upward leaders without any preceding flash activity. The upward flashes that were preceded by nearby lightning activity were defined as “other-triggered” (formerly Type 2) whereas the upward lightning that initiated from the tall objects without preceding lightning activity were defined as “self-initiated” (formerly Type 1). Negative upward flashes, where an upward propagating positive leader initiates from the tall object and effectively brings negative charge downward, was the dominate upward flash polarity.Zhou et al.4 reported on 205 upward flashes from the Gaisberg Tower from 2005 to 2009. Only a small minority, 26/205 (13%), were triggered by preceding lightning activity. Of those 26 lightning-triggered upward lightning flashes, 15 upward positive leaders were triggered by a preceding positive cloud-to-ground (+CG) flash and 9 upward positive leaders were triggered by a preceding intracloud (IC) flash. One of the 9 upward positive leaders triggered by a preceding IC flash experienced a reversal of current (negative then positive) during the flash and was therefore a bipolar upward flash. Furthermore, one upward negative leader (UNL) was triggered by a negative cloud-to-ground (−CG) flash and one upward negative leader was triggered by an IC flash. Therefore, 24 of the 26 lightning-triggered upward lightning events (92%) began with the development of an upward positive leaders and only two (8%) began with the development of an upward negative leaders.Jiang et al.5 reported on 4 lightning-triggered upward flashes from a 325 m tall tower in Beijing China during two different storms in 2012. In two cases, an upward positive leader was preceded by a nearby +CG flash, and the other two cases by intracloud lightning flashes.6. Assets included a 10-station Lightning Mapping Array (LMA), 8-station slow antenna network, 2 electric field meters (EFMs), multiple high- and standard-speed video cameras, in situ lightning current transient measurements for all turbines, and National Lightning Detection Network (NLDN) data. Rison et al.7 reported on three observed negative upward flashes. For one case, an IC flash resulted in negative leaders passing over the windfarm and a single UPL initiated from one wind turbine coincident with the passage of the leaders. For the two other cases, +CG flashes resulted in the initiation of UPLs from multiple turbines during the same flash.In 2013, a comprehensive field campaign at a windfarm in Kansas, USA sought to address wind turbine lightning attachment from both downward and upward lightninget al.8 observed upward lightning cases in both Brazil and the United States and found that 61 out of 72 (84%) and 15 out of 28 (53%), respectively, were caused by a +CG flash within 1 s before and after the initiation of the upward lightning, at a distance within 80 km. Yuan et al.9 found that 14 out of 19 (75%) of upward lightning flashes from a 325-m tower in Beijing (from 2012 to 2016) were triggered by +CG lightning flashes.Saba et al.10 reported a three-dimensional development characteristics of an upward lightning triggered by +CG flash. Wu et al.11 reported on a multiple lightning-triggered upward flash from 7 tall structures preceded by a very intense +CG flash.Recently, Qi The present work describes which triggering flash type is more effective and the various processes during flash activity that can trigger upward leaders from tall objects in Brazil and USA.The data presented here were obtained during the upward lightning studies in Rapid City, South Dakota, USA and in S. Paulo, Brazil from 2012 to 2016. Description of our experimental setup is found in the section titled “Methods” below.T-leader), and the initiation time of the upward leaders from the towers. In some cases, the exact initiation time of the upward propagating leaders was not possible due to the low luminosity of the initial developing leader and/or poor visibility due to rain or back lighting. In these cases, an initiation time was estimated based on the leader’s position and speed once its luminosity increased to detectable levels. Once upward leaders developed, the optical assets provided further information on the development and behavior of these leaders in relation to the triggering flash. The fast antenna systems provided the time of the first field change associated with triggering flashes and the electric field change polarity and magnitude preceding upward leader initiation and during the development of the upward leaders. The challenge of interpreting field change data during lightning-triggered upward flash is that once an upward leader develops, the field change is influenced by both the ongoing triggering flash and the upward leaders that are developing from the towers. The placement of fast electric-field sensors and of electric field meters (EFMs) at multiple locations at varying distances from the towers helped to differentiate the relative contribution of each. The LMA provided the time and location of triggering flash initiation and its spatial development relative to the towers. Particularly at the moment upward leaders initiated from the towers as determined by the optical assets.A timeline of events for each upward flash was created by combining data from each of the recording assets (the description of a case is presented below as an example). The high- and standard-speed optical assets provided the time of initial brightness increase associated with a triggering flash, leader spatial propagation behavior when visible below the cloud base  data was analyzed comparatively to the timeline created from the other assets. LDN event timing, polarity and magnitude (i.e. estimated peak current) were compared with fast antenna data and high-speed optical observations to further characterize impulsive events that took place during triggering flashes and upward leader development. LDN location data for these events further quantified the spatial development of these flashes.All the upward leaders from the towers in Rapid City and in S. Paulo during the summer observations developed in response to preceding lightning activity. Based on the analysis of the data, each triggering flash was assigned a flash type of either intracloud (IC) or cloud-to-ground (CG). Once a flash type was assigned, a triggering flash component responsible for the initiation of the upward propagating leaders was determined based on the spatial and temporal analysis.T-leader or the return stroke of the triggering flash. The propagation timeframe for these two possible triggering flash components (T-leaders or return strokes) are quite different. Leaders propagating in virgin air travel at speeds in the 104–105 ms−1 range and can cover large distances exceeding 100 km13. Therefore, this component can have a timeframe typically in the 10 s or 100 s of milliseconds lasting up to a second or more for an entire flash. On the other hand, a return stroke travels in the 107–108 ms−1 regime and is confined to the T-leader network formed prior to its initiation. Thus, the timeframe for a return stroke flash component is limited from tens of microseconds up to a couple of milliseconds for the case of a horizontally extensive T-leader network12. If the upward leader starts 3 milliseconds after the return stroke, the triggering component assigned is the continuing current. This threshold used is based on high speed video data of past studies on continuing current and it avoids contamination of what could just be return-stroke pulse tails15.In this study, we observed that the flash components that trigger the upward leader are either the propagation of a To summarize, for each of the 110 upward flashes recorded, a triggering flash type, and the triggering flash components were assigned based on analysis of the correlated data .T-leader activity increased in altitude to 6 km while traveling northeast. At 02:17:14.747 UT, the NLDN registered a 23 kA estimated peak current +IC event 19 km south of Tower 4. A fast electric field antenna measurement .After applying a similar analysis, as mentioned above, for the whole dataset, we observed that the flash components that trigger the upward leader are either T-Leader  does not reach the threshold for upward leader initiation, this threshold may be reached by the return stroke that subsequently traverses the T-leader network. The return stroke’s rapid electric field change serves as the triggering mechanism for upward leader initiation.  during an intracloud flash; (b) before a return stroke of a CG flash, and (c) during the occurrence of a continuing current event. When the electric field change associated with the leader activity that propagated near the towers before the return stroke does not reach the threshold for upward leader initiation, a return stroke that traverses a previously formed T-leader may cause the triggering of an upward flash. Both in USA and in Brazil, most of the upward flashes were triggered by leader propagation. In almost half of the cases the T-leader propagation occurs during the continuing current that follows a return stroke.Based on the analysis of 110 cases of triggered upward flashes in US and in Brazil, we observed that the majority of triggering flash types were +CG flashes and that the component of +CG flash that causes the required electric field change to initiate upward leaders from towers are either the nearby The instrumentation used during the upward lightning studies in Rapid City, South Dakota, USA and in S. Paulo, Brazil were composed of optical instruments, electrical field sensing and some auxiliary instrumentation described in this section.Optical assets included high- and standard-speed video cameras along with digital still image cameras. In South Dakota, USA, up to five high-speed cameras operating between 1,000 and 35,000 images per second (ips) were used to record lightning flash activity. Cameras were grouped into two mobile platforms, which were positioned at various locations from 600 m to 9 km from the towers. The two vehicles were spatially separated so that recordings could provide some level of 3-dimensionality when comparatively analyzed. Four of the most common locations used by the two vehicles are shown in Fig. Three fixed optical sites located on opposite sides of Rapid City captured continuously-recorded wide field of view standard-speed video at 60 ips as well as digital still images. Distances from these sites to each of the towers ranged from 2.5 km to 11 km.In São