However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca2+, and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca2+ release events in FRD vs. CD WT cardiomyocytes. [3H]-Ryanodine binding assay revealed higher [3H]Ry binding in FRD than CD WT hearts. O2 consumption at State 4 and hydrogen peroxide (H2O2) production rate were increased, while respiratory control rate (RCR) and Ca2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca2+ leak by RyR2 activation which in turn increases mitochondria Ca2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca When cardiomyocytes are stimulated by an action potential, depolarization of the plasma membrane allows Ca2+ entry through L-type Ca2+ channels (LTCC). Ca2+ binds to ryanodine receptors (RyR2) at the sarcoplasmic reticulum (SR) to produce Ca2+-induced-Ca2+-release (CICR)1, which subsequently activates myofilaments for muscle contraction. Relaxation occurs when the SR Ca2+ ATPase (SERCA2a) reuptakes Ca2+, lowering cytosolic Ca2+ concentration in combination with Ca2+ extrusion via the Na+-Ca2+ exchanger (NCX)2. The sarcolemma Ca2+-ATPase and mitochondrial calcium uniporter (MCU) also participate in decreasing systolic Ca2+ concentration, although to a much lesser extent than the previously mentioned transporters3. Mitochondrial Ca2+4 is essential for ATP and reactive oxygen species (ROS) production/elimination, warranting cell functioning, and survival5. Indeed, mitochondrial Ca2+ is one of the major activators of Ca2+-dependent dehydrogenases in the tricarboxylic acid (TCA) cycle, which in turn can produce an increase in ATP or reactive oxygen species (ROS) production7. However, mitochondria can also determine the progression of cell death with great relevance in cardiovascular (patho)physiology. In this scenario, Ca2+ also plays a pivotal role in opening mitochondria permeability transition pore (mPTP) when reaching a threshold level, enabling the release of ions and solutes from the mitochondrial matrix and thereby triggering programmed cell death8.In adult cardiomyocytes, excitation-contraction-bioenergetics coupling (ECB) is the process that links electrical stimulation, at the cell surface, with myofilament interaction, that drives contraction and with mitochondria energetics, that fulfills cardiac ATP demands. In each one of these processes, calcium ions , located in the outer mitochondrial membrane (OMM), and optic protein atrophy 1 (Opa1), in the inner mitochondrial membrane (IMM) are major recognized players. On the other hand, dynamin-related protein 1 (Drp1) is the most important protein involved in fission11. Despite these described canonical roles, these proteins can exert non-canonical functions, i.e. Mfn2 and Drp1 may participate as a tether14 and Opa1 as a maintainer of cristae structure15. Both fission and fusion work in concert to sustain mitochondria physiological function, including mitochondrial DNA stability, respiratory capacity, apoptosis, response to cellular stress, and mitophagy16. In this context, the mitochondrial balance between fusion and fission is essential in mammals, and even mild defects in mitochondrial dynamics are associated with diseases. Besides, fission is usually associated with deleterious processes and fusion with compensatory mechanisms, although both processes are necessary during development18.It is well known that mitochondria are dynamic organelles, which constantly change their size, location, and shape, by fusion and fission. This two complementary processes21, we found that Ca2+ handling was altered, inducing cardiac arrhythmias and apoptosis due to Ca2+/calmodulin kinase II (CaMKII) hyperactivity and phosphorylation of RyR222. It has been described that in type 2 diabetes mellitus (T2DM) hearts (db/db mice), arrhythmia susceptibility is due to cardiac sympathetic dysfunction23, but also cellular mechanisms are involved in developing arrhythmogenic pattern in the overt T2DM, such as early and delayed afterdepolarization, EAD and DAD, respectively24. Moreover, we also observed that apoptosis in FRD hearts was linked to mitochondria swelling and mitochondria membrane potential changes as well as to a closer distance between mitochondria and SR22.In previous studies performed on early diabetic animals induced by fructose-rich diet (FRD), a standardized prediabetic model characterized by impaired glucose tolerance and insulin resistance2+ leak through RyR2 hyperactivity and increased SR-mitochondria tethering, alters mitochondrial metabolism and biodynamics, favoring fission processes. The present experiments were undertaken to explore this hypothesis, with the conviction that knowledge of the molecular aspects underlying the metabolic disturbances of cardiomyocytes in the prediabetic stage should be useful in developing strategies to prevent, avoid, or even reverse the structural and functional cardiac consequences of the overt disease.Based on these results, we hypothesized that prediabetes-induced CaMKII SR-Ca2+ mishandling, increases apoptosis, and produces mitochondria-SR remodeling in mice fed with FRD (fructose-rich diet). This remodeling was suppressed in AC3-I mice, with cardiac-targeted CaMKII inhibition22. Since phosphorylation of the S2814 site increases RyR2 activity in a HEK293 cell line exposed to high glucose22, we hypothesized that AC3-I mice prevent the increased RyR2 activity and the induction of SR-Ca2+ leak. Confocal microscopy and [3H]Ry binding assay are shown in Fig. 2+ release events (SCaRE) in FRD AC3-C cardiomyocytes as compared to the CD AC3-C. In contrast, FRD treatment does not increase the SCaRE in cardiomyocytes from AC3-I mice  to compare with the CaMKII inhibited (AC3-I) mice. Moreover, the increased SR Ca2+ leak in FRD is the ignition step of the following events, even in rats21 and all the mice lines used previously22, we decide to continue the next experiments in WT mice (Please see discussion below). Figure 3H]Ry binding (Bmax) in FRD WT mouse hearts compared to CD WT hearts. Oppositely to WT littermate controls, AC3-I hearts exhibited a reduced Bmax with CD and Bmax did not significantly increase with FRD , producing an ideal pathway for Ca2+ traffic between both organelles14. Therefore, we sought to investigate whether these proteins, Grp75, Mfn2, and VDAC, were involved in the prediabetic-induced increase in SR-mitochondria proximity.It is well known that mitochondria and SR/ER (Sarcoplasmic/Endoplasmic Reticulum) are in tight contact2+ leak is associated with enhanced SR-mitochondria proximity, a condition anticipated to favor effective Ca2+ trafficking between both organelles, potentially could contribute to mitochondrial Ca2+ overload. To test this hypothesis, we performed experiments in isolated mitochondria to measure the Ca2+ retention capacity (CRC). Figure 2+ in isolated mitochondria from FRD mice compared to CD WT mice (left) was observed. These data could be the consequence of an increased MCU uptake and/or decreased NCLX (Na+/Ca2+/Li+ exchanger) Ca2+ efflux27. Overall results on the right show that mitochondria CRC was significantly higher in CD vs. FRD WT mitochondria. These results suggest that mitochondria Ca2+ load in FRD WT hearts is closer to the threshold at which mPTP could be opened, in agreement with the disturbance in mitochondrial membrane potential reported previously by our group22.Figure 2+ mishandling. Based on the differences in mitochondrial-SR ultrastructure and SR-Ca2+ handling, we next asked if FRD induced changes in mitochondrial metabolism, energetics, and morphology.Although optimal SR-mitochondria proximity is likely necessary and beneficial for mitochondrial function, it may turn to be detrimental, affecting mitochondrial properties as well as whole-cell metabolism under conditions of intracellular Ca2 consumption, ATP, and H2O2 production rate in freshly isolated mitochondria. In WT mice, FRD significantly increased mitochondrial O2 consumption in State 4  without significant changes in State 3  when compared to CD  , which rapidly dismutates to H2O228. These findings reveal a mild uncoupling of electron transport change (ETC), likely responsible for the increase in H2O2 production through O2·− dismutation29.We measured mitochondrial O30  from CD and FRD WT hearts specimens. Figure ria Fig. B. The fi16. We, therefore, investigated whether these proteins were modified in FRD WT hearts. Figure 11, would be involved in its non-canonical function as a tethering protein between SR and mitochondria14.The decreased mitochondrial size, the increased mitochondrial density, and the impairment of mitochondrial metabolism described above strongly suggested the occurrence of mitochondrial fragmentation, which in turn induces vacuole formation. The fragmentation may result from increased fission, decreased fusion, or both. Opa1 and Mfn1/2 have been described to be essential for mitochondrial fusion, whereas Drp1 is considered the “master regulator” of mitochondrial fission, responsible for constricting the mitochondrial membrane2+ overload  with respect to the CD Fig. A. Accordoad Fig. C. Taking2O2 with no changes in ATP production rate. In contrast, mitochondria bioenergetics were not altered by FRD when compared to the CD group in AC3-I mice , autoimmune, and neurodegenerative diseases. In the heart, Ca2+ is particularly indispensable for muscle contraction but also for energy production as a cofactor of NAD dehydrogenases. Therefore, Ca2+ handling is finely tuned and its alterations, even at restricted subcellular microdomains, may trigger cardiac remodeling which can evolve to heart failure (HF) and arrhythmias. Indeed, intracellular Ca2+ depletion leads to impaired contractility while an excess of Ca2+ activates phosphatases and kinases involved in reprogramming gene transcription and unbalancing reactive oxygen/nitrogen species production which may give rise to malignant arrhythmias and cell death.Ca2+ mishandling22. In the present study, we moved forward to further investigate the mechanisms by which Ca2+ mishandling defects alter cardiac function in prediabetic hearts. We hypothesized that the FRD-induced CaMKII-dependent increase in SR-Ca2+ leak promotes a dysregulated interplay between Ca2+ handling and mitochondrial bioenergetics that may constitute the first steps to cardiac failure in the diabetic heart. Our results revealed that SR-Ca2+ leak decreases mitochondria Ca2+ buffering, through a CaMKII dependent activation of RyR2. It was further shown that there is a CaMKII dependent ultrastructure remodeling at the mitochondria-SR microdomain which reduces the distance between these organelles and may contribute to increasing mitochondria Ca2+ load. This study also provides evidence that CaMKII activity and the enhanced mitochondria Ca2+ load are involved in mitochondria metabolism alterations and the increased fission process present in prediabetic hearts.We previously described that prediabetic hearts present arrhythmias and apoptosis associated with Ca2+ release in association with increased proximity of SR-mitochondria and that both alterations are mediated by CaMKII. Yet, the molecular basis of this increased interaction is not known. Ca2+ transfer from SR/ER to mitochondria is a well-characterized function of tethering zones. Localized Ca2+ released from the SR/ER stimulates mitochondrial Ca2+ uptake. VDAC at the OMM, and principally MCU at the IMM, drive Ca2+ from microdomains to the mitochondrial matrix32. Moreover, in different tissues, Grp75 is required to form VDAC/Grp75/IP3R channel complexes34. Mfn2, which resides at the OMM, is essential  for mitochondrial fusion10, but also has been reported to be expressed in the ER, forming ER-mitochondrial tethers via homodimerization or