The extracellular matrix plays an important role in cellular balance, and collagen fibers are its most important component. Over the last few years, second harmonic generation (SHG) microscopy has been used for the analysis of collagen fibers in several types of gynaecological cancers, such as breast and ovarian cancer. The value of collagen parameters obtained with this technique to gain insights on the physiopathology and on the prognostic evaluation of cancer has been advocated. Herein, we have characterized the collagen fibers in squamous cell carcinoma (VSCC) and preneoplastic lesions using the SHG microscopy. Collagen parameters, quantity, organization, and uniformity, of VSCC, adjacent skin of VSCC or preneoplastic lesions were compared with values obtained in normal tissue of healthy control. There was an evident decrease in the values of collagen fiber parameters in the VSCC. Increased quantity and uniformity of tumor associated collagen fibers were associated with the presence of lymph node metastases, which suggest a prognostic value of such parameters in the evaluation of vulvar cancer. It represents only about 5% of all tumors of the lower genital tract2 and is thus considered a rare cancer. Among the different histological types of vulvar cancer, squamous cell carcinoma (VSCC) is the most common type (95%), followed by melanoma (<5%), sarcoma (1–2%), and basal-cell carcinoma (2%). VSCC has two mechanisms of development, which arise via preneoplastic lesions called vulvar intraepithelial neoplasia (VIN): (1) infection with the human papillomavirus (HPV), which affects younger women (<50 years), and (2) HPV-independent development (approximately 80%), which appears after menopause (50–80 years) and originates from lichen sclerosus6.Vulvar cancer is the fourth leading gynaecological cancer worldwide8.VIN is graded in a similar way as cervical intraepithelial neoplasia: VIN 1, VIN 2, or VIN 3. In VIN 1, atypical cells are limited to the lower third of the vulvar squamous epithelium; in VIN 2, atypical cells compromise the deeper two thirds of the epithelium; and in VIN 3, atypical cells affect the entire squamous epithelium. In all three grades, the basal membrane remains intact, i.e., the lesions are not invasive. VIN 1 is not considered a precursor of vulvar cancer, as it has low malignant potential and low probability of evolving into VIN 2 and VIN 3. These last two grades instead are considered high-grade lesions with real risk to transform into malignancy10. However, its role surpasses the structural aspect, and it is considered to influence tumor invasiveness and metastasizing10.Over the last few years, studies on cancer development have focused on the role of remodeling of the extracellular matrix (ECM), more specifically on collagen fibers (COL), to tumor progression. COL are major components of the ECM and are important for the maintenance of tissue and organ structure and integrity12.The study of COL in histology laboratories goes from the traditional stains, such as Masson’s trichrome, Movat pentachrome, and picrosirius red staining, to the more specific detection methods, as immunohistochemistry, the wide-field-of-view polarization, and the second harmonic generation (SHG) imaging. The SHG has been increasingly being used to study COL, due to its high sensitivity and specificity. It is a quantitative nonlinear optical microscopy technique that captures images from biological tissues having polarized anisotropic structures, such as COL13. In the present study, SHG images were used to determine the quantity, uniformity, and organization of COL in vulvar cancer and its preneoplastic lesions. It is intended to determine the clinical value of COL evaluation by this method, not previously reported in vulvar neoplasia.The association of increased COL expression in vulvar carcinoma and higher tumor aggressiveness has been recently reported, but the evaluation of COL was made using the picrosirius stainingWe made the OrientationJ plug-in and an ImageJ for angle analysis the orientation, energy, and coherency maps, hue-saturation-brightness (HSB) color-coded map and orientation histogram in normal tissue of healthy control , and the Multiuser Laboratory of the “Gleb Wataghin” Physics Institute . A survey was undertaken on patient records with the diagnoses of VSCC and VIN, between 2000 and 2010. The formalin fixed paraffin embedded tissue blocks from the patients with both diagnoses were retrieved from the archives of the Pathology Department of Hospital de Cancer de Pernambuco, Recife, Brazil.The study included a total of 52 Brazilian patients older than 18 years, of which 40 patients had been diagnosed with VSCC and 12 with potential precursor lesions (VIN 2 and VIN 3). Adjacent tissues to the tumor with no neoplastic and/or preneoplastic morphological features were used as controls (17 patients). The metastases considered in the study were all to the lymph nodes, confirmed by histopathology of the surgical specimens  in the stromal areas adjacent to VIN, in intratumoral stroma, and in normal adjacent tissues. In each case, three areas were selected, and these areas were submitted to SHG analyses at the INFABiC – Unicamp13. SHG microscopy was performed on a confocal microscope Zeiss LSM 780-NLO  using a lens with an objective of 40×/1.3 EC Plan-Neofluar with immersion oil. The highest numerical aperture (1.3) was necessary to allow an adequate spatial resolution to observe the fibrils. The excitation wavelength was 780 nm, with a pulse of approximately 100 fs at a repetition frequency of 80 MHz, supplied by a Tsunami Ti:Sapphire laser . The objective lens laser power was