Paulo, Brazil, optical assets included two high-speed cameras operating between 1,000 and 10,000 ips and a single digital still camera and two standard-speed video cameras recording in standard-definition that provided 60 ips continuous video at extremely wide field of views. All cameras were grouped into a platform, which was positioned at 5 km from two towers Fig. .Figure 9In South Dakota, USA, the National Institute for Space Research (INPE) operated two flat-plate, fast antenna systems with a sample rate of 5 million samples/second and a lower and upper frequency bandwidth of 306 Hz and 1.5 MHz respectively. A high-gain and low-gain fast antenna were placed together approximately 5 km west of the towers Fig. . At thisIn São Paulo, Brazil, the same high-gain and low-gain fast antenna system as well as an EFM (sampling the ambient electric field at 50 samples/sec) were placed together approximately 11 km from the towers Fig. .The field meters were factory calibrated but not calibrated to account for local field enhancement at each individual sensor site. Therefore, they were not absolutely calibrated so that electric field magnitude could only be compared relatively between the three different site locations. Data obtained from the EFMs provided insight into the initial electric field polarity as well as direction and magnitude of change during upward flashes at varying distances and directions from the towers.12 will be used when referring to the electric field and its change. A positive electric field is an upward pointing field when the ground is positively charged and there is negative charge overhead. A positive field change would result if there were an increase in negative charge overhead.The physics sign convention18.The LMA centroid was not located over the towers due to additional research objectives not related to the Upward Lightning Triggering Study (UPLIGHTS)19. This required that it be placed further eastward onto the high-plains. This location did, however, provide good 3-dimensional resolution of lightning generated source points for triggering and upward flashes from the towers. The LMA system recorded using 80 µs time windows20.For the 2014 observation season in USA, a nine station Lightning Mapping Array (LMA) provided by New Mexico Tech was installed with the centroid approximately 50 km east-southeast of the towers22.For the 2011 observation season in Brazil, a ten station Lightning Mapping Array (LMA) also provided by New Mexico Tech was installed with the centroid approximately 50 km east-southeast of the towers24 for a 200 km radius around the towers was obtained and analyzed for this study. Analysis provided timing and location of impulsive events detected by the networks during triggering and upward flashes in Brazil and in USA.Lightning detection network stroke data


Here we tested a novel hypothesis that hypoxia-induced RyR-mediated Ca2+ release may, in turn, promote mitochondrial ROS generation contributing to hypoxic cellular responses in PASMCs. Our data reveal that application of caffeine to elevate intracellular Ca2+ concentration ([Ca2+]i) by activating RyRs results in a significant increase in ROS production in cytosol and mitochondria of PASMCs. Norepinephrine to increase [Ca2+]i due to the opening of inositol 1,4,5-triphosphate receptors (IP3Rs) produces similar effects. Exogenous Ca2+ significantly increases mitochondrial-derived ROS generation as well. Ru360 also inhibits the hypoxic ROS production. The RyR antagonist tetracaine or RyR2 gene knockout (KO) suppresses hypoxia-induced responses as well. Inhibition of mitochondrial Ca2+ uptake with Ru360 eliminates N- and Ca2+-induced responses. RISP KD abolishes the hypoxia-induced ROS production in mitochondria of PASMCs. Rieske iron–sulfur protein (RISP) gene knockdown (KD) blocks caffeine- or NE-induced ROS production. Taken together, these findings have further demonstrated that ER Ca2+ release causes mitochondrial Ca2+ uptake and RISP-mediated ROS production; this novel local ER/mitochondrion communication-elicited, Ca2+-mediated, RISP-dependent ROS production may play a significant role in hypoxic cellular responses in PASMCs.Mitochondrial reactive oxygen species (ROS) cause Ca A series of studies have revealed that ryanodine receptors (RyRs), the Ca2+ release channels on the Endoplasmic reticulum (ER), are important for the hypoxic increase in [Ca2+]i and contraction in PASMCs i in PASMCs has been thought to be attributed to the enhanced production of intracellular reactive oxygen species (ROS) due to their specific effects on ion channels  and [Na+]  with 2 μM coelenterazine w . After incubation, PASMCs were treated with different concentrations of caffeine and norepinephrine for 5 min. Luminescence intensities were recorded by luminometer. Luminescence unit was calibrated into Ca2+ concentration by following previous publications mito concentration and quantified as dose–response curve. As shown in 2+]mito in a dose-dependent manner. We assumed that caffeine- or NE-induced Ca2+ release might cause mitochondrial Ca2+ uptake, and accordingly mediate Ca2+-dependent mitochondrial ROS generation. To test this assumption, PASMCs were treated with Ru360 (1 µM), which is a mitochondria calcium uniporter (MCU) inhibitor i in PASMC [2+]i and contractile responses in PASMCs; however, the role of RyR2 dominates over that of RyR1 and RyR3 i may give rise to an increase in mitochondrial Ca2+ uptake, leading to ROS production in mitochondria. Consistent with our conjecture, treatment with Ru360, a specific mitochondrial Ca2+ uptake inhibitor i in PASMCs and pulmonary vasoconstriction in PAs i in pulmonary arteries and right ventricular systolic pressure [2+- and caffeine-induced ROS production in mitochondria from PASMCs. We have also shown that RIPS plays an important role in hypoxia-induced increase in [Ca2+]i in PASMCs. However, further experiments are needed to determine whether Ca2+ may produce a direct or an indirect effect on RISP-mediated mitochondrial ROS production in PASMCs.In support, Waypa et al. have also found that RISP gene depletion abolishes hypoxia-induced increase in [Capressure . In line2+ release from the ER via RyRs or IP3Rs can increase [Ca2+]i, mitochondrial Ca2+ uptake, and RISP-dependent mitochondrial ROS production (2+-induced ROS production may play an important role in the overall hypoxia-induced mitochondrial RISP-dependent ROS production, thereby contributing to Ca2+ and contractile responses in PASMC.Mitochondrial ROS are generated in both physiological and pathological conditions. On the one hand, moderate levels of ROS are involved in cell signaling by affecting the redox state of signaling proteins, Under physiological conditions, the balance between ROS generation and ROS scavenging is highly controlled . Howeveroduction . This no2+ release increases ROS production in mitochondria of PASMCs.Agonist-induced ER Ca2+ causes ROS production in isolated mitochondria. RYR2 antagonist or Ryr2 KO, mitochondrial Ca2+ uptake inhibitor, RISP KD all prevent the hypoxic ROS production.Exogenous Ca2+-mediated, RISP-dependent ROS production may significantly contribute to hypoxic cellular responses in PASMCs.ER/mitochondrion communication-elicited, CaClick here for additional data file.


This series on immunotherapies in acute myeloid leukemia (AML) aims to give readers new insights on established but also emerging immunotherapeutic approaches for AML patients. The therapeutic landscape in AML is rapidly changing, and several drugs have been developed and approved such as first and second generation FLT3 inhibitors ,2,3, IDHTherefore, immunotherapeutic approaches might be an option to prevent disease relapse and to eliminate leukemic cells or leukemic stem cells (LSC) that survive intensive treatment approaches. The efficacy of immunotherapeutic approaches has become ever more evident in solid tumors, especially immune-checkpoint inhibitors that are routinely used in several solid tumor entities, but also lymphoma ,12. Our focus in this special issue is different strategies of immunotherapeutic approaches in AML.Some of the immunotherapies in the treatment of AML, such as allogeneic hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI), have been part of routine clinical practice in the treatment of AML for a long time, whereas other immunotherapeutic approaches have only recently entered clinical practice or need to be further developed. A key aspect is the mechanisms underlying the cure of AML patients, which are based on the graft-versus-leukemia (GvL) effect, in which allogeneic T cells recognize target antigens on malignant cells by T cell approaches including DLI. An effective and well-tolerated regimen for HSCT in patients with AML and MDS is the FLAMSA-RIC regimen, and therefore novel data of this approach are presented in this issue .It is very appropriate to utilize DLI after allogeneic HSCT to prevent relapse, to prolong progression-free survival, to establish full donor chimerism, and to restore the GvL effect in patients with hematological malignancies. There are different strategies to use DLI in a therapeutic setting for the treatment of morphological relapse, and also for prophylactic use in AML/MDS and DLI administered preemptively. There is also the approach of antigen-directed immunogenic and specifically stimulated and modified DLI as well as virus-specific donor T cells and third-party DLI .DC-based immunotherapies also have the potential to bring about demonstrable clinical responses in AML patients, although there has not been a complete breakthrough for this type of therapy until today. Van Acker et al. have highlighted different DC strategies in AML .Leukemia-associated antigens (LAAs) represent immunogenic structures to target LSC ,17, and In this special issue, we also elucidate antibody-based therapies in AML, such as T cell activating antibodies including immune-checkpoint inhibitors and diverse monoclonal antibodies ,12,24. IChimeric antigen receptor T cells (CARs) are highly effective in the treatment of refractory and relapsed acute lymphoblastic leukemia, to some lower extent in aggressive lymphoma, but also in multiple myeloma . HoweverAll of these aspects emphasize the high potential of immunotherapeutic approaches to improve the survival of AML patients in the future, where combination therapies utilizing immunotherapeutic drugs could represent further innovation strategies to further improve the treatment of AML.