heterodimerization with Mfn1 at the OMM 12. In addition, other studies have elegantly shown that in the absence of Mfn2, the enhanced distance ER-mitochondria, slowed mitochondria Ca2+ uptake35. Supporting the essential role of this inter-organelle communication, Seidlmayer et al.36 showed that when the physical link between SR and mitochondria by Mfn2 was disrupted in ventricular cardiac myocytes of Mfn2 KO mice, the SR-mitochondrial metabolic feedback mechanism and ATP production were highly compromised. Similar results were obtained in Grp75 KO mice37. In line with these previous findings, the present study demonstrated that Mfn2, VDAC, and Grp75, all proteins critically involved in SR/ER-mitochondria interaction, are overexpressed in prediabetic WT mice (FRD treated mice) by a mechanism also dependent on CaMKII. Our results, therefore, indicate that the enhanced SR-mitochondria proximity observed in FRD WT hearts, further increases Ca2+ trafficking , between both organelles. Since the mitochondrial Ca2+ uptake CaMKII-dependent through the MCU is controversial41, we cannot ensure that CaMKII is responsible for MCU activity. In fact, Luczak et al.39, have recently described that the AC3-I mice do not express the inhibitory peptide inside the mitochondria. If mitochondrial CaMKII (mtCaMKII) was activating the MCU complex, FRD AC3-I mice should have mitochondrial Ca2+ overload with respect to CD AC3-I. In this context, increased Ca2+ at the microdomains by SR-Ca2+ leak is responsible for decreased mitochondrial CRC. Therefore, we propose that the increased Ca2+ levels at the SR-mitochondria microdomains, in addition to the increased proximity between organelles, favors the mitochondrial Ca2+ uptake and consequently, disturbing mitochondrial function. However, further investigations are needed to identify the role of mitochondrial transporters, as MCU complex, mitochondrial RyR1, and NCLX45, that could participate in mitochondrial Ca2+ overload in prediabetic model. Despite there have been described that the MCU is downregulated in DM47, in others pathologies, as heart failure, its inhibition provide cardioprotection48. Nevertheless, mitochondrial Ca2+ handling in prediabetic models is an unexplored field.The present results confirm that FRD evokes an enhanced SR-Cain vitro evidence suggesting that hyperglycemia induces mitochondrial fragmentation and mitochondrial ROS accumulation in H9c2 cells, a process that was suggested to be Drp1-dependent and that would lead to cell death by apoptosis49. Besides, mitochondrial fragmentation was associated with tissue disorder and a higher number of vacuoles in FRD vs CD WT mice, which is not evident in FRD vs CD AC3-I mice. The vacuole formation has been linked to a final state of mitochondria degradation, which begins with an IMM compromised (cristae onion-like), continues with OMM alterations and finally, the entire degeneration of IMM and OMM results in vacuole formation30.The present results reveal that prediabetes increases mitochondria fission. Indeed, the significant decrease in mitochondria size and increase in mitochondria sphericity and density observed in FRD WT mice associated with the Drp1 overexpression, greatly support this contention. Significantly, both phenomena, mitochondrial morphological changes and Drp1 overexpression did not occur in FRD AC3-I mice, in which CaMKII activity was absent. These data support the notion that CaMKII is involving in Drp1 overexpression and mitochondria fission in prediabetic hearts. The results are consistent with previous 1-F0-ATPase activity. In this scenario, it has been shown that mitochondria dynamics are relevant to different physiological as well as pathological processes, like HF, T2DM, and apoptosis. In previous experiments, we demonstrated that an increase in apoptosis was also a prominent finding in prediabetic hearts22. Indeed, mitochondria are the primary source of ATP but also of ROS production, which in turn can trigger oxidative stress, thereby determining the fate of the cell 50. The rate of ETC is Ca2+-dependent, which activates the TCA cycle51, generating NADH and activating ATP52, and ROS production that are eliminated by different mechanisms7. This mitochondrial triangle Ca2+-ATP-ROS is highly regulated and any misbalance in one of them may result in changes in the other two53. The increased O2 consumption and decreased RCR in prediabetic mice indicate that ETC is highly demanded with the acceleration of NADH and NADPH consumption. This process may have two possible consequences: (1) Increased O2 consumption and H+ extrusion with a rise in the rate of ATP production if the ETC is coupled or (2) Increased O2 consumption and H+ extrusion without any associated ATP production and enhanced O2·− and H2O2 generation if the ETC is un-coupled54. Our results strongly suggest that the second one is the case of prediabetic hearts; the increase in SR-Ca2+ leak and the enhanced SR-mitochondria proximity, are responsible for the decreased mitochondrial CRC observed, which in turn may trigger the increased H2O2 production described, unbalancing ROS production and elimination, typical of oxidative stress. Accordingly, in previous results, we have demonstrated that an increase in lipid peroxidation was associated with apoptosis and arrhythmias22. Moreover, scavenging ROS avoids CaMKII activation and, therefore, apoptosis22. It worth to mention that either increased Ca2+ or ROS can generate mPTP opening, and that have been described in several pathological conditions55. In fact, the decreased CRC and increased holes area in mitochondria are related to previous results in which prediabetic mitochondria swells22. Altogether, these results reveal the major role of mitochondrial-ROS production on prediabetic heart pathogenesis, non-previously demonstrated.Mitochondria fission is related to an impaired ETC and F57. However, although cardiac pathophysiological alterations are relatively well characterized in fully developed diabetes , information about prediabetes is limited due to its poor diagnosis. Cardiac dysfunction in diabetic cardiomyopathy has been attributed to numerous causes and different pathways . Although it has been described that the prediabetic state induces mild diastolic dysfunction, cardiac consequences of prediabetes and their molecular mechanism are still unclear. The prediabetic model used in the present study was validated by previous works from our22 and other laboratories20, and the period of treatment to achieve this model is extremely short (21 days). Therefore, the findings reported in this and the previous studies22 seem to occur very early in the disease’s development. These facts imply that the pathways running in the overt diabetic cardiomyopathy started when glucose mishandling was just at the beginning, emphasizing the emergency of routinely testing of prediabetes in the population. Actually, health organizations have not uniform criteria for screening prediabetes in individuals that do not present risk factors for T2DM 62; although this is not a translational study, our prediabetic mice model did not show any apparent comorbidity to suspect prediabetes but did show several and serious heart perturbations. The results from this and our previous studies indicate that prediabetes is a silent process, with activation of harmful molecular pathways, extremely aggressive from the beginning, that should be stopped on time to prevent the progression to a more deleterious stage of the disease.Prediabetes has a growing prevalence and worldwide incidence64. In this regard, Luo et al.63 described that oxidized CaMKII (ox-CaMKII) was significantly increased in pacemaker tissues from diabetic patients compared with that in non-diabetic patients after myocardial infarction. In that work, the authors described that Streptozotocin-treated mice had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by myocardial infarction63. Several years before, in 2007, the group of Xiao65 already described that CaMKII is a central key of several apoptotic stimuli that activates cell death pathways in cardiac muscle. In addition, CaMKII activation by oxidation seems to be a redox sensor that determines health and on-off disease switch, as well the sensitivity to ROS levels across the life span66. Moreover, other authors attribute CaMKII-deleterious effects over myocardium through phosphorylation of histone deacetylases68 and to the activation of other kinases 69. PKD is a member of the CaMK superfamily and has been reported to be inhibited by AC3-I67; therefore we could not discard a PKD effect in our results. Nevertheless, in previous results from our group22 we found several harmful effects in the heart due to ox-CaMKII hyperactivity. Those effects were prevented in SR-AIP and S2814A, two mice lines that specifically presents the lack of CaMKII phosphorylation of its targets at SR downstream the kinase activation. The previous results are beyond PKD inhibition and support the data present in the actual work in AC3-I mice. The role of CaMKII in prediabetes has taken a relevant place just in the last years. In our laboratory, using this short-term treatment of fructose-rich diet (FRD), we discovered that prediabetic mice present ventricular arrhythmias that were dependent on CaMKII phosphorylation of RyR221. In the scenario of FRD treatment, CaMKII is activated by increased oxidative stress 21, although we do not discard other ways of activation that could be masking the oxidation of the enzyme, i.e. O-GlcNAcylation64. Later, we described that the same pathway produces apoptosis in the prediabetic heart involving the participation of mitochondria22. In the present study, we continue characterizing the prediabetic heart and revealed that CaMKII is also involved in connecting the SR-Ca2+ leak to the mitochondria Ca2+ overload and disturbed mitochondria metabolism. Moreover, mitochondria fission observed in prediabetic hearts is also linked with CaMKII activation. Once more, CaMKII hyperactivity results in deleterious outcomes to the heart function apparently not compensated by other mechanisms that may counteract these hazardous effects.As we mentioned before, several cardiac alterations in diabetic cardiomyopathy have been attributed to CaMKII activation2+ leak and mitochondria fission. In Fig. 2+ leak by RyR2 activation in FRD AC3-C mice, which in turn decreases mitochondrial CRC due to, at least in part, the enhanced SR-mitochondria tethering. Although these experiments indicate an increased mitochondrial Ca2+, further precise experiments are needed to confirm the rise of mitochondrial Ca2+ load. Moreover, the ETC is partially un-coupled and H2O2 production is increased in association with mitochondria fragmentation in prediabetic hearts. In AC3-I mice, in which myocardial CaMKII is inhibited, these events are prevented. The overexpression of the tethering and fission proteins described are CaMKII-dependent. Since CaMKII inhibition avoids SR-Ca2+ leak and the following steps described as the underlying signaling , we propose that enhanced Ca2+ at the microdomains is the pivotal event in the pathogenesis of prediabetic hearts. However, further investigations across the time are required to asseverate that this molecular signaling in the prediabetic state is the basis of diabetic cardiomyopathy.In the present work, we have described for the first time that as earlier as in a prediabetic state, the heart SR-mitochondria microdomains suffer a CaMKII remodeling, involving SR-CaFull descriptions of methods used are available in the “Supplementary materials” section.All experiments were performed in accordance to the Guide for Care and Use of Laboratory Animal and approved by the Ethics committee