Pectis brevipedunculata (EOPB), a Brazilian ornamental aromatic grass, is characterized by its high content of citral , limonene (4.7%) and α-pinene (3.4%). Vasodilation induced by EOPB and isolated citral was investigated in pre-contracted vascular smooth muscle, using thoracic aorta from Wistar Kyoto (WKY) rats which was prepared for isometric tension recording. EOPB promoted intense relaxation of endothelium-intact and denuded aortic rings with the concentration to induce 50% of the maximal relaxation (IC50) of 0.044% ± 0.006% and 0.093% ± 0.015% (p < 0.05), respectively. The IC50 values for citral in endothelium-intact and denuded rings were 0.024% ± 0.004% and 0.021% ± 0.004%, respectively (p > 0.05). In endothelium-intact aorta, EOPB-induced vasorelaxation was significantly reduced by l-NAME, a nitric oxide synthase inhibitor. The vasodilator activity of citral was increased in the KCl-contracted aorta and citral attenuated the contracture elicited by Ca2+ in depolarized aorta. EOPB and citral elicited vasorelaxation on thoracic aorta by affecting the NO/cyclic GMP pathway and the calcium influx through voltage-dependent l-type Ca2+ channels, respectively.The essential oil of  Pectis is a genus composed by small herb plants of the daisy family generally considered as weeds in current usage [Pectis are native to the Americas, including the Caribbean, being found also in the Pacific Islands [Pectis elongata have been popularly utilized in tea and as spices in French Guyana to replace the lemongrass species, Cymbopogon citratus, Poaceae [Pectis species have traditional use as infusions or juice drink preparations for hypertension, stomach disorders and colds [Pectis tea preparations [In Brazil, many plant species are known as lemongrass due to the citric fragrance from their volatile constituents. nt usage . Some sp Islands . These s Islands . Due to  Poaceae . In soutnd colds . Calmatiarations ,7.Pectis brevipedunculata, a Brazilian ornamental aromatic grass, is one of the “lemongrass odor” correlated species. Its essential oil content is characterized by the high percentage of citral (up to 78%). This monoterpene aldehyde fraction is normally composed by a mixture of the two geometric cis- and trans-isomers: geranial and neral. Many biological activities were described to this aldehyde monoterpene fraction. Bacteriostatic and fungistatic properties have already been related to essential oils rich in citral [Cymbopogon citratus [Cymbopogon citratus and citral induced vasodilation in isolated thoracic rat aorta and demonstrated that citral may affect the intracellular calcium concentration on vascular smooth muscle cells [Pectis brevipedunculata (EOPB) and its major constituent citral on rat isolated thoracic aorta and elucidated the mechanism underlying this activity.l up to 7%. This mn citral ,10,11. Icitratus . In paticitratus . Recentlle cells . The preP. brevipedunculata was 0.4% and the chemical analysis of the investigated EO is presented in P. brevipedunculata volatile fractions consists of monoterpene compounds, hydrocarbons, sesquiterpenes, alcohols and aldehydes. The GC-FID and CG/MS methods were used to identify and quantify these compounds. The essential oil was characterized by a high percentage of citral  of P. brevipedunculata EO occurring in lemon grass (C. citratus) where citral is found up to 70.0% in the mixture. In addition, the presence of limonene in the EO of P. brevipedunculata contributes to enhance the lemon fragrance of the composition.The relative amount (%v/w) of the essential oil (EO) of fresh aerial parts of 50) in the phenylephrine-induced contraction of endothelium-intact and denuded rings from WKY rats were 0.044 ± 0.006% (n = 4) and 0.093 ± 0.015%, respectively  and 1.33 ± 0.18 mM, respectively  to 0.081 ± 0.013% . Considering the endothelial involvement in the relaxation of EOPB in aortic rings, we investigated the possible involvement of the NO/cyclic GMP pathway in the endothelium-dependent mechanism of EOPB. The incubation of aortic rings with l-NAME  signific+-induced contraction was investigated. When endothelium-denuded aortic rings were contracted with high K+ extracellular concentration (80 mM), the relaxation after exposure to citral was increased .In another set of experiments, the effect of citral on the Kncreased . Under t2+ influx through voltage-dependent Ca2+ channels. Concentration-response curves for Ca2+ were obtained in the absence and presence of citral, in depolarized aorta from WKY rats. Citral effectively attenuated the contractile response to Ca2+ in aortic rings. Maximal contraction induced by 10 mM CaCl2 was reduced to 53.38 ± 5.33% of the control  or completely abolished when in the presence of 0.6 mM and 6 mM of citral  is used in the folk medicine as a high blood pressure treatment. Many hypertensive patients drink Cymbopogon citratus tea daily due to its calmative, anxyolitic as well as anti-hypertensive properties. The citrus scent of C. citratus essential oil is mainly a results of the high content of neral and geranial  in its composition, followed by geraniol. The main components of the volatile fractions in the Pectis brevipedunculata EO are neral and geranial  followed by nerol and geraniol as minor derivatives. The EO of P. brevipedunculata contains 80%–90% of citral, placing this native Brazilian plant as a potential natural source of citral-rich essential oil. Similar fragrances common to Pectis species could explain the traditional use of that lemongrass. In agreement with this similarity, ethnopharmacological studies have reported the traditional use of infusions of P. brevipedunculata and P. jangadensis by some communities in Brazil to produce anxyolisis [P. elongata Kunt and P. linifolia L. var. linifolia have been used in the folk medicine to treat hypertension and gastric diseases, while P. oligocephala (Gardner) Sch.Bip. is usually indicated to treat cold [P. brevipedunculata are quite similar, showing citral as main compound in the mixture  in both species. This fact could explain their use in folk medicine for the same purposes. Unfortunately, no data is available in literature concerning the phytochemical and pharmacological profile of tea drinks extracted from Pectis species. However, some ethnopharmacological data are available for citral and tea preparations from C. citratus. Despite the strong popular indication, some discrepant results can be found. Caluscusin has found that twice-a-day intake of lemongrass decoction had a significant effect on the mean arterial pressure, probably due to a diuretic action of the tea [In Brazil,  citral >0.0% in ieat cold ,5. The q the tea . On the  the tea ,18,19 an the tea . That co the tea .C. citratus such as saponins, sesquiterpenes, lactones, alcacaloids, tanins, steroids, triterpenes and flavonoids [Additionally, it should be considered that the effective dose of EO is still inconsistent with the amount of fresh leaves frequently used to make a cup of tea (2 to 10 g). Considering the average extraction yield of 0.5%, roughly 200 g of fresh leaves should be required for EO doses of 1.0 g/kg. Moreover, differences have been found in dosage regimen, typically in repeated or chronic intake of tea in comparison to acute treatment tested experimentally. In addition, different proportions of the EO could be found in tea preparation or infusions. Several secondary metabolites were already found in aqueous solutions of avonoids ,23.Shimono and co-workers reported that inhalation of a synthetic citral formulation reduced blood pressure. The antihypertensive agent comprises citral and linalol and has a mass ratio of citral to linalol of not less than 10:1 and not greater than 40:1. This formulation could be used as antihypertensive agent for medical drugs, foods, beverages and feeds. The mass ratio of 20:1 is preferred because the greatest effect was exhibited .Although citral is reported as an antihypertensive agent, it is suggested that the concentration found in the small quantity of plant material used in a cup of tea could not be sufficient to the antihypertensive activity. Therefore, the reduction of blood pressure in humans observed in some studies may be related to the presence of other compounds in lemongrass tea.Cymbopogon citratus [Cymbopogon citratus and citral on isolated thoracic rat aorta suggested an impairment of the phenylephrine-induced contraction in Ca2+-free solution and a reduction of the CaCl2-induced contraction in endothelial intact and denuded aortic rings pretreated with citral [2+ influx from the extracellular space possibly via receptor-operated calcium channels or the Ca2+ release from intracellular storage sites.Transient hypotension and bradycardia were previously reported in rats treated with the citral-rich essential oil obtained from citratus . Additiocitratus . Recent h citral . These f+ and phenylephrine-induced contractions. Moreover, the vasodilator effect of citral on KCl-induced contraction was more potent than on phenylephrine-induced contraction. It is well known that high K+-induced contraction in vascular smooth muscle is mediated by cell membrane depolarization and consequently an increase in Ca2+ influx through voltage-dependent l-type Ca2+ channels [2+ influx through l-type Ca2+ channels, (2) Ca2+ influx through non-l-type Ca2+ channels such as receptor-operated calcium channels (ROC) [2+ sensitivity [2+ release from sarcoplasmic reticulum (RS) [1-adrenergic receptors on vascular smooth muscle, leading to the formation of second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 promotes Ca2+ release from the RS and DAG activates the protein kinase C (PKC) which, in turn, phosphorylates and (1) activates ROC channels and promotes calcium influx; (2) inhibits K+ channels and leads to cellular depolarization which opens l-type calcium channels; (3) activates Raf–MEK–MAPK and RhoA/Rho-kinase pathways, enhancing the myofilament force sensitivity to Ca2+. Thus an l-type Ca2+ channel inhibitor induces less potent inhibition on phenylephrine-induced contraction than on depolarization-induced contraction. Secondly, citral impaired extracellular Ca2+ influx through voltage-dependent Ca2+ channels by reducing the contraction mediated by CaCl2 in depolarized vascular smooth muscle. Taken together, these results suggest that citral inhibits the extracellular Ca2+ influx through the blockade of voltage-dependent l-type Ca2+ channels.In the present study, we demonstrated an endothelium-independent vasodilator action elicited by citral on thoracic aorta from WKY rats. Citral concentration-dependently inhibited high Kchannels ,26. In cls (ROC) , (3) enhsitivity , and (4)lum (RS) . Phenylel-type Ca2+ channels, the reduction of intracellular calcium concentration via receptor-operated calcium channels or the Ca2+ release from RS has already been described [2+. Citral could decrease the myofilament sensitivity by activating the myosin light chain (MLC) phosphatase, which causes MLC dephosphorylation and reduces actin-myosin interaction. Alternatively, citral could inhibit the MLC kinase with consequent reduction of phosphorylated MLC and actin–myosin interaction. And also, could interfere with the formation of Ca2+-calmodulin complex and thus with the vascular smooth muscle contraction. Finally, citral could inhibit Raf-MEK-MAPK or RhoA/Rho-kinase pathways, decreasing myofilament force sensitivity and leading to vasorelaxation [In addition to the mechanism of citral described in the present work, which is related to the inhibition of escribed . Therefolaxation .Pectis brevipedunculata (EOPB). This essential oil also induced a concentration-dependent vasodilation on thoracic aortic rings from WKY rats. Endothelium removal and the pretreatment with l-NAME caused partial inhibition of the EOPB vasorelaxant response. Nitric oxide is likely to be the endothelium-dependent component, since inhibition of the EOPB response by l-NAME was comparable to that by endothelium removal. Our findings suggest that EOPB-mediated vasorelaxation contained an endothelium-dependent component, involving the NO/cyclic GMP pathway. Additionally, in the presence of l-NAME, higher concentrations of EOPB which contains high concentration of citral, induced the same relaxation of the control indicating that the endothelium-independent component of EOPB-induced vasodilation could be related to the l-type calcium channels blockage. At high concentrations of EOPB, the endothelium-independent vasorelaxation should predominate in relation to the endothelium-dependent pathway.We also investigated the vascular effects of the essential oil of et al. analyzed the anti-proliferative effect of lemongrass essential oil on three evolutive forms of Trypanosoma cruzi, which was effective against T. cruzi trypomastigotes and amastigotes. In this study, no cytotoxic effects were observed when mouse peritoneal macrophages were incubated with lemongrass essential oil at concentrations corresponding to the IC50 for trypomastigotes, suggesting a lack of cytotoxic effects against mammalian cell type [et al., citral was cytotoxic on tumoral cells (human cervix epithelioid carcinoma cells) but not on non-tumoral cells (african green monkey kidney cells) [Hayes and Markovic demonstrated that citral has antimicrobial activity against a range of Gram positive and Gram negative bacteria, but cytotoxicity assays suggest that citral is toxic to human liver- derived cells, skin cells and skin fibroblasts. Despite the described cytotoxic activity of citral, a product containing 1% lemon myrtle oil  was significantly less toxic to human skin cells and fibroblasts . On the ell type . Finallyy cells) . ConsidePectis bervipedunculata were collected in Rio de Janeiro, RJ in April 2011. The botanical vouchers were identified by Dr. Roberto L. Esteves and kept at the Herbarium (HB) of the Rio de Janeiro National Museum under number 1007 (R). The fresh aerial parts were submitted to hydrodistillation for 2 h in a modified Clevenger-type apparatus. The obtained essential oil (EO) was dried over anhydrous sodium sulphate, yielding 0.4% w/w which was immediately stored in closed dark vials at 4 °C until analysis.Aerial parts (100 g) of −1 was used as carrier gas. The percentages of the compounds were obtained by GC-FID analysis.Quantitative and qualitative analysis were carried out on a Shimadzu GC 2010 machine equipped with a ZB-1MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness). The operating temperatures used were: injector 260 °C, detector 290 °C and column oven 60 °C up to 290 °C (10 °C/min). Hydrogen at 1.0 mL·min−1 was used as carrier gas for GC/MS. The essential oil components were identified by comparison of their retention indices and mass spectra with published data [n-alkanes.Qualitative analyses were carried out on a GC-QP2010 PLUS Shimadzu machine with a ZB-5MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm film thickness). The operating temperatures used were: injector 270 °C, detector 290 °C and column oven 60 °C up to 290 °C (3 °C/min). Helium at 1.0 mL·minhed data  and compFor HSCCC, we selected a two-phase solvent system composed of hexane/ACN . The distribution of the components in the EO was estimated by thin-layer chromatography  with hexane/EtOAc (3:2) as the eluting solvent. The separation of compounds was observed under a UV lamp at 254nm and by spraying sulfuric acid/methanol reagent , followed by heating to assist visual estimation of the relative distribution of the compounds in each phase. The solvent mixture was hand mixed and thoroughly equilibrated in a separation funnel at the same temperature as in the vessel of HSCCC and the two phases were separated shortly before use. The sample solutions were prepared by diluting the essential oil in the mixture solution of lower and upper phases  of the used solvent system for HSCCC separation and vigorously hand mixing before the TLC analysis.A CCC , consisting of a PTFE 80-mL coil, a HPLC pump , a low-pressure injection valve , and a PTFE 5-mL sample loop were used. This system was coupled to a fraction collector  programmed to collect at 1-min intervals. Appropriate volumes of solvents hexane/ACN  were vigorously hand-mixed in a separatory funnel, transferred to a flask, degassed (ultrasonic bath) for 30 min. Isocratic elution was conducted in a tail-to-head manner, with the acetonitrile (lower phase) as stationary phase . The coil was entirely filled with the lower phase of the solvent system with no rotation. Rotation of the coil was then started at 860 rpm, and the upper organic phase was pumped at a flow rate of 1.0 mL/min. Hydrodynamic equilibrium was attained prior to sample injection. The stationary phase retention was about 72.5%. The crude essential oil (1 mL) was dissolved in the biphasic solvent system and injected into to the apparatus separately. Fractions (240) of 1 ml each were collected in 200 min. The collected fractions were analyzed by TLC, GC – FID, GC-MS. About 400 μL of citral was obtained in high level of purity >98.0%.The protocols used in the present study were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro.2 1.2, KH2PO4 1.2, glucose 11.5, NaHCO3 15.5, CaCl2 1.2; bubbled with 95% O2/5% CO2 and maintained at 37 °C. After a 2 h equilibrium period of 1 g resting tension, the aortic rings were contracted with phenylephrine (10 µM), followed by exposure to acetylcholine (10 µM) to test the integrity of the endothelium. Acetylcholine-induced relaxation more than 80% denoted the presence of intact endothelium. In some experiments, EOPB or citral was tested in aortas in which the endothelium had been mechanically removed [+ solution (80 mM KCl) was made by substituting NaCl with equimolar KCl.Thoracic aortas were removed from 15- to 17-week-old male Wistar Kyoto rats (WKY), cleaned of connective tissue and prepared for isometric tension recording, as previously described . Aortic  removed . To inve2+ influx in the smooth muscle, denuded rings were equilibrated in Ca2+-free saline solution for 15 min. The solution then was replaced with Ca2+-free/high-K+ solution (80 mM). Aortic rings were kept in this depolarized state for 15 min, in the absence or presence of 0.6 or 6 mM citral, after which increasing concentrations of CaCl2 (10−6–10−2 M) were added.To investigate the effects of citral on Caω-nitro-L-arginine methyl ester (l-NAME) [In other experiments, endothelium-intact aortic rings from WKY rats were incubated for 20 min with 100 μM N(l-NAME) , a selecω-nitro-L-arginine methyl ester (l-NAME) were purchased from Sigma Chemical Co. . All compounds were dissolved in distilled water. EOPB and citral  were dissolved in distilled water. Phenylephrine, acetylcholine and N50) was determined for each experiment. The concentration-response curve was fitted to the following equation: ymax = ymin + a/(1 + e−x−x0/b), where y is the percentage of isometric tension; a = ymax − ymin; b = slope; x0 = IC50. The variable x is the concentration of EOPB or citral to produce relaxation. Differences between 2 groups were determined with the unpaired Student’s t-test and were considered significant when p was <0.05.All data are expressed as the mean ± standard error of the mean (SEM). The concentration necessary to reduce the phenylephrine-induced contraction by 50%  elicited vasorelaxation of thoracic aorta by affecting the NO/cyclic GMP pathway. In addition, our results suggest that citral reduced the calcium influx by the blockade of voltage-dependent l-type Ca2+ channels. Thus Pectis brevipedunculata, a Brazilian native plant, could be used to reduce high blood pressure.In this work, we have demonstrated that