of the Faculty of Medicine, La Plata, Argentina Institutional Animal Care and Use Committee (CICUAL no. P03-02-2017). All experiments were performed following the recommendation of ARRIVE guidelines.70, generously supplied by Dr. Mark Anderson , was reproduced and genotyped in our laboratory. As control animals, we used either C57bl/6 or inactive scrambled control peptide (AC3-C) mice. The AC3-C mice were used only for SCaRE measurements since the AC3 mice line co-express the GFP protein which interferes with the Ca2+ signal. The rest of the experiments were performed with C57bl/6 (WT) mice as control. Animals were divided into two groups: control diet group (CD), fed with a standard commercial diet, and fructose-rich diet group (FRD), which received the same diet plus 10% (w/v) fructose in the drinking water for 3 weeks. The FRD is an already validated model of prediabetes in which animals develop impaired glucose tolerance, hyperinsulinemia, and insulin resistance21.Male transgenic mice with cardiomyocyte-delimited transgenic expression of a CaMKII inhibitor peptide, AC3-I3H]Ryanodine binding assays, myocyte isolation, or mitochondria isolation, fully described in Supplementary Material Section.After treatment, animals were sacrificed by cervical dislocation and the heart was immediately excised by central thoracotomy. At that moment, hearts were assigned for biochemical studies, transmission electron microscopy (TEM), [71.Cardiomyocytes were isolated by enzymatic digestion as previously described2+ sparks, waves, and spontaneous contractile activity were captured in line scan mode.Confocal images of Ca72.Binding assays were carried out following a modified version of a protocol previously described73. The analysis of the immunoblots was performed by Fiji program. For sake of clarity, representative blots were selected to be shown in the main figures, and the full-length gels are presented in Supplementary Figure Hearts were freeze-clamped, pulverized, and processed as previously described74 by differential centrifugation. The isolated mitochondria were used in fluorescence spectroscopy using a microplate reader  to measure different mitochondrial parameters. For ATP production rate75 we used the luciferin/luciferase assay, for H2O2 production rate Amplex Red were used76, and for calcium retention capacity (CRC)77 we performed sequential addition of 10 μM CaCl2 until the mitochondria reached a CaCl2 saturation point. Mitochondrial O2 consumption was measured with a Clark-type O2 electrode for high-resolution respirometry74.Animals were anesthetized, and hearts were immediately excised. Heart mitochondrial purified fractions were obtained as described earlier3 samples. Tissue samples were fixed in 2% glutaraldehyde at 4 °C and processed as previously described22. TEM images were used to measure mitochondria roundness, diameter, SR-mitochondria distance, and mitochondria density22.Strips from the middle of the left ventricle wall, avoiding the apex and the base area, were cut in 1 mmGraphPad Prism 8.0.1 program. The assumption of normality was examined using the Shapiro-Wilk test. When P > 0.05 distribution of data was considered normal. Continuous variables were expressed as mean ± SEM and were evaluated with either an unpaired Student’s t-test, or a Mann Whitney test for not normal populations.The statistical analyses were performed using P < 0.05 value was used to indicate statistical significance. The exact P value was indicated in the figures. The number of experiments and the corresponding number of animals, between parenthesis, were indicated either in the figures or its legends.A All authors gave their consent for all data publication.Supplementary Information.


In the proliferation stage, increases in intracellular Ca2+ concentration through voltage-dependent Ca2+ channels, P2Y2 channels, transient receptor potential (TRP) channels, store-operated Ca2+ channels (SOCCs), and IP3 receptors and a decrease in intracellular Ca2+ concentration through plasma membrane Ca2+ ATPases and secretory pathway Ca2+ ATPases (SPCA) activate breast cancer cell proliferation. TRPM7, SOCC, inositol trisphosphate receptor (IP3R), ryanodine receptor (RyR), and sarco-/endo-plasmic reticulum Ca2+-ATPase (SERCA) increase the expression of epithelial-to-mesenchymal transition (EMT)-related proteins; meanwhile, SPCA and the Na+/Ca2+ exchanger (NCX) control the activation of EMT-related proteins. Increased Ca2+ through TRPC1, TRPM7/8, P2X7, and SOCC enhances breast cancer cell migration. The stromal interaction molecule (STIM)-Orai complex, P2X7, and Ca2+ sensing receptors are involved in invadopodia. Various pharmacological agents for Ca2+ channels have been proposed against breast cancer and have provided potential strategies for treating metastatic processes.Intracellular Ca2+ signaling and the overexpression pattern of Ca2+ channels have been regarded as oncogenic markers of breast cancer. In other words, breast tumor development can be mediated by inhibiting Ca2+ channels. Although the therapeutic potential of inhibiting Ca2+ channels against breast cancer has been demonstrated, the relationship between breast cancer metastasis and Ca2+ channels is not yet understood. Thus, we focused on the metastatic features of breast cancer and summarized the basic mechanisms of Ca2+-related proteins and channels during the stages of metastatic breast cancer by evaluating Ca2+ signaling. In particular, we highlighted the metastasis of breast tumors to the brain. Thus, modulating Ca2+ channels with Ca2+ channel inhibitors and combined applications will advance treatment strategies for breast cancer metastasis to the brain.Metastatic features of breast cancer in the brain are considered a common pathology in female patients with late-stage breast cancer. Ca Cancer metastasis occurs in several stages, including proliferation, epithelial-to-mesenchymal transition (EMT), invasion, transport, colonization, and angiogenesis  1]. In . In 1]. 2+ signaling molecules are introduced, and the processes involved in the fundamental modulation of Ca2+ signaling modules and potential strategies against breast cancer are addressed.In this review, we elucidate the essential processes of metastasis in breast cancer. In particular, Ca2+ signaling is commonly known to include muscle contraction to crosslink actin, myosin, and muscle fibers i) in the resting state is sustained up to 100 nM, allowing the use of evoked Ca2+ for the signaling pathways involved in cellular functions i [+/Ca2+ exchanger (NCX) is activated by changes in Ca2+ concentration to control [Ca2+]i by exchanging Ca2+ and Na+ i . Excepti and Na+ . LGCCs iceptors) ,43,44. Lceptors) . IP3R, Ric drugs . In partic drugs . Stimulaic drugs . Activatic drugs . Each sichannels ,51. In achannels ,53. Furtchannels . In the ed SOCCs .2+ plays a crucial role not only in malignant proliferation but also in cancer metastasis i through IP3R3 induces MCF-7 breast cancer cell growth ex) induce overexpression of the TRPC1 channel and increase proliferation through epidermal growth factor receptor (EGFR) signals with ERK1/2 phosphorylation in MCF-7 cells [2+]i induces cell proliferation through TRPV6-mediated Ca2+ influx i, which is induced by phospholipase C, activates the ERK 1/2 signaling pathway and subsequently stimulates MDA-MB-231 breast cancer cell migration ex and [Ca2+]i also regulate the intravasation of breast cancer cells through the extracellular Ca2+-sensing receptor (CaSR) with mitogen-activated protein kinases [2+]ex activates CaSR and subsequently stimulates EGFR to induce invasion, whereas CaSR knockdown attenuates the intravasation of MDA-MB-231 cells e kinases . Increas31 cells .2+ signaling has not been clearly elucidated. The mitochondrial Ca2+ uniporter is located at the mitochondrial membrane of breast cancer cells and regulates tumor proliferation i signaling is critical for cellular functions regardless of the cellular type (tumor vs. nontumor). In other words, the application of Ca2+ signaling blockers for anticancer drugs requires in-depth studies of the basic mechanisms underlying Ca2+ signaling and cancer cells. Thus, we summarized the studies that have used Ca2+ channel blockers for breast cancer medication to understand the associated mechanisms  and pimion drug ) inhibitl growth .2+ signaling with the voltage-independent Ca2+ channel inhibitor carboxyamidotriazole reduces MCF-7 proliferation by arresting G2/M phase cell cycle, decreasing BCL-2  expression, and increasing p21 expression, which induces apoptotic signaling [As mentioned in ignaling . Furtherignaling , which iignaling . In addiignaling .2+ channel blockers enhance the therapeutic effect of traditional drugs or overcome resistance to insignificant drugs. In an attempt to improve their therapeutic effect on breast cancer, mibefradil enhanced the apoptotic effect of the anticancer drug 2-deoxy-D-glucose (2-DG) by arresting the cell cycle in MDA-MB-231 cells [2+ channel blocker that arrests the cell cycle at the G1 phase and evaluates glucose metabolism [2+ channel blockers and traditional anticancer drugs should be conducted. These combined treatments are suggested as novel therapeutic strategies against breast cancer and breast-to-brain metastatic cancer.In addition, Ca31 cells . Further31 cells . Mibefratabolism . The apptabolism . The comtabolism , which itabolism ,167. Doxtabolism . They hatabolism . Additiotabolism ,172,173.tabolism ,175. Thetabolism . The com2+ channel blockers have pharmacological potential. However, the therapeutic application of Ca2+ channel blockers is challenging, as each reagent does not act on a single channel or transporter. The TRPM7 inhibitor 2-APB inhibits IP3R [2+ influx [2+ channel blocker clotrimazole can inhibit Ca2+-activated potassium channel 3.1 [2+ through SOCE [2+ channel blockers are pharmacologically complicated to use as therapeutic strategies, the specific mechanisms of Ca2+ channel blockers need to be clarified.As mentioned above, Caits IP3R , Orai1/2its IP3R , and othits IP3R . In cont+ influx . Additionnel 3.1 , which dugh SOCE . Althoug2+ channels and breast cancer has been assessed for several decades; however, the effect of Ca2+ channels on the metastasis of breast cancer to the brain requires further investigation. The treatment of breast cancer by modulating Ca2+ channel expression and its activity has been considered a cancer therapeutic strategy using various Ca2+ channel blockers. Although Ca2+ signaling is closely related to cancer metastasis in various organs, the application of Ca2+ channel modulation for breast cancer metastasis has not been sufficiently studied. Based on the scope of metastatic breast cancer in this review, several studies have shown that Ca2+ channels have the potential to control metastatic stages and the movement of metastatic breast cancer cells to the brain by modulating adhesive function and permeability. Over the past several years, the number of cases of brain metastases from breast cancer has increased, and the entire metastatic process has not been fully elucidated. In addition, other metastatic processes should be highlighted beyond adhesive and invasive processes. For example, cellular-secreted processes and gene transcription activities are associated with Ca2+ signaling. In other words, communication between cancer cells and other tissues will commence with the untact mode, such as cytokine release. This mode builds up prior to the contact mode, which includes adhesion. As mentioned at the beginning of this article, Ca2+ is an attractive source of the untact mode for transferring the on-mode of metastatic signals through simple mobilization from abundant sources. Therefore, blocking Ca2+ channels as gatekeepers and modulating Ca2+ signaling can be attractive candidates for therapeutic approaches, and suitable combination therapies are suggested as relevant options for metastatic breast cancer therapy.The relationship between Ca