Here we provide an overview of changes in Ca2+ homeostasis in diabetic ventricular myocytes and discuss the therapeutic potential of targeting Ca2+ handling proteins in the prevention of diabetes-associated cardiomyopathy and arrhythmogenesis.A rapid growth in the incidence of diabetes and obesity has transpired to a major heath issue and economic burden in the postindustrial world, with more than 29 million patients affected in the United States alone. Cardiovascular defects have been established as the leading cause of mortality and morbidity of diabetic patients. Over the last decade, significant progress has been made in delineating mechanisms responsible for the diminished cardiac contractile function and enhanced propensity for malignant cardiac arrhythmias characteristic of diabetic disease. Rhythmic cardiac contractility relies upon the precise interplay between several cellular Ca Diabetirtension .Diabetes is a chronic metabolic disorder characterized by hyperglycemia and reduced glucose utilization due to defective insulin secretion or action . Type 1 2+ concentration [Ca2+], leading to rhythmic contraction and relaxation of cardiomyocytes in response to variable metabolic demand . Depolarization of the sarcolemma and activation of voltage-dependent L-type Ca2+ channels (LTCCs) leads to Ca2+ influx into the cytosol. This small influx subsequently triggers a much larger Ca2+-induced Ca2+ release . The significant global increase in cytosolic [Ca2+], known as the Ca2+ transient, activates contractile machinery and leads to muscle contraction. For relaxation to then occur, Ca2+ must be sequestered and the intracellular [Ca2+] decreased. This is primarily by extrusion from the cytosol via the Na+/Ca2+ exchanger (NCX1), or resequestration into the SR via the sarco/endoplasmic reticulum-ATPase (SERCa2a).In the heart the process of excitation-contraction (EC) coupling drives cyclic changes in intracellular Cac demand . Cardiacse CICR;  from the2+ cycling machinery has been implicated in reduced contractility and proarrhythmic electrical instabilities in a variety of inherited and acquired cardiac diseases including HF and diabetic cardiomyopathy  of sarcolemma implicated in initiation of triggered activity in the heart  hyperglycemic rodents, while obesity can also be induced by high fat diet (HFD) or high-sucrose diet  during action potential initiates Ca2+ release from the sarcoplasmic reticulum (SR). The LTCC consists of the pore forming subunit α1c, and regulatory subunits α2/δ and β2  were linked to hereditary Long QT syndrome type 8 and 14 , the decrease in ICa originates from a reduced number of channels in the sarcolemma. Similar results were obtained in the Akita mouse model  directly associates with and regulates RyR2 channels ) and phosphatases  that dynamically and reversibly modulate its phosphorylation state , a condition characterized by pathogenic mutations in RyR2  on RyR2 are significantly increased in diabetic heart tissue, and this increase could be partially attenuated with insulin treatment  overexpression. This revealed increased Ca2+ spark activity compared to controls that could be reduced by application of antioxidant mitoTEMPO. A significant increase in mitochondrial oxidative stress was suggested to increase RyR2 oxidation and subsequent SR Ca2+ release. In 8-week mice with HFD-induced obesity, 2+], whereby channels were much more active to those isolated from control mice. No changes were observed in RyR2 expression levels or phosphorylation status at S2808 or S2814 sites. Instead, this phenomenon was attributed to significantly increased RyR2 oxidation in HFD-mice, implicating the diabetes-related increase in oxidative stress in abnormal Ca2+ handling. Changes in RyR2 in HF and diabetes are summarized in Table Abnormal intracellular lipid concentration is a hallmark of both obesity and diabetes, and 2+] is decreased primarily via sequestration into the SR by SERCa2a, the primary cardiac SERCa isoform  or CaMKII at Threonine 17 (T17), relieves SERCa2a inhibition and increases activity  rats. No changes in protein expression were observed in the db/db mouse, but rather an increased PLB expression was posited to account for diminished SR Ca2+ uptake in this model. 2+ uptake in db/db mice. Conversely, increased expression of SERCa2a and reduced PLB mRNA was observed in ZDF rats, which could be further increased with insulin treatment in a concentration-dependent manner  is activated by intracellular Ca2+ in submicromolar range transporting one Ca2+ ion in exchange to thee Na2+ ions  prediabetic mice. The authors showed increased activity of RyR2 manifested in enhanced frequency of spontaneous Ca2+ waves in field-stimulated myocytes. Mice expressing SR-targeted CaMKII inhibitor peptide AIP showed less ectopic activity that WT FRD mice, and in rats FRD-mediated increase in pro-arrhythmic spontaneous Ca2+ release was eliminated by incubation of myocytes with pharmacological inhibitor of CaMKII KN93 or antioxidant Tempol. The authors conclude that RyR2 phosphorylation by ROS-activated CaMKII at CaMKII site S2814 plays a major role in diabetes-related arrhythmogenesis. Interestingly, the authors did not find changes in RyR2 phosphorylation at PKA site S2808. Earlier work by 2+-dependent triggered activity in rats. The authors proposed that acute hyperglycemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc), resulting in prolonged activation of CaMKII and subsequent increase in RyR2 CaMKII phosphorylation and thereby its activity.The enhanced propensity to ventricular tachyarrhythmias in diabetic and obese patients is well established . The arr2+ of mitochondrial function and SR Ca2+ handling machinery by mitochondrial ROS  and PKA  . Angiote and PKA . Angiote and PKA . Antagon and PKA , while aeostasis .2+ handling proteins , carvedilol, dantrolene and tetracaine analogs have previously been tested for therapeutic potential . However2+ leak .2+ homeostasis and attenuates arrhythmogenesis (2+ release events, blunting diastolic dysfunction and arrhythmogenesis in vivo (2+ leak (Stabilization of RyR2-mediated Ca release can be achieved indirectly by targeting CaMKII, given chronic activity in cardiac disease has been linked to RyR2 channel dysfunction . It has ogenesis , includiogenesis , but thiogenesis . Alterna in vivo . Antioxi(2+ leak . While i(2+ leak .2+ transient amplitude of isolated rat cardiomyocytes (2+ release, abolishing ventricular arrhythmias observed in a mouse model of CPVT.Targeting accessory proteins of RyR2 also offers therapeutic potential. Adenoviral overexpression of sorcin in the hearts of diabetic mice improved contractile function and increased the Camyocytes . In rece2+ homeostasis vary across different models of diabetes and obesity. Diabetes is a progressive disease and therefore results from different laboratories using the same model can differ. Longitudinal studies are warranted to resolve the ongoing discrepancies. Although there are many similarities with HF, there are substantial differences in Ca2+ handling in diabetic cardiomyopathy so the treatment strategies could be different.In conclusion, changes in cardiac Ca2+ cycling sufficient to increase arrhythmic potential appear very early during disease progression of diabetes. The most consistent finding even at very early stages of disease is enhanced RyR2 activity, making it an attractive therapeutic target. Future studies are needed to identify the most suitable approaches for RyR2 stabilization in diabetes. SERCa2a is also attractive target, especially at the later stages of disease progression and cardiomyopathy development. However, it remains unclear whether SERCa2a function is depressed at the early stages when arrhythmic risk is already high. The emerging concept of a mitochondria-SR microdomain and its potential as a therapeutic target may warrant investigation, given mitochondrial dysfunction is a well-established in diabetes. However, levels of mitochondrial ROS emission should be confirmed at different stages of disease, given work of some laboratories suggesting there may be an adaptive improvement in metabolism of fatty acids and thus a reduction in ROS emission. Furthermore, there remains a need for studies using larger animal models of type 2 diabetes with physiology more analogous to that of humans.Abnormalities in CaSH and DT obtained funding, conceived of, and wrote the 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.


Otoferlin, a multi-C2 domain protein, is proposed to regulate Ca2+-triggered exocytosis at this synapse, but the precise mechanisms of otoferlin function remain to be elucidated. Here, performing whole-cell voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from SGNs in otoferlin mutant mice, we investigated the impact of Otof disruption at individual synapses with single release event resolution. Otof deletion decreased the spontaneous release rate and abolished the stimulus-secretion coupling. This was evident from failure of potassium-induced IHC depolarization to stimulate release and supports the proposed role of otoferlin in Ca2+ sensing for fusion. A missense mutation in the Otof gene (pachanga), in which otoferlin level at the IHC plasma membrane was lowered without changing its Ca2+ binding, also reduced the spontaneous release rate but spared the stimulus-secretion coupling. The slowed stimulated release rate supports the hypothesis that a sufficient abundance of otoferlin at the plasma membrane is crucial for the vesicle supply. Large-sized monophasic EPSCs remained present upon Otof deletion despite the drastic reduction of the rate of exocytosis. However, EPSC amplitude, on average, was modestly decreased. Moreover, a reduced contribution of multiphasic EPSC was observed in both Otof mutants. We argue that the presence of large monophasic EPSCs despite the exocytic defect upon Otof deletion supports the uniquantal hypothesis of transmitter release at the IHC ribbon synapse. Based upon the reduced contribution of multiphasic EPSC, we propose a role of otoferlin in regulating the mode of exocytosis in IHCs.Sound encoding relies on Ca Excitatory postsynaptic currents (EPSCs) recorded from type I spiral ganglion neurons (SGNs) show remarkable variability in amplitude and shape. This EPSC heterogeneity led to the hypothesis of synchronized multiquantal release (MQR) at hair cell synapses , Pga/PgaOtof and -/-Otof mice and investigated exocytosis at the levels of single synapses and single release events. Analyzing the spontaneous and stimulated release rates as well as the amplitude and shape of EPSCs, we find evidence for roles of otoferlin in Ca2+-dependent fusion and replenishment of vesicles. Moreover, we propose an additional role of otoferlin in regulating the exocytic mode of IHCs to facilitate multiphasic EPSCs, potentially by controlling the vesicle fusion pore during uniquantal release at the IHC ribbon synapse.In order to further examine the otoferlin’s function we performed postsynaptic voltage-clamp recordings from afferent boutons of type I SGNs in wild-type  as well as National Rehabilitation Center for Persons with Disabilities animal experimentation committee (Japan).Pga/PgaOtof (-/-Otof (+/+Otof ), which were not littermates of Pga/PgaOtof or -/-Otof mice, were employed. In total, 33 mice  were used in the present study. After decapitation under deep carbon dioxide inhalation anesthesia, the apical coils of organ of Corti were harvested out of the cochlea.Postnatal day (P) 8–11 mice of either sex were used. Generation and general description of fPga/Pga  and Otof for both dissection and recording contained (in mM): 5.8 KCl, 155 NaCl, 0.9 MgCl2, 1.3 CaCl2, 0.7 NaH2PO4, 5.6 D-glucose, and 10 HEPES, pH 7.4. In the high K+ extracellular solution to depolarize presynaptic IHCs, 40 out of 155 mM NaCl were replaced with equimolar KCl. In most recordings, tetrodotoxin (1–2 μM) was added to block voltage-gated Na+ channels. Currents were low-pass filtered at 5–10 kHz and sampled at 20–50 kHz. EPSCs were recorded at a holding potential of -90 mV  at room temperature (21–24°C).Whole-cell voltage-clamp recordings from postsynaptic boutons of mouse type I spiral ganglion neurons in apical coils of freshly dissected organ of Corti were performed as previously described for rats  and for All chemicals were purchased from Sigma-Aldrich  except for tetrodotoxin . The EPC-10 amplifier controlled by Patchmaster software  as well as an upright microscope with differential interference contrast optics  was used.s, 40 ± 2 MΩ for 13 +/+Otof  SGNs, 41 ± 3 MΩ for 7 Pga/PgaOtof SGNs, 40 ± 2 MΩ for 13 -/-Otof SGNs), membrane capacitance  and membrane input resistance  were calculated as previously described  not to ignore very low-frequency auditory nerve fibers. Data is shown as mean ± SEM. Statistical significance was evaluated by Student’s t-test or one way ANOVA followed by post-hoc Tukey’s test.For detection and analysis of EPSCs, MiniAnalysis software  was used with a detection threshold set at 3–5 times greater than the root mean square (rms) of the baseline noise. To classify EPSCs into mono- or multiphasic, the methods introduced by escribed . Recordi+/+Otof , Pga/PgaOtof (missense mutation with reduced abundance of otoferlin) and -/-Otof mice. At rest (1.3 mM Ca2+ and 5.8 mM K+ in aPL), SGNs of all three genotypes steadily exhibited spontaneous EPSCs  and 0.14 ± 0.04 Hz for -/-Otof SGNs . Hence, unlike disruption of synaptotagmin I or II at the central synapses, which is thought to unclamp spontaneous release  was bath-applied onto IHCs to increase the open probability of their CaV1.3 L-type Ca2+ channels. The frequency of EPSCs in +/+Otof  SGNs was increased from 0.25 ± 0.11  to 9.67 ± 2.90 Hz  was undervalued due to our preferential application of high K+-containing aPL onto low-frequency synapses, since remaining more active synapses  provided a sufficient number of EPSCs for the analyses of EPSC size and shape even in the normal K+-containing aPL. In Pga/PgaOtof SGNs, high K+ stimulation increased the frequency of EPSCs in 4 out of 5 Pga/PgaOtof SGNs from 0.20 ± 0.10 (control) to 1.86 ± 0.57 Hz , indicating that the missense Otof mutation in Pga/PgaOtof mice spares stimulus-secretion coupling in IHCs. The reduced rate of stimulated release in Pga/PgaOtof IHCs (p = 0.032 compared to +/+Otof ) is compatible with impaired vesicle replenishment , indicating that Otof deletion abolishes stimulus-secretion coupling in IHCs. The difference in stimulated release rate between +/+Otof  and -/-Otof SGNs again showed significance (p = 0.010).Next, we examined the stimulus-secretion coupling in IHCs. High Knishment . In contOtof disruption on the size and shape of EPSCs. As reported in previous studies using the organ of Corti of rats  or the fraction of multiphasic EPSCs (41.9 ± 3.8 % for control vs. 41.1 ± 1.7 % for high K+). Moreover, EPSC size and kinetics distributions were unaltered by high K+ stimulation in each phenotype SGNs . There was a trend to smaller EPSC amplitudes also for Pga/PgaOtof SGNs, which, however, did not reach significance. EPSC kinetics , on the other hand, was not different among those three groups . Thus, monophasic EPSCs dominate transmission in the absence of otoferlin, suggesting that otoferlin regulates the mode of spontaneous and stimulated release at the first auditory synapse.Notably, the scatter plot of EPSC amplitude versus charge shows the predominance of monophasic EPSCs clustering around the unity line in SGNs of both s Figure . The fra-/-Otof SGNs. As Ca2+ influx and vesicle availability on a morphological level are maintained at the AZs of -/-Otof IHCs (2+ no longer efficiently drives fusion in the absence of otoferlin. This supports the role of otoferlin as a Ca2+ sensor of fusion (2+ sensor whose capacity is saturated at resting [Ca2+]i and (2) a Ca2+-independent release process. For (1), the activation range of the remaining Ca2+ sensor should be below the resting [Ca2+]i of a few tens of nanomolar (2+ sensor for fusion is the C2 domain protein Doc2b that promotes membrane fusion at submicromolar Ca2+ concentrations (but higher than 100 nM)  .2+-evoked release that is triggered by rare openings of CaV1.3 channels. We consider it likely that the reduction of spontaneous release reflects a combination of disrupted Ca2+-triggered spontaneous release of IHCs and reduced vesicle replenishment.Interestingly, the absence of otoferlin did not increase the spontaneous EPSC frequency, which is in contrast to the effect of ablation of synaptotagmin I  or II P in the mPga/PgaOtof mice, wherein otoferlin membrane abundance is attenuated down to 3% of +/+Otof  mice , which alter its Ca2+-binding (2+-sensing of otoferlin is critical for both steps of exocytosis (2+-sensing capacity but also plasma membrane level of otoferlin is essential for IHC exocytosis.Previous studies showed that otoferlin abundance at the IHC plasma membrane correlates with the presynaptic function and sound encoding . OtofPga+/+ mice , displays Figure  suggests-binding  but pres-binding , impairsocytosis . Thus, n2+]i conditions break EPSCs down into unitary events. In contrast, at the rodent IHC ribbon synapse the EPSC amplitude remains sizable despite the massive buffering of presynaptic Ca2+ (2+ influx (The heterogeneity of EPSC amplitude and shape is a hallmark of ribbon synapses, but the underlying mechanisms are not well understood. Notably, the mammalian IHC ribbon synapse differs from other ribbon synapses such as the amphibian and reptile hair cell synapses  as well tic Ca2+  or aboli+ influx . This an+ influx  activate+ influx .-/-Otof IHC afferent synapse (Figure Otof disruption. Besides, either hypothetic mechanism might be modulated by altered Ca2+ signaling at the release site as might have resulted from the scaffolding function of Otoferlin for Ca2+ channels (2+ currents in Otoferlin-lacking IHCs (2+ signals at the individual IHC AZs in Pga/PgaOtof mice (Given the presence of large monophasic EPSCs at the e Figure , i.e., uchannels  or attening IHCs . HoweverPga mice . FurtherPga/PgaOtof SGNs (Figure A smaller fraction of multiphasic EPSCs was also found in s Figure  and we ss Figure . It is ns Figure  may conts Figure . By promHT and TM designed the study. HT and TO-T performed experiments and analyzed the data. HT, TO-T, and TM wrote the manuscript. The experiments were performed at the University Medical Center Göttingen and Research Institute of National Rehabilitation Center for Persons with Disabilities.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.