D-Ribose (2dDR) was first identified in 1930 in the structure of DNA and discovered as a degradation product of it later when the enzyme thymidine phosphorylase breaks down thymidine into thymine. In 2017, our research group explored the development of wound dressings based on the delivery of this sugar to induce angiogenesis in chronic wounds. In this review, we will survey the small volume of conflicting literature on this and related sugars, some of which are reported to be anti-angiogenic. We review the evidence of 2dDR having the ability to stimulate a range of pro-angiogenic activities in vitro and in a chick pro-angiogenic bioassay and to stimulate new blood vessel formation and wound healing in normal and diabetic rat models. The biological actions of 2dDR were found to be 80 to 100% as effective as VEGF in addition to upregulating the production of VEGF. We then demonstrated the uptake and delivery of the sugar from a range of experimental and commercial dressings. In conclusion, its pro-angiogenic properties combined with its improved stability on storage compared to VEGF, its low cost, and ease of incorporation into a range of established wound dressings make 2dDR an attractive alternative to VEGF for wound dressing development.2-deoxy- D-Ribose (2dDR) is a D-isomer of a deoxy pentose monosaccharide in which the hydroxyl group at the C-2 position is replaced by a hydrogen atom. 2dDR was discovered in 1930 by Phoebus Levene during his studies revealing DNA structure . S. S Pseudlantarum , and Selinantium  ferment Escherichia coli and Salmonella enterica are capable of utilising the 2-deoxy-d-ribosyl moiety of 2′-deoxyribonucleosides as a carbon source [In addition, n source . This liStaphylococcus epidermidis, Enterococcus faecalis and the anaerobe Cutibacterium acnes. The response of the bacteria to 2dDR both as a sole substrate and as released by the dressing is being investigated in mono- and mixed species. To date, addition of 2dDR to media appears to have either no effect or a negative effect on growth of any species tested (As above, a wound dressing ideally should not encourage growth of the microbiota either in an infected wound or on surrounding skin. To that end, experiments are underway in our laboratories to measure the utilisation of a broader range of skin dwelling bacteria and wound pathogens, including s tested B.For sterilisation, our group demonstrated that gamma irradiation was very effective without affecting the stability of 2dDR ,34 but fFirst identified in the structure of DNA in the 1930s, and then as a degradation product of it due to TP activity, 2dDR has more recently been investigated as a stimulator of angiogenesis. Other small sugars have been reported to be pro-or anti-angiogenic, and in particular, of the two isomeric forms of this sugar, the D-form appears to be predominantly pro-angiogenic and the L-form to be anti-angiogenic. While it remains difficult to propose any simple structure–activity relationship between these sugars and their angiogenic activity, the biological response to 2dDR is strong and clearly demonstrable in a series of in vitro and in vivo bioassays, and in animal experiments of cutaneous wound healing.Incorporation of 2dDR into a variety of experimental and commercial dressings showed good results in all cases. The sugar can be loaded and released over a few days at a concentration which drives angiogenesis in the CAM bioassay and accelerates wound healing in rats.Its biological profile mimics that of the addition of VEGF with 2dDR achieving 80–100% of the biological actions of VEGF. The sugar is more stable and inexpensive than VEGF, and preliminary results suggest that it can be stored in a variety of potential dressings.We suggest that the addition of 2dDR to conventional, well-accepted wound dressings may provide a low-cost and simple-to-use pro-angiogenic dressing for use in the treatment of chronic non-healing wounds. This review aims to contribute to the field of angiogenesis by summarising the latest knowledge on a novel pro-angiogenic agent, 2dDR, and introducing it to the scientists working in this field to encourage them to contribute to the further exploration of 2dDR as a pro-angiogenic agent.


The past decades have seen tremendous progress in our understanding of the function of photoreceptors and olfactory sensory neurons, uncovering the mechanisms that determine their properties and, ultimately, our ability to see and smell. This progress has been driven to a large degree by the powerful combination of physiological experimental tools and genetic manipulations, which has enabled us to identify the main molecular players in the transduction cascades of these sensory neurons, how their properties affect the detection and discrimination of stimuli, and how diseases affect our senses of vision and smell. This review summarizes some of the common and unique features of photoreceptors and olfactory sensory neurons that make these cells so exciting to study. Photoreceptors and olfactory sensory neurons (OSNs) have highly specialized structures that enable them to capture their respective stimuli of light and odorant ligands. Both photoreceptors and OSNs have evolved highly specific abilities to detect and discriminate light wavelengths or odors. They use intricate transduction mechanisms to convert sensory stimuli into electrical signals. Their transduction cascades not only are able to greatly amplify the signal but also to enhance the signal to noise, enabling these cells to detect and distinguish minute stimuli within very noisy backgrounds conditions. Such transduction mechanisms provide for modulation at multiple steps to adapt the sensory neurons to different background stimulation and optimize the capture of useful information about the surrounding world.In this review, we summarize some of the key structural and functional features of vertebrate rod and cone photoreceptors and of OSNs, and the molecular mechanisms that underlie their function. While describing features of both cell types, we emphasize the similarities and differences between photoreceptors and OSNs and the unique features of each cell type that make them perfectly suited to perform their function.Vertebrate rod and cone photoreceptors as well as OSNs are ciliary neurons  with spevia the cell body.The similar morphological structure of rods, cones, and OSNs, with a ciliary part able to detect the respective stimuli and an adjacent cell body, allows similar electrophysiological approaches to record stimulus-induced responses in these cell types. The cell body of a photoreceptor or an OSN can be sucked into the tip of a recording pipette by using a loose-patch (or suction pipette) recording configuration . As a result, rod photoreceptors can reliably detect single photons , middle wavelength , or short wavelength  opsin Nathans, . When boSimilar to photoreceptors, the ligand specificity of the OSNs is also dictated by the expression of OR genes in their cilia. As photoreceptors, each OSN expresses generally only one receptor gene so that its ligand specificity is determined by the structure of the OR expressed in that particular cell. However, photoreceptors typically use no more than five opsin genes to cover the visible spectrum, while OSNs can use hundreds, in the case of humans, to thousand and more, for rodents and dogs, OR genes to cover the odor space Malnic, . The samDetermining the ligand specificity of ORs is an ongoing endeavor  from its β/γ complex (Tβγ), Tα then binds to the cGMP phosphodiesterase (PDE) complex, relieving the inhibition of its catalytic α- and β-subunits by its inhibitory γ-subunits  , causingolf, causing its dissociation into active Gαolf and olfactory β- and γ-subunit, Gβγolf. In contrast to transducin, however, Gαolf is part of the Gαs protein family and binds to adenylyl cyclase 3 (AC3), activating it. As a result, the synthesis of cAMP in the olfactory cilia is upregulated, causing its rapid increase and the opening of cAMP-gated channels . This would cause a large increase in osmotic pressure and also would prevent Ca2+ clearance via the olfactory Na+/Ca2+, K+ exchanger (see below) with greatly deleterious effects. In contrast, high intracellular Cl− is maintained throughout the OSN so that its local depletion in the cilia upon ligand activation is rapidly reversed by diffusion from the cell soma. Both these issues do not exist for photoreceptors as they are embedded in the interstitial fluid of the eye and photoreceptors are sufficiently large and their transduction currents are sufficiently small that ion concentration changes due to changes in transduction current are relatively small  are GAPs that modulate the lifetime of an activated G protein as described above. RGS2, instead of functioning as a GAP, directly inhibits AC3 to control the size of the odorant response  has been identified as a GTP exchange factor (GEF) expressed in OSNs, which facilitates the exchange of GDP for GTP on GαAdaptation plays a critical role in the capacity of our sensory neurons to remain able to detect stimuli above the background in a complex and rapidly changing environment. For instance, in constant light conditions, the dynamic range for both rods and cones is only 100-fold, spanning a range from threshold stimulation to saturation . However2+ upon stimulation. This change is sensed by several Ca2+-binding proteins that trigger a negative feedback on the vision and olfaction transduction cascades by modulating several of their steps. In the outer segments of rods and cones and in olfactory cilia, Ca2+ levels are controlled by the balance of influx via the CNG channels, whose current is carried in part by Ca2+, and efflux via Na+/Ca2+, K+ exchangers (NCKXs) that use the electrochemical gradient for Na+ and K+ to extrude Ca2+ —GCAP1 and GCAP2. When Ca2+ in the outer segments is high, Ca2+-bound GCAPs bind to and partially inhibit the activity of GC. Upon photoactivation and the decline in Ca2+, GCAPs become Ca2+-free and release from GC, resulting in upregulation of cGMP synthesis which restores the dark current after photostimulation and modulates the activation of the transduction cascade in the presence of background light , with these genes also being under purifying selection . These events include head or face trauma, inhalation of toxic chemicals, or viral infection (as SARS-CoV2), and, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Attems et al., The past several decades have brought tremendous progress in our understanding of the function of photoreceptors and OSNs on the cellular and molecular levels. The development of new experimental tools, from electrophysiological recordings to genetic manipulation, advanced imaging, and genome analysis have produced a wealth of information about these exciting sensory neurons. While many questions have been answered, many more remain and will be actively investigated over the coming years. In the case of photoreceptors, one exciting area of rapidly expanding research is focused on the differences in the structure and function of foveal photoreceptors in the central part of the retina and peripheral photoreceptors (Sinha et al., OSNs offer different kinds of complexities compared to photoreceptors, because of the much greater variety of ORs in the olfactory system, the continuous renewal of OSNs, which also necessitates their successful incorporation of OSN axons into existing olfactory circuits, and their direct exposure to the outside environment. These processes are still not well characterized and our understanding of the molecular mechanisms that regulate many of these functions, comparatively, still lags behind. As the visual system, the olfactory system is also affected by aging, causing a progressive decline of sensory abilities. The olfactory epithelium undergoes regeneration throughout the entire lifetime, but these regenerative abilities are limited. In mice, it has been shown that aging reduces the rate of regeneration of OSNs (Child et al., The many parallels between photoreceptors and OSNs have helped us understand the general principles by which sensory neurons detect stimuli, amplify their signals, and adapt. Their differences have also informed us and lead us to understand how these two sensory systems deal with the specifics of the stimuli they have to transduce, the environment they operate in, and how they communicate our outside world to the brain.FG, JR, and VK wrote the article. FG prepared the figures. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To compare the long-term safety, efficacy, predictability, and refractive stability following SMILE versus SMILE combined with accelerated cross-linking (SMILE XTRA), and to specifically study the regression patterns following the two procedures.  This retrospective study included 54 eyes of SMILE and 54 eyes of SMILE XTRA treated for normal and borderline cases of myopia/myopic astigmatism, respectively, based on certain predefined topographic features and risk factors. Patients in both the groups were matched for age and refractive error. The mean postoperative follow-up for the SMILE group was 22.18 ± 10.41 months and the SMILE XTRA group was 21.81 ± 10.19 months.  At the end of follow-up, the mean sphere, cylinder, and SE reduced to −0.03, −0.09, and −0.08 D in the SMILE group and −0.06, −0.15, and −0.13 D in the SMILE XTRA group. 96% and 93% eyes remained within ±0.50 D in SMILE and SMILE XTRA groups, respectively, and 94% eyes maintained an UDVA of 20/20 or better in the SMILE as well as SMILE XTRA groups. Safety and efficacy indices for the SMILE group were 1.03 and 1.00. For the SMILE XTRA group, the safety and efficacy indices were 1.00 and 0.99. No eye in either group had postoperative ectasia or enhancement performed for significant residual refractive error.  Both the SMILE and SMILE XTRA groups exhibited comparable visual outcomes, safety, and efficacy. Contrary to the belief, combination of prophylactic CXL with SMILE did not result in a hyperopic shift in the long term. No eye in either group encountered postoperative ectasia; however, further follow-up is suggested to establish the long-term effects on refractive and corneal stability following SMILE XTRA, as all the eyes treated in this group were borderline. Despite the potential advantages of SMILE over femtosecond laser-assisted in situ keratomileusis (FS-LASIK) and photorefractive keratectomy (PRK), the procedure is characterized by a steeper learning curve, during which intraoperative complications may occur. Suction loss, black spots, dense opaque bubble layer, lenticule tears, incision tears, and inability to find the lenticule are some of the intraoperative complications of SMILE that were reported earlier. –5.SMILE was proposed to be biomechanically more stable compared to LASIK and PRK . HoweverThis class of refractive surgeries, popularly known as “XTRA procedures,” can be combined with PRK, LASIK, and SMILE and is typically performed in cases where the topographic/tomographic indices or the clinical history is suggestive of “at risk” corneas.However, many refractive surgeons are reserved to combine corneal refractive procedures with cross-linking due to reasons such as potential risk of haze, overcorrection, or hyperopic outcome due to progressive flattening as a result of cross-linking, additional cost, and lack of knowledge and experience, etc. However, evidence is growing that a refractive surgery with simultaneous cross-linking is safe and effective in preventing ectasia without any significant side effects –28. EspeThe present retrospective study was approved by institutional ethics committee of Nethradhama Super Speciality Eye Hospital, Bangalore, and adhered to the tenets of Declaration of Helsinki. Data were collected from electronic medical records of all patients who had refractive surgery performed for correction of myopia or myopic astigmatism with SMILE or SMILE XTRA procedure from January 2017 to December 2018. Only those patients who had a minimum follow-up of 12 months were included in the study.Corneal thickness <480 micronsResidual bed thickness between 250 and 280 micronsRefractive Error >−6.00 D spherical equivalent (SE)D-value >1.65Pentacam criteria: Belin Ambrosio display final Corvis-ST criteria: Corvis Biomechanical Index (CBI) >0.5 and Tomographic Biomechanical Index (TBI) >0.29Additional risk factors = age <30 years, family history of keratoconus, or history of eye rubbingPreoperative evaluation was performed using the combined corneal tomography  and biomechanics . Based on the tomographic and biomechanical evaluation and patient's age, refractive error, and additional risk factors, eyes were categorized into “normal” or “borderline” based on the following criteria :Corneal If none of the above criteria were present, eyes were classified as “normal”, whereas if 3 or more of the above criteria were present, eyes were classified as “borderline” for SMILE surgery. All eyes in the “normal” group underwent a routine SMILE procedure, whereas in the “borderline” group, some eyes underwent SMILE XTRA and some eyes underwent only SMILE. The decision regarding the procedure in the “borderline” eyes was influenced by factors such as additional cost, surgeon's intuition, and patient's willingness. Patients who did not undergo SMILE XTRA due to any reason were strictly advised against eye rubbing and were called for 6-monthly follow-ups. They were also asked to report earlier if they noticed any drop/change in their vision. Only eyes with a minimum follow-up of 12 months were included in the study.During treatment planning, a similar nomogram (10% overcorrection) was used for both the SMILE and SMILE XTRA groups.As regards the surgical procedures, all procedures were performed by 2 experienced SMILE surgeons (SG and SB). SMILE was performed with the VisuMax FS laser  using the following parameters: a cap thickness of 100–120 microns, an optical zone of 6-7 mm, energy cut index between 28 and 32 (140–160 nJ), and a superior access incision of 2-3 mm.2 for 75 s, delivering a total energy 3.4 J/cm2.For SMILE XTRA, the surgical steps were as follows: (1) SMILE performed following the standard protocol (described above); (2) 0.22% riboflavin in saline  or 0.23% riboflavin  injected into the interface and allowed to diffuse for 60 s, followed by irrigation of the interface with balanced salt solution; and (3) UV-A irradiation through the cap using a power of 45 mW/cmPostoperative medication regimen consisted of topical 0.3% ofloxacin  4 times/day for 3 days after SMILE and 7 days after SMILE XTRA, 0.1% prednisolone acetate eye drops  4 times/day for 4 weeks (tapering weekly), and lubricants 4 times/day for 4 weeks or more following both procedures.t-tests and intragroup comparisons were performed using the paired t-tests. A p value of 0.05 or less was considered statistically significant.Microsoft excel statistical tool pack was used to analyze the data and perform the statistical analysis. Data were checked for normality before subjecting to statistical tests. Based on the results of normality tests, parametric or nonparametric tests were applied. Intergroup comparisons were performed using the independent n = 27 patients in the SMILE group, and n = 27 patients in the SMILE XTRA group) undergoing a bilateral refractive surgery for myopia or myopic astigmatism correction were included in study. Both groups were comparable with respect to preoperative age, sphere, cylinder, SE, and corneal astigmatism; however, the SMILE XTRA group had a significantly thinner central pachymetry and steeper keratometry (both K1 and K2)  compared to the SMILE group   . Regardi 8.25 μ) .p=0.45.The mean follow-up in the SMILE group was 22.18 ± 10.41 (range 12–54) months and in the SMILE XTRA group was 21.81 ± 9.19 (range 12–52) months, n = 51) in the SMILE group as well as in the SMILE XTRA group  (No significant difference in the mean postoperative UDVA was observed between both the groups (p=0.56) .The efficacy index (postoperative UDVA/preoperative CDVA) was 1.00 and 0.99 for the SMILE and SMILE XTRA groups, respectively.n = 51) in the SMILE group had postoperative CDVA same or better, compared to 93% (n = 50) in the SMILE XTRA group. No eye in either group had loss of 2 lines or more  was 1.03 and 1.00 for the SMILE and SMILE XTRA groups, respectively.n = 52) eyes in the SMILE group and 93% (n = 50) eyes in the SMILE XTRA group had postoperative SE predictability between ±0.5 D. All eyes in the SMILE group were within ±1.00 D, whereas all eyes in the SMILE XTRA group were within ±1.50 D   .n = 52) in the SMILE group versus 93% eyes (n = 50) in the SMILE XTRA group were within ±0.50 D, and all eyes in both the groups were within ±1.00 D of cylinder correction .Mean regression in the SMILE group was 0.08 D, which was less, compared to the SMILE XTRA group (0.13 D), the difference between the two groups, however, was not statistically significant  at the last follow-up. However, no patient complained of any visual disturbances due to this. When asked about the spectacle independence and quality of vision through a subjective questionnaire, the mean score of overall satisfaction (out of 100) was 98.2% in the SMILE group, and 95.4% in the SMILE XTRA group .Inspired by the reports of safety and efficacy of LASIK XTRA, in 2015, we explored SMILE XTRA as a potential treatment option for borderline cases . HoweverThe current literature on SMILE XTRA suggests that combined SMILE and prophylactic accelerated cross-linking does not affect the safety and efficacy of the procedure . In 2015The protocol of SMILE XTRA used in our series appears to be effective for preventing ectasia in susceptible eyes, as all eyes remained stable by the end of the follow-up. Recent studies, however, report using different riboflavin concentration, soak time, UV-A irradiation power, and duration to perform combined SMILE and accelerated cross-linking However, none of the eyes which underwent SMILE XTRA in the previously published studies progressed to ectasia. The present study, with a follow-up ranging from 1 to 4 years in both the groups, provides a substantial anecdotal evidence regarding the potentially enhanced stability provided by the simultaneous accelerated CXL, as no eye progressed to ectasia in this series.Studies have found that collagen cross-linking results in , 30, 31 Nevertheless, our study adds to the existing knowledge on the refractive surgery and simultaneous accelerated cross-linking, especially related to the SMILE XTRA procedure by observing no ectasia, any significant haze or any hyperopic over correction in the borderline eyes treated and followed up for a mean duration of 21 months and longest duration of 4 years. However, since all these eyes were borderline and “at risk” for postoperative ectasia, they definitely call for even longer and closer follow-ups, as the prophylactic CXL may just be delaying the onset of ectasia, which may occur over subsequent course of time. Thus, the long-term safety, efficacy, stability, and effects on corneal stabilization following SMILE XTRA when used to treat borderline corneas, still remain to be established.