Leukemia, specifically acute myeloid leukemia (AML), is a common malignancy that can be differentiated into multiple subtypes based on leukemogenic history and etiology. Although genetic aberrations, particularly cytogenetic abnormalities and mutations in known oncogenes, play an integral role in AML development, epigenetic processes have been shown as a significant and sometimes independent dynamic in AML pathophysiology. Here, we summarize how tumors evolve and describe AML through an epigenetic lens, including discussions on recent discoveries that include prognostics from epialleles, changes in RNA function for hematopoietic stem cells and the epitranscriptome, and novel epigenetic treatment options. We further describe the limitations of treatment in the context of the high degree of heterogeneity that characterizes acute myeloid leukemia. Acute myeloid leukemia is a common, heterogeneous, and aggressive hematopoietic malignancy , which cPML) gene, which encodes an eponymous tumor-suppressor protein (RARA) gene on chromosome 17, resulting in the PML-RARA fusion gene  inhibits regularly induced cellular apoptosis  chromosomal abnormality is a translocation that fuses the promyelocytic locus  translocation is reciprocal – it generates both  fusions , with bo fusions  – the PMportance . The PMLleukemia , a subtyleukemia .PML-RARa product works in tandem with DNA methyltransferases (DNMTs) to induce hypermethylation in PML-RARa targets, particularly RARβ  (RUNX1 gene encodes runt-related transcription factor 1 (or acute myeloid leukemia factor 1/AML1) and plays a regulatory role in hematopoietic development , a transcription factor involved in cell cycle regulation and myeloid differentiation  chromosomal translocation fuses the ML1-ETO) . The RUNelopment , includielopment . Mutationtiation . The AMLpression , playingpression .In the AML1-ETO fusion protein, the ETO domain aids in the recruitment of histone deacetylases , epigenevia inhibition of the lysine acetyltransferase p300  may affect leukemic potential  contributes to the leukemic phenotype: AML1-ETO product-mediated recruitment of a DNA methyltransferase to the BASP1-promoter region leads to promoter methylation and ultimate silencing of BASP1, the expression of which regulates the cell cycle and arrests the cellular proliferation .KIT gene, can confer chemoresistance  cytogenetic abnormality show generally favorable response to conventional chemotherapy and have a relatively favorable prognosis , accompasistance  and negasistance .CBFB) gene to the myosin heavy chain 11 (MYH11) gene, producing a CBFB-MYH11 fusion gene and resulting in the expression of a CBFB-SMMHC fusion protein (The inv(16) chromosomal inversion is an inversion that fuses the core-binding factor subunit beta  is thought to be largely irrelevant to leukemogenesis  reciprocal translocation, in which both the ogenesis , the recogenesis , with thanscript . Functioanscript , and it anscript . Furtheranscript .A driver of the leukemic phenotype in inv(16) leukemia is the histone deacetylase 8 (HDAC8)-mediated deacetylation of the p53 protein . HDAC8 eMN1) oncogene (It seems that comparatively little work has been done to elucidate the mechanisms of epigenetic dysregulation in the inv(16) subtype. However, it has been shown that inv(16) AMLs tend to feature a hypomethylated phenotype relative to non-inv(16) AMLs, with the bulk of this hypomethylation occurring in the meningioma 1  in glioblastoma . FurtherDNMT3A, TET1/2, and IDH1/2 . The prognostic significance of epigenetic shift was preserved  in a subsequent multivariate analysis controlling for additional clinical variables including age, white blood cell count at diagnosis, and gender. None of these additional clinical variables was singularly significantly associated with time-to-relapse in the studied AML cohort, and Li et al. additionally found no significant association between the degree of somatic mutations and time-to-relapse  of blast cells collected from 344 AML subjects, clustering them clusters , each chclusters ; inv(16)clusters ; and t with t positive patients, the epigenetic cluster contains individual cases that are not positive for the cluster’s characteristic t abnormality. Despite the cytogenetic difference, there is no significant difference  in survival between cluster 3 patients with t and cluster 3 patients without t, indicating that the underlying methylation signature is the primarily discriminative feature that predicts survival   cytogenetic class, also contains individual leukemia cases that do not feature the cytogenetic abnormality characteristic of their cluster’s class. Though cluster 3 is defined as a t cluster due to being significantly enriched  .PML-RARa fusion oncogene , which, as described above, is often induced by a oncogene . Specifioncogene  in compaoncogene .PML-RARa-binding sites were generally lacking of aberrant methylation signatures, despite the PML-RARa fusion being the hallmark initiating cytogenetic event for acute promyelocytic leukemia . ,b and teleukemia . Singh eoic acid .via chromatin remodeling. Methylation of these genes was found in all of the AML samples analyzed. The second group was comprised of highly heterogeneous hypo- and hyper-methylated samples. These methylation patterns demonstrate the true complexity of methylation in AML  proteins, a class of proteins which contributes to epigenetic silencing of genes n in AML .As the role of DNA methylation, histone acetylation, and related epigenetic events is, as described above, relevant to AML pathophysiology, the use of epigenetically focused therapies for AML is an area of active research. Several treatments targeting the epigenetic mechanisms and heterogeneity of AML are currently being explored.Histone methyltransferases can regulate gene transcription through the addition of a methyl group to a lysine  or arginEnhancer of Zest Homologue 2 (EZH2) is a histone lysine methyltransferase that catalyzes the Polycomb Repressive Complex 2 (PRC2) . PRC2 isex vivo analysis of high-risk acute promyelocytic leukemia (APL), Singh et al. found that the administration of MC2884 directly reduced the expression of EZH2 and induced apoptosis with implications that MC2884 reduced both methylation and acetylation at H3K27. The potential of this inhibitor to shape the leukemic epigenome and upregulate apoptosis has important implications for APL treatment  is a histone methyltransferase that targets the histone H3 lysine 79 position, or H3K79 . Uniquelll cycle , indicatHOXA9 and HOXA7 , acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML) . In partnd HOXA7 .Due to the suspected role of DOT1L in oncogenesis, inhibition of this histone methyltransferase has been of interest. While multiple DOT1L inhibitors such as EPZ04777  and SGC0There exist additional epigenetic modalities of treatment, including histone demethylase inhibitors , bromodoHistone deacetylase (HDAC) inhibitors are posited to treat cancers by preventing excessive deacetylation of histones and transcription factors, particularly those that regulate tumor-suppressor genes . AlternaGenerally, HDACs deacetylate lysine residues on proteins, including those that regulate cell death and cell proliferation, and are classified into one of four classes based on their similarity to yeast HDACs . The clap53 knockout cells and in cells treated with anti-apoptotic agents  and azacytidine (5-azacytosine) are cytosine analogues (azanucleosides) that inhibit the function of DNA methyltransferase, an enzyme responsible for catalyzing the methylation of DNA . As the p51 tumor-suppressor gene, aiding in the restoration of normal cell cycle and function (via methylation of the BASP1-promoter region) in acute myeloid leukemias characterized by the t translocation  and DNA methyltransferase remain covalently bound to one another until the methyltransferase enzyme is eventually degraded or removed , which have been implicated in the inhibition of this metabolic process . Thus, ivia the genetic amplification  and histone deacetylases (HDACs) along with other transcriptional machinery to specific sites in the chromatin (Bromodomains (BRDs) are protein domains involved in the regulation of gene expression hromatin .The bromodomain and extra terminal (BET) protein family includes the proteins BRD2, BRD3, BRD4, BRD9, and BRDT . BET proc-Myc  – a BRD2, BRD3, and BRD4 inhibitor – was demonstrated to have a stronger anti-proliferative effect than JQ1 . In a siMyc transcription, the induction of cellular proliferation, and the inhibition of hematopoietic differentiation -2-hydroxyglutarate (2HG) (Approximately 6–10% of patients with acute myeloid leukemia (AML) have mutations in the isocitrate dehydrogenase 1 (IDH1) gene . IDH1 iste (2HG) . This exte (2HG) .Furthermore, 2HG has been shown to inhibit histone demethylation , a problIn 2018, the Food and Drug Administration (FDA) approved ivosidenib (Tibsovo®), an inhibitor of IDH1/2HG production, for adult AML patients with the IDH1 mutation , followiLysine-specific demethylase 1 (LSD1) is an enzyme responsible for the demethylation of histone H3 lysine 4, playing an important role in epigenetic regulation . LSD1 exvia adjustments to dosage and via blood transfusions , which enhances cellular differentiation, in conjunction with tranylcypromine (an LSD1-inhibitor) – decreased tumor burden more substantially than the use of either agent alone . Furthert extent . This, at extent , points Cellular heterogeneity is increasingly implicated in oncogenesis. Tumors evolve clonally through a series of stepwise, somatic mutations . Like moLike in other cancers, heterogeneity in acute myeloid leukemia is likely maintained due to the increase in fitness associated with a heterogeneous population. In an environment with fluctuating selective pressures (such as one exposed to therapeutic drugs and radiation), it is beneficial for the population to employ a bet-hedging strategy . AlthougFLT3 and NPM1, display both homogeneity and heterogeneity in AML populations and appear to be undergoing convergent evolution , in which the RUNX1-binding domain is fused to the ETO regulator, and t, in which the RUNX1-binding domain is fused to the EVI1 regulator], Loke et al. determined that these two subtypes each displayed a unique epigenetic landscape and gene expression profile. Each type had a different transcriptional network, which likely explains the differing clinical outcomes for the two types . ClearlyFLT3 mutations and TET2 mutations has been shown to confer a gain-of-function DNA methylation, leading to differentiated gene expression , and lesser epigenetic heterogeneity and higher mutational burden . The genetically driven cluster increases in epigenetic heterogeneity as the disease progresses ,b. As reIDH2) gene, which commonly lead to hematopoietic differentiation arrest, are a progenitor to AML. Enasidenib is a selective small-molecule inhibitor of IDH2; mutant IDH2 inhibition promotes leukemic cell differentiation. Many patients treated with enasidenib relapse due to selection for resistant clones and subsequent clonal evolution. Quek et al. recently used enasidenib as a model pathway to develop a method to reveal how cancerous subclones respond to therapeutic agents via clonal mapping throughout disease progression  offer promising avenues for the optimization of AML therapies by taking the characteristic heterogeneity of acute myeloid leukemia into account, thereby improving outcomes for this malignancy.Acute myeloid leukemia is a complex disease characterized not only by significant genetic mutation but also by deleterious epigenetic differentiation, which has motivated the development of therapies that target epigenetic modifications in AML. However, as these epigenetic modifications are heritable and highly heterogeneous, acute myeloid leukemia undergoes significant clonal evolution, culminating in therapy resistance and eventual relapse. New animal models are being explored that can begin to tease out aspects of these diseases, such as in canine and primates , and theSG, CH, and MK equally authored the significant portions of the work, and SG and CH directed the development of the manuscript. AS, YJ, and EA contributed to individual sections of the work. EA provided feedback. CM contributed to various sections throughout the manuscript and provided guidance and feedback regarding the overall direction of the work.CM is a cofounder and a board member of Biotia and Onegevity Health, as well as an advisor or a compensated speaker of Abbvie, Acuamark Diagnostics, ArcBio, BioRad, DNA Genotek, Genialis, Genpro, Karius, Illumina, New England Biolabs, QIAGEN, Whole Biome, and Zymo Research.The remaining 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.