BCL2 mutation was not present in our cohort, indicating that, in contrast to chronic lymphocytic leukemia, this BCL2 mutation is dispensable for acquired VEN resistance in AML. Instead, we found that reconstructed existing mutations, especially dominant mutation conversion , rather than newly emerged mutations , mainly contributed to VEN resistance in AML. According to our results, the combination of precise mutational monitoring and advanced interventions with targeted therapy or chemotherapy are potential strategies to prevent and even overcome acquired VEN resistance in AML.Venetoclax (VEN) plus azacitidine has become the first-line therapy for elderly patients with acute myeloid leukemia (AML), and has a complete remission (CR) plus CR with incomplete recovery of hemogram rate of ≥70%. However, the 3-year survival rate of these patients is < 40% due to relapse caused by acquired VEN resistance, and this remains the greatest obstacle for the maintenance of long-term remission in VEN-sensitive patients. The underlying mechanism of acquired VEN resistance in AML remains largely unknown. Therefore, in the current study, nine AML patients with acquired VEN resistance were retrospectively analyzed. Our results showed that the known VEN resistance-associated The online version contains supplementary material available at 10.1186/s40364-021-00288-7. To the EditorBCL2Mut in AML, remain largely unknown ) ; second,e of VEN 00 mg/qd Additional file 1: Table S1. Differences in the mutational landscape between patients with VEN-I and VEN-R AML.


The voltage-dependent anion channel (VDAC) functions as a passageway for the exchange of ions, including Ca2+, across the outer mitochondrial membrane. In cardiomyocytes, genetic or pharmacological activation of isoform 2 of VDAC (VDAC2) effectively potentiates mitochondrial Ca2+ uptake and suppresses Ca2+ overload-induced arrhythmogenic events. However, molecular mechanisms by which VDAC2 controls mitochondrial Ca2+ transport and thereby influences cardiac rhythmicity remain elusive. Vertebrates express three highly homologous VDAC isoforms. Here, we used the zebrafish tremblor/ncx1h mutant to dissect the isoform-specific roles of VDAC proteins in Ca2+ handling. We found that overexpression of VDAC1 or VDAC2, but not VDAC3, suppresses the fibrillation-like phenotype in zebrafish tremblor/ncx1h mutants. A chimeric approach showed that moieties in the N-terminal half of VDAC are responsible for their divergent functions in cardiac biology. Phylogenetic analysis further revealed that a glutamate at position 73, which was previously described to be an important regulator of VDAC function, is sevolutionarily conserved in VDAC1 and VDAC2, whereas a glutamine occupies position 73 (Q73) of VDAC3. To investigate whether E73/Q73 determines VDAC isoform-specific anti-arrhythmic effect, we mutated E73 to Q in VDAC2 (VDAC2E73Q) and Q73 to E in VDAC3 (VDAC3Q73E). Interestingly, VDAC2E73Q failed to restore rhythmic cardiac contractions in ncx1 deficient hearts, while the Q73E conversion induced a gain of function in VDAC3. In HL-1 cardiomyocytes, VDAC2 knockdown diminished the transfer of Ca2+ from the SR into mitochondria and overexpression of VDAC2 or VDAC3Q73E restored SR-mitochondrial Ca2+ transfer in VDAC2 deficient HL-1 cells, whereas this rescue effect was absent for VDAC3 and drastically compromised for VDAC2E73Q. Collectively, our findings demonstrate a critical role for the evolutionary conserved E73 in determining the anti-arrhythmic effect of VDAC isoforms through modulating Ca2+ cross-talk between the SR and mitochondria in cardiomyocytes.Mitochondria critically regulate a range of cellular processes including bioenergetics, cellular metabolism, apoptosis, and cellular Ca In particular, a tight cross-talk between mitochondria and the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) was associated with important physiological and pathophysiological processes  in the outer mitochondrial membrane, which critically regulate transport of ions, substrates, and metabolites across the outer mitochondrial membrane. VDAC dysfunction has previously been linked to pathophysiological outcomes  and in iPSC-derived cardiomyocytes from a CPVT patient , whereas VDAC3 contains a glutamine (Q73), a residue that was previously reported to be involved in VDAC regulation. Strikingly, substitution of VDAC3 Q73 for a glutamate confers a gain of function ability to restore rhythmic cardiac contractions in tremblor/ncx1h mutants while the converse exchange in VDAC2 abolishes its phenotype rescue ability. Finally, using cellular assays, we show that the glutamate at position 73 of VDAC proteins is essential for mediating mitochondrial Ca2+ uptake.Overexpression of VDAC2 allows ofibrils , 2017, iProtein sequences used for phylogenetic analyses were downloaded from the NCBI database.Human and zebrafish VDAC protein sequences were downloaded from the NCBI database and their percent identity was determined using a standard protein BLAST (blastp). Pairwise comparisons were visualized in R v3.6.3 using the ggcorrplot package.E73Q and VDAC3Q73E were inserted by SOE-PCR.Plasmids containing the full-length zebrafish VDAC1, 2, and 3 cDNA were purchased from Open Biosystems and cloned into pCS2+ or pCS2+3xFLAG plasmid for mRNA synthesis. Point mutations VDAC2For HeLa cell transfections, coding regions of VDAC genes were cloned into the pCS2+ vector together with the NLS-EGFP fragments and the viral T2A sequence. The T2A sequence enables bicistronic expression of VDAC proteins and NLS-EGFP for the identification of VDAC-overexpressing cells.Plasmids for the generation of transgenic lines were generated using the Tol2Kit . Plasmidtremblor (tc318dtre) mutant line (ZDB-ALT-980203-1756) was bred and maintained as previously described , Tg, and Tg, 1 μM tebufenozide (TBF) was added to the embryo media at 1 day post-fertilization . mRNA was injected into one-cell stage embryos collected from crosses of Total RNA of embryos was isolated using Trizol Reagent (Life Technologies) and cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). Sequences for forward and reverse primers used for RT-PCR are provided in in situ hybridization was performed as previously described (in vitro transcription was performed using the DIG RNA labeling kit (Roche).Whole-mount escribed  using VD2, 10 mM glucose, and pH adjusted to 7.4 with NaOH) for 1 h at 15°C followed by an additional 30 min incubation in wash buffer  at 37°C to allow de-esterification of cytosolic AM esters. Prior to imaging, cells were permeabilized with 100 μM digitonin for 1 min at room temperature. After approximately 10 s of baseline recording, cells were exposed to an external Ca2+ pulse . Confocal images were recorded in internal buffer  every 0.6 s (Nikon Eclipse Ti microscope) at 545 nm excitation using 20x objective to monitor mitochondrial Ca2+ dynamics. Confocal images were analyzed and quantified using ImageJ .HeLa cells were transfected with expression plasmids using Lipofectamine2000 (Life Technologies). 24 h after transfection, cells were loaded with 5 μM Rhod2-AM (Life Technologies) in loading buffer  were a gift from William Claycomb (Louisiana State University) and were cultured as previously described . To knoc2+ transfer from the SR into mitochondria was measured as described previously , cells were washed with internal solution before measurements. Rhod-2 fluorescence was monitored at excitation wavelength 540 ± 9 nm and emission wavelength 580 ± 20 nm with an Infinite® 200 PRO multimode reader . After recording 30 s of baseline fluorescence, 10 mM caffeine was added to release Ca2+ from the SR.Caeviously . In briet-test unless otherwise specified. *Significance levels are expressed as *p < 0.05; **p < 0.01; ***p < 0.001; and NS not significant.Data are expressed as mean ± s.e.m. Statistical testing was carried out using student In vertebrates, the VDAC gene family consists of three paralogs: VDAC1, VDAC2, and VDAC3 . We examInterestingly, while some plants and fungi possess multiple VDAC paralogs, only a single VDAC protein is present in the majority of the invertebrate animal species surveyed . This sutremblor/ncx1h mutant embryos at the 1-cell stage allows tremblor/ncx1h mutant hearts to establish rhythmic Ca2+ transients and consequently preserves the integrity of myofibrils and maintains coordinated cardiac contractions  or tebufenozide (TBF) treatment after 1 day of development, at which time the primitive heart has already formed and the tremblor mutant hearts are fibrillating  and vice-versa , the amino acid at position 73 (with respect to zebrafish VDAC2) differs between VDAC1 and 2, which exhibit anti-arrhythmic activity, and VDAC3, which does not ,B. ResidInterestingly, E73 also appears in some fungi and in several protists (including choanoflagellates) that are believed to be closely related to animals , suggest2+ uptake restores cardiac contraction in tremblor/ncx1h mutant embryos  significantly enhanced mitochondrial Ca2+ uptake, demonstrating that E73 is a key determinant of VDAC2’s mitochondrial Ca2+ uptake activity  abrogated its ability to promote SR-mitochondria Ca2+ transfer , while a glutamine is present at this position in VDAC3 (Q73). Using multiple model systems, we demonstrate that only isoforms with E73 are able to promote mitochondrial Ca of VDAC  and is ahis site . Also suysosomes . Given tysosomes , it is uchannels . An indichannels  and thatchannels  and medichannels . Though 2+ signals  can be found in the article/The animal study was reviewed and approved by University of California Los Angeles Animal Care and Use Committee.HS, SH, AL, LC, JH, KW, FW, and JS performed the experiments. JS and J-NC conceptualized the study. HS, JS, TG, and J-NC acquired the funding. HS, AL, LC, JS, and J-NC wrote the manuscript. All authors commented on 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Opposite emotions like fear and reward states often utilize the same brain regions. The bed nucleus of the stria terminalis (BNST) comprises one hub for processing fear and reward processes. However, it remains unknown how dorsal BNST (dBNST) circuits process these antagonistic behaviors. Here, we exploited a combined Pavlovian fear and reward conditioning task that exposed mice to conditioned tone stimuli (CS)s, either paired with sucrose delivery or footshock unconditioned stimuli (US). Pharmacological inactivation identified the dorsal BNST as a crucial element for both fear and reward behavior. Deep brain calcium imaging revealed opposite roles of two distinct dBNST neuronal output pathways to the periaqueductal gray (PAG) or paraventricular hypothalamus (PVH). dBNST neural activity profiles differentially process valence and Pavlovian behavior components: dBNST-PAG neurons encode fear CS, whereas dBNST-PVH neurons encode reward responding. Optogenetic activation of BNST-PVH neurons increased reward seeking, whereas dBNST-PAG neurons attenuated freezing. Thus, dBNST-PVH or dBNST-PAG circuitry encodes oppositely valenced fear and reward states, while simultaneously triggering an overall positive affective response bias (increased reward seeking while reducing fear responses). We speculate that this mechanism amplifies reward responding and suppresses fear responses linked to BNST dysfunction in stress and addictive behaviors. Responding to environmental stimuli that predict positive and negative outcomes with appropriate affective behaviors is critical to survival. Fearful stimuli elicit defensive coping strategies , while rewarding stimuli trigger reward-seeking behaviors. Typically, research delineates the neuronal processing of one affective state at a time, i.e., fear LeDoux,  and rewaThe bed nucleus of the stria terminalis (BNST) constitutes a key relay and integration point  and European  legislation on animal experimentation and covered by the license M58/002220/2011/9.All experiments were conducted using 2 to 6-month-old male mice . They were group housed by 2–5 and in a temperature- and humidity-controlled room under a 14-h light/10-h dark cycle, an alternative cycle commonly used in rodent studies. Before the experiments, they had ®, Abbot Laboratories, North Chicago, IL, USA; induction, 2.3%; maintenance, 1.5–2%; airflow, 180 ml/min) and placed in a stereotactic frame . Gentamicin ointment  was used to protect the animals' eyes, and their body temperature was maintained at 36°C with a heating pad controlled by a rectal thermometer . After injecting 0.1 ml of Lidocaine  under the skin as analgesia, the skull was exposed and perforated with a stereotactic drill at the desired coordinates relative to Bregma  were implanted bilaterally 1 mm above the dBNST  under a 15° angle toward the midline in the coronal plane to avoid damaging the lateral ventricle. All implants were fixed to the skull with dental cement .For optogenetic experiments, mice were injected bilaterally into the dBNST  with GFP  or ChR2 . A Micro4 Micro Syringe Pump controller  was used to regulate injection volumes with a rate of 5–10 nl/min. After the injection was completed, the glass needle was left in place for supplemental 5–10 min to guarantee complete injection and diffusion of the virus. The mice were then implanted with optic fiber(s) above either PVH or the l/vl PAG. One optic fiber  per mouse was implanted 0.5 mm above the PVH . Placing a single fiber at the midline ensured bilateral illumination of PVH neurons, which accumulated close to the midline on both sides of the 3rd ventricle. Two optic fibers  per mouse were implanted 0.5 mm above the l/vl PAG  under a 10° angle toward the midline in the coronal plane to allow enough space between the two fibers, which were implanted relatively close to the midline.2+ indicator . For calcium recording in dBNST-projecting neurons to PVH or PAG, another cohort of mice was injected unilaterally with a retrograde canine-adenovirus expressing Cre-recombinase into the PVH or the l/vlPAG  and with an AAV carrying a Cre-dependent Ca2+ indicator into the dBNST . To ensure localization and visualization of the CAV2.Cre injection during histology, CTB-Alexa Fluor 555  was also injected with the virus (one-part CTB for one-part CAV2.Cre). At least 4 weeks after injection, a microendoscope  was implanted above the dBNST. After a 1-week recovery period, a baseplate was cemented onto the skull  and covered .For the calcium imaging experiments, mice were injected unilaterally into the left or right dBNST with an AAV carrying a Cahttp://www.audacityteam.org) with a maximal sampling frequency of 192 kHz and played from a Terratec sound card , and a video camera  monitored the animal's behavior. Port training, reward conditioning, and test sessions utilized the original experiment chambers combined with a grid floor , cleaned with 70% ethanol before each mouse, with the house light turned on and with access to a port located in the center of the right wall. In the port, delivery of the solution was controlled by opening an electronic valve  from a syringe located outside the chamber until a drink tube in the port. Photocell sensors mounted at the entry of the port  and on either side of the drink tube  measured port visits and licking/drinking via beam breaks of the animal's head and tongue, respectively. Fear conditioning occurred in a custom-made triangular cartridge inserted to the experiment chambers, combined with a stainless-steel shock floor , cleaned with lemon-scented diluted cleaning solution before each mouse, with the house light turned off and no access to the port. The day before the start of the experiments, body weight was measured, and water deprivation prompted mice to seek liquid reward. Throughout the duration of the training, mice were weighed before each session and were given water after training to maintain 82–90% of their free drinking weight. The mice were trained, and then during a 50-min port training session to collect a drop of sucrose  randomly delivered 36 times on a 100 ± 30 s interstimulus interval. Only after successful port training, the mice underwent eight sessions of reward conditioning where, after a baseline of 1 min, reward-CS presentation  was immediately followed by sucrose delivery. Each session lasted 50 min and consisted of 24 trials with a randomized intertrial interval (ITI) of 110 ± 20 s. The day after the last session of reward conditioning, the mice were submitted to a fear-conditioning session, where after a baseline of 2 min, fear-CS presentation  was immediately followed by a footshock  delivered to the floor via an external shocker . The session lasted 10.5 min and consisted of 5 trials with a randomized ITI of 100 ± 30 s. The next day(s), the mice were tested for the expression of the correct behavioral response during the presentation of fear-CS and reward-CS in the absence of shock or sucrose delivery. After a baseline of 1 min, the mice received 4 presentations of fear-CS and reward-CS with a randomized ITI of 60 ± 10 s within the same session. For the inactivation experiment, the test first consisted of 4 presentations of reward-CS, and then four presentations of fear-CS. Reward-CS were presented before the fear-CS to avoid any fearful states during the reward-CS trials. For optogenetic experiments, reward-CS and fear-CS presentations were associated with laser illumination during the test. One week later, a second test was repeated after retraining to reward conditioning to confirm the results of the first test . Infusions were performed using internal cannulas  inserted into the guide cannulas. The internal cannulas were attached with polyethylene catheter tubing to 1-μl Hamilton syringes , which were controlled by an infusion pump . The syringes were fixed in a constant rate infusion pump (100 nl/min). The cannulas were left in place for an additional 3 min before removing them to guarantee drug diffusion away from the injection site. Behavioral testing started 15 min after infusion.The mice were handled and habituated to intracerebral injection procedures for several days prior to the behavioral experiment. On the day of testing, the animals were brought to the infusion room and received intra-dBNST bilateral infusions (100 nl per side) of artificial cerebrospinal fluid  or the GABADorsal BNST neurons projecting to the PVH and PAG were retrogradely labeled using cholera toxin subunit B (CTB) conjugated with a different fluorophore. All the mice were between 10 and 12 weeks at the time of surgery, following previously described procedures. Each mouse received bilateral CTB injections into the PVH and l/vlPAG. To label the projections, 10 nl (per injection) CTB-Alexa Fluor 488 (Invitrogen) was injected into the PVH, and 10 nl (per injection) CTB-Alexa Fluor 647 (Invitrogen) was injected into the l/vlPAG. For both regions, an injection speed of 5 nl/min was used. One week later, the mice were deeply anesthetized with a mixture of 10 mg/ml ketamine  and 0.8 mg/ml medetomidine hydrochloride  in 1 × PBS. They were then intracardially perfused with heparin solution , followed by cold 4% PFA. Brains were immediately removed, post-fixed overnight in 4% PFA at 4°C, and transferred to 1 × PBS. The brains were cut into 60-μm thick coronal slices at a vibratome. Sections were rinsed in 1 × PBST (0.1% Triton X-100 in 1 × PBS), incubated for 2 h at room temperature with DAPI  and mounted onto microscopy slides with Fluorescence Mounting Medium . Images of CTB labeling in the dBNST were acquired with a confocal microscope . A region of interest (ROI) was defined and cropped following the anatomical landmarks of the dBNST  to the fiber implants for several days prior to behavioral experiments. ChR2 activation was performed with 473 nm or a 457-nm laser, delivering 5-ms pulses with a frequency of 20 Hz, at an intensity of 8–10 mW at the fiber tip, measured with a power meter . The laser was triggered by custom MATLAB scripts  during test sessions throughout CSs presentations only. From a total of 37 mice, 6 animals did not complete the behavioral task because of sickness developed after surgery or loss of their optic fiber(s). Moreover, 5 animals were excluded from the analysis due to incorrect viral expression and/or fiber(s) placement.2+ signals was conducted using the nVista HD System v2.0.32  at 20 fps. During each session of the behavioral protocol and for each CS trial, Ca2+ recording started 15 s before the CS onset and finished 15 s after the CS offset. Data were analyzed with Mosaic v1.2.0 software (Inscopix). The videos were concatenated per session, down-sampled 2 × 2 (time × space), motion corrected, and the Ca2+ signal was calculated as the relative change of fluorescence over the entire recording session [ΔF/F0 = (Ft-F0)/F0]. The individual neurons and their Ca2+ traces were extracted by applying PCA-ICA analysis. Spatial filters obtained by PCA-ICA were manually selected to avoid duplicates or false units in further analysis. Ca2+ traces were then low-pass filtered at 0.5 Hz, and Ca2+ events were automatically detected with an event threshold >5 s.d. and τoff >0.5 s. Exported events were further analyzed with Neuroexplorer software v5.114 . Neuronal events were exported as peri-event time histogram  and z-scored per recording session. Only data within −8 to 18 s relative to the CS onset were considered and were binned at 1 s. Average z-scores were aligned with CS or the behavior onset . Responders dBNST cells were defined as showing a positive response  within the first 4 s (1-s bin) of the fear-CS, reward-CS, or the behavior onset.Several days prior to the experiments, the mice were