The development of allogeneic hematopoietic-stem-cell transplantation has improved the prognosis of younger acute myeloid leukemia (AML) patients. However, the outcome of older AML patients remains poor. The majority of AML patients are elderly. For elderly AML patients unfit for intensive chemotherapy, less toxic single agent that targets a specific gene mutation or combination therapy with a single agent is needed. The role of chromosomal abnormalities and genetic mutations in leukemia has become more apparent, and detailed prognostic stratification based on the type of genetic mutation has been established. Next-generation sequencing (NGS) has been used for gene analysis of AML. In the future, the evaluation of biologically homogeneous population on the basis of chromosomal abnormalities and gene mutations will lead to a paradigm shift that will help in the development of optimized therapy. As rapid diagnosis of gene mutations is required by the clinical physicians to decide on induction therapy, it is important to have a swift turnaround time for comprehensive DNA sequencing to provide actionable data to clinical physicians. It is required to conduct a feasibility study to evaluate the turnaround time from sending the specimens to receiving the results while maintaining the quality of the specimens contributing to gene analysis. To detect infrequent gene mutations, investigators need to perform multicenter studies and/or cooperative-group trials with a certain sample size to examine the frequency of the gene mutations in elderly AML patients, enabling sufficient statistical power for meaningful comparisons. Acute myeloid leukemia (AML) is a genetically heterogeneous malignant disorder of the haemopoietic stem cells. In Japan, the annual incidence of AML is 5.6/100,000  in AML [IDH1-mutated AML in the relapsed and refractory setting.Isocitrate dehydrogenase (IDH) inhibitors have been also developed for both utations –27. The ] in AML . AnotherIDH2 was shown in IDH2-mutated AML [IDH2. The Overall response rate (ORR) and CR rate (CRR) for all relapsed/refractory AML patients was 40.3% and 19.3% in a phase 1/2 study to assess the maximum tolerated dose (MTD) and clinical activity of enasidenib in patients with mutant-IDH2 relapsed/refractory myeloid malignancies [IDH2 mutated AML.The promising activity of AG-221 (enasidenib) against mutated ated AML –27. Precated AML , 30. AG-gnancies . In thisIDH1R132. The ORR of the relapsed and refractory AML was 33% (7/21) in a phase 1 study enrolling patients with myeloid malignancies, gliomas, and solid tumors [IDH305 is a targeted inhibitor against mutated At present, targeting agents are being typically developed and approved in the setting of relapsed or refractory AML. However, targeting agents are also in demand for untreated AML in older patients who are unfit for intensive chemotherapy. If a targetable mutation is detected in older AML patients, they should be considered for frontline treatment with a targeting agent that is less toxic than intensive chemotherapy in the context of a clinical trial.The majority of AML patients are elderly. For elderly AML patients, intensive chemotherapy, which is standard treatment for younger AML patients, results in the addition of only a few months of prolonged lifespan as compared to palliative care. The number of phase III studies in which the subjects have been elderly patients in the last decade is less than 10 and the long-term survival rate of elderly AML patients has remained at 10–25%. It is hard to say if the standard treatment is effective in elderly patients with AML. Therefore, there is an unfulfilled medical need for an effective treatment for the elderly AML patients. In the future, for elderly AML patients unfit for intensive chemotherapy, less toxic single agent that targets a specific gene mutation or combination therapy with a single agent might become the standard therapy.As described above, gene mutations can influence the prognosis of patients with newly diagnosed AML. As rapid diagnosis of gene mutations is required by the clinical physicians to decide on induction therapy, it is important to have a swift turnaround time for comprehensive DNA sequencing to provide actionable data to clinical physicians. It is required to conduct a feasibility study to evaluate the turnaround time from sending the specimens to receiving the results while maintaining the quality of the specimens contributing to gene analysis, especially when a physician would like to conduct a comprehensive genomic profiling overseas.Many studies on the genetic abnormalities of AML have been reported from Western countries. However, the impact on the mutation frequency of each gene in AML differs with race. Although JALSG in Japan have reported genetic abnormality of AML, the subjects for this study were limited to less than 65 years of age . Gene muGiven the scarcity of certain gene mutations in AML, enrollment of patients for trials is often difficult. To detect infrequent gene mutations, investigators need to perform multicenter studies and/or cooperative-group trials with a certain sample size to examine the frequency of the gene mutations in elderly AML patients, enabling sufficient statistical power for meaningful comparisons. HM-SCREEN-Japan is a genetic screening project to establish precision medicine for AML patients conducted by National Cancer Center Hospital in collaboration with other institutions. The purpose of this project is to develop novel effective drugs against AML and to facilitate rapid clinical introduction of new diagnostic techniques such as multiplex somatic mutation diagnostic agents. HM-SCREEN-Japan 01 is an actionable mutation profiling multicenter study of patients with newly diagnosed acute myeloid leukemia unfit for the first standard treatment or relapse/refractory acute myeloid leukemia supported by Chugai Pharmaceutical Co., Ltd.The objective of this study is to evaluate the frequency or the characteristics of cancer-related genome alterations in acute myeloid leukemia using a comprehensive genome profiling assay .Histologically diagnosed with AML with bone marrow aspiration.①Patients with newly diagnosed AML unfit for standard treatment.②Patients with relapse/refractory AML.Either of the following was fulfilled.The specimens provided are fit for genetic analysis or not.Obtained written informed consent.The subjects enrolled for the HM-SCREEN-Japan 01 fulfilled the following criteria:https://www.umin.ac.jp/ctr/index.htm).The primary outcome is to estimate the frequency of each leukemia genome alteration. The secondary outcome is to evaluate the association between each leukemia genome alterations and clinicopathological characteristics or prognosis and quality of the specimens contributing to gene analysis. This trial has been registered at the UMIN Clinical Trials Registry as UMIN UMIN000035233 (F1H was developed by Foundation Medicine Inc. (FMI) and provides a comprehensive genomic profile that applies next-generation sequencing with hybrid capture-based target enrichment approach to identify somatic genomic alterations in genes known to be unambiguous drivers of hematologic malignancies  as well as sarcomas using formalin-fixed, paraffin-embedded specimens. Each profile simultaneously sequences the complete coding region of 406 genes as well as selected introns of 31 genes involved in rearrangements. F1H also investigates the RNA sequences of 265 commonly rearranged genes to better identify gene fusions (including de-novo and rare gene fusions). In addition to detecting rearrangements, F1H detects all classes of genomic alterations, including base substitutions, insertion and deletions, and copy number alterations using a small, routine clinical sample. All F1H samples were simultaneously profiled for tumor mutation burden (TMB) status. TMB is determined by measuring the number of somatic mutations occurring in the sequenced genes on the F1H profile and extrapolating to the genome as a whole.Evaluation of F1H for use in HM-SCREEN-Japan 01 enables analysis of cancer-associated genes which can be used as therapeutic targets and fusion genes which have been rarely identified so far in AML. The data on these genetic abnormalities are important epidemiological information for the development of novel therapeutics. It is also expected to lead to efficient patient enrollment in the early phase studies.