habituated to the microscope-mounting process using a dummy microscope. On recording days, the microscope (Inscopix) was attached to the baseplate before the start of the behavioral experiment. Acquisition of CaAll the mice in the inactivation experiment received, after completion of the behavioral procedure, intra-dBNST bilateral infusions (100 nl per side) of muscimol conjugated with a fluorophore . The mice were rapidly decapitated 45 min later, and their brains were immediately placed in 4% paraformaldehyde (PFA) in 1 × PBS (pH 7.4) for fixation at 4°C overnight. The brains were then stored in 1 × PBS and cut on a vibratome into 60-μm coronal sections. The sections were mounted on slides and observed at Zeiss fluorescence stereomicroscope for verifying the location of the Muscimol-BODIPY intracerebral infusion.The mice in the optogenetic and calcium experiments were killed after completing the behavioral procedure, and their brains were processed for histological analysis as described above. After vibratome sectioning, the sections were counterstained with DAPI and mounted on slides. Images were acquired using a confocal microscope and a Mirax slide scanner.post-hoc analyses as needed . The chi-squared test was used to compare cell numbers among the CTB-projecting neurons. Two-tailed paired or unpaired t-tests were used to evaluate the statistical differences between two groups. Differences between groups were considered statistically significant at a value of p < 0.05.Statistical analyses were performed using GraphPad Prism software . ANOVAs were used with 1 or 2 dependent factors, repeated or non-repeated, followed by We adapted a combined Pavlovian reward and fear-conditioning paradigm  events from their Ca2+ traces as a rise in Ca2+ signal > 5 s.d.  of affective processing. We next examined whether these different response types map to different dBNST output channels. There are several candidate downstream regions, which are known to regulate affective responding, in particular, fear responses via PAG or stress, energy homeostasis, and feeding via PVH LeDoux, . Thus, w2+ events a few seconds after reward delivery in dBNST-PVH neurons , but not fear  expressing Cre in the PVH or l/vlPAG and with an AAV expressing Cre-dependent GCaMP6f in the dBNST . Using t neurons , but not neurons . Interes neurons  showed art visit  and the rt visit . During rt visit , suggestrt visit . HoweverAs dBNST-PVH and dBNST-PAG activity is biased for reward or fear, respectively, we wondered how these circuits then control affective responding. So, we probed whether optogenetic activation of those circuits modulated reward and fear behavioral responses. We implanted optic fibers above PVH or l/vlPAG and bilaterally injected the mice with an AAV construct either carrying GFP or ChR2 into the dBNST for subsequent optogenetic activation . We utilWe, therefore, reconditioned the mice 2 weeks later to the Fear-CS using a low-intensity setting  and tested them again the next day. This modified procedure unmasked a partial decrease in freezing behavior during optogenetic activation of the dBNST-l/vlPAG circuit , right, The same brain region can encode opposite affective states  in response to natural positive and negative USs (shock and sucrose), which extends findings of increased BNST Ca2+ signals in response to positive and negative odor stimuli  but lead to an overall behavioral change that reflects a decreased negative valence. Indeed, our results show that Fear-CS activates PAG-projecting dBNST neurons. However, optogenetic activation of this projection decreases freezing behaviors. This finding implies that BNST-projecting neurons to PAG encode Fear-CS, as modulating freezing is consistent with the role of PAG, especially the l/vlPAG, in freezing control , whereas dBNST neurons projecting to the l/vlPAG are excited by the Pavlovian Fear-CS. We demonstrate a dichotomy between valence (fear vs. reward) and Pavlovian features (stimulus vs. behavior). Identifying neural correlates of opposing emotions often utilizes discriminating between positive vs. negative valence , which bias the emotional experience and modulate behavioral outcomes toward positive valence, either by attenuating negative fear states or by intensifying positive reward-related states . This biThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All animal care and behavioral tests were conducted in agreement with the Austrian  and European  legislation on animal experimentation and covered by the license M58/002220/2011/9.NK and WH conceived the project. NK, SA, and MH performed experiments and data analysis. NK, SA, and WH wrote the manuscript. All authors contributed to the article and approved the submitted version.WH was supported by a grant from the European Community's Seventh Framework Programme (FP/2007–2013)/ERC grant agreement No. 311701, the Research Institute of Molecular Pathology (IMP), Boehringer Ingelheim, and the Austrian Research Promotion Agency (FFG). NK was supported by long-term postdoctoral fellowships from Fyssen Foundation, EMBO (Grant No. 1214–2012), and Marie Curie Actions (Grant No. 331015).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nonactivated aziridine with an electron-donating group at the ring nitrogen should be activated to an aziridinium ion prior to being converted to cyclic and acyclic nitrogen-containing molecules. This review describes ways to generate aziridinium ions and their utilization for synthetic purposes. Specifically, the intra- and intermolecular formation of aziridinium ions with proper electrophiles are classified, and their regio- and stereoselective transformations with nucleophiles are described on the basis of recent developments. Aziridines are three-membered cyclic organic heterocyclic compounds with one nitrogen atom in the ring. They are valuable and versatile due to the reactive three-membered ring ,6,7,8,9.A or B, with the assistance of electrophiles after the formation of aziridinium ions. Experimental and theoretical studies have shown that nonactivated aziridine is possibly not serving as a 1,3-dipole, as shown in C and D, while activated aziridines can be utilized for a cyclization reaction with cleavage of the C–C bond for dipolarophile  methylphosphonothioate .,40.11) wThis method is quite good to introduce a mild nucleophile that is not reactive enough to break the aziridine ring. In addition, TMS used for the activation of aziridine through an aziridinium ion is removed after addition of a nucleophile to replace iodine when the reaction proceeds. This method has been used for the synthesis of many biologically important molecules in our lab . 16) by treating starting aziridine (15) with methyl triflate, taking advantage of the extremely high nucleofugality of the triflate anion in a highly efficient manner. The formation of methylated aziridinium ion was observed using 1H- and 13C-NMR spectra. This methylated aziridine easily reacted with proper nucleophiles, whose regiochemical pathways were dependent on starting substrates. With simple alkyl substituents R, the ring opening proceeded via pathway “a” for 17, while the starting material with vinyl or acyl at R gave product (18) from ring opening via pathway “b”  bN-methylative aziridinium ring opening” gives us an opportunity to obtain biologically important molecules including MeBMT ..33) was Recently, a report revealed that the same strategy is applicable to realize stereoselective and regioselective synthesis of α-amino-β-fluorophosphonates . Most aforementioned cases for the formation of an aziridinium ion took ionic pathways. Recently, photo-induced single-electron transfer for olefin-diamination with alkylamines was successful  61]. A . A 61]. 36) from an amine (34) and olefin (35) via in situ activation with N-chlorosuccinimide (NCS), protonation, and photo-induced SET (single-electron transfer) reduction in the presence of a Brønsted acid and Ru(bpy)3(PF6)2 (1 mol%) as the photocatalyst in CH2Cl2 solvent under blue light irradiation at 0 °C. The following ring opening by another amine gives rise to vicinal diamine compounds .The synthetic protocol consists of the formation of an aziridinium ion (40) through displacement of a suitable leaving group at the side chain, as shown in (39) by nucleophilic aziridine amine  tThis convenient approach renders the construction of various types of aza-rings with substituents from nucleophilic attacks either at the bridgehead or at bridge positions of bicyclic aziridinium ions without loss of the substrate’s stereochemistry . This sy43) with an appendage of hydroxymethyl at the α-position of amine engenders the bicyclic aziridinium ion (44) with removal of the leaving group including a hydroxy group. This was further reacted with an applicable nucleophile to yield new aza-ring products (45 and/or 46) via ring opening at the bridgehead (a) or the bridged (b) position of the bicyclic aziridinium ion (44) (a” or “b” depends on nucleophiles .,74.52) f55 and 59) via intramolecular or intermolecular addition of diazoacetate (54 or 58) to aziridine (54 or 57) with metal catalysts. Subsequent ring openings initiated by the anion at the α-position of carboxylate give ring-expanded products (56) and (60) ..67) was 69) to alkyne (70) can make an aziridinium ylide (71) whose anionic olefin can attack the vinyl group, thus giving rise to various substituted benzazepines (72) (It has been disclosed that the addition of electron-rich nitrogen at the nonactivated aziridine (nes (72)  81]..69) to a74) from the reaction of N-propargyltetramethylpiperidine (73) with trans-alkenyl-B(C6F5)2 compounds via trans-1,2-amine/borane addition to a carbon–carbon triple bond. Subsequent alkenylborate attack with ring opening to the activated three-membered aziridinium ion afforded a stable boronated alkenyl piperidine resonance between (75) and (76), as shown in Recently, an interesting reaction was developed to generate a reactive zwitterionic aziridinium intermediate (Nonactivated aziridines are valuable starting materials for the synthesis of densely functionalized nitrogen-containing compounds in a highly regio- and stereocontrolled manner. Their chemical diversity stems from the intrinsic properties of nonactivated aziridines to be activated with electrophiles including acyl halide, TMS halide, alkylhalide, sulfonyloxyalkanes, protic acid, and a Lewis acid. In addition to the backbone of starting substrates and applicable nucleophiles, the chemical reactivity and regiochemical pathways depend highly on the electronic characteristics of aziridinium ions. In this review, we summarized the ways of generating and classifying various types of aziridinium ions for synthetic purposes. The hope is that readers find the value of nonactivated aziridines and their synthetic utilities.