approximately 80 mW, with circular polarization. The acquisition time for each image was approximately 60 s.SHG is a nonlinear optical process, in which two photons with the same frequency interact with a nonlinear material and generate a new photon with twice the energy and half the wavelength of the initial photons. Fibrillar collagen has high nonlinear susceptibility, with SHG being the ideal method to evaluate its structure25; this was followed by filtering with long-pass filters LP490 at 45° to avoid two-photon excited fluorescence (TPEF), and SP430 at 90° to filter only the SHG signal. Two detectors were used to capture the TPEF and SHG signals, namely a photomultiplier tube detector (PMT) and non-descanned detector (NDD), respectively. Of these, only the SHG signal was considered for the present study. The emission spectrum analysis between 350 and 430 nm showed only a narrow peak at 390 nm consistent with the SHG signal. The TPEF signal given by eosin could also be detected at 530 nm.Only the SHG forward signal at 390 nm was collected using a condenser lens 0.55 NA – WD 26 mm. A short-pass SP690  was used to filter the excitation wavelength at 780 nmet al.25 was used, with daily optical alignments performed by obtaining a normalization factor, which consisted of the SHG image of a standard specimen , at the beginning of each experiment. The vision field of this objective was 212 × 212 cm, but to avoid the figure margins in which the SHG intensity was weaker, we used a zoom to select a more homogeneous area of 177 × 177 cm. To eliminate background signals, a “blind” observer, with a degree in physics and without medical training, applied a common threshold to all images, comparing them with the control sample of human aorta. After this, all background signals unrelated to COL were eliminated.The procedure described by Burke http://imagej.nih.gov/ij/) and the OrientationJ plug-in software26. Nine representative areas (150 × 150 pixels) were randomly selected from each image to evaluate COL quantity, uniformity, and organization, as described below.SHG signals were stratified according to COL organization and structure in the stroma, using image-pattern analysis methods. Quantitative analysis of COL parameters was performed on SHG images using ImageJ  and organization (coherence). Uniformity (energy) reflects the density of COL, i.e., whether fibers are farther from  or closer to  each other. Organization (coherence) reflects the spatial direction of COL, with values between 0  to 1 (highly oriented fibers). From the perspective of mathematical definitions, these parameters should be correlated. The relationship between COL parameters for vulvar lesions, normal adjacent tissues, and VIN was added to the calculation27.COL quantity was determined as the sum of SHG signal intensities in 1024 × 1024 pixel images, which was directly related to the quantity of collagen molecules. These values were obtained using the integrated density function of ImageJ software. Using the OrientationJ plug-in, as previously describedDescriptive statistics for categorical variables are presented in tables as the distribution of absolute and relative frequencies, whereas continuous variables are presented as measures of central tendency . Normality was tested using the D’Agostino-Pearson test. The variables quantity and uniformity of collagen fibers were log-transformed, because they depend upon the square of the SHG signals. Continues values were plotted as the median and interquartile range (25th–75th). The Mann-Whitney nonparametric test was used to analyze numerical variables between two groups. The Kruskal-Wallis test was used to analyze numerical variables between three groups. Data analysis was performed using GraphPad Prism v 8.0 software . All comparisons were considered statistically significant at p < 0.05.


Intracellular Ca2+ concentration increases during systole and falls in diastole thereby determining cardiac contraction and relaxation. Normal cardiac function also requires perfect organization of the ion currents at the cellular level to drive action potentials and to maintain action potential propagation and electrical homogeneity at the tissue level. Any imbalance in Ca2+ homeostasis of a cardiac myocyte can lead to electrical disturbances. This review aims to discuss cardiac physiology and pathophysiology from the elementary membrane processes that can cause the electrical instability of the ventricular myocytes through intracellular Ca2+ handling maladies to inherited and acquired arrhythmias. Finally, the paper will discuss the current therapeutic approaches targeting cardiac arrhythmias.Calcium ions (Ca Of particular importance is the fact, that within cardiac myocytes, [Ca2+]i changes must be tightly regulated, so that the heart can beat rhythmically. This means that during the cardiac systole, [Ca2+]i has to increase to certain levels to make contraction occur and must fall in diastole, allowing the muscle to relax and prepare for the next cardiac cycle. E-C coupling has been reviewed in detail (Excitation-contraction coupling (E-C coupling) of the cardiac myocytes is a well studied phenomenon. We know that the calcium ion  , in which Ca2+ is released from the sarcoplasmic reticulum (SR) into the cytoplasm via ryanodine receptors (RyR), allowing Ca2+ to bind to the myofilament protein troponin C, activating the contractile machinery. Normal cardiac function also requires relaxation to occur; this results from a decrease of free cytoplasmic Ca2+ levels. Several Ca2+ transport pathways are involved in this process, as Ca2+ reuptake into the SR by the SR Ca2+-ATPase (SERCA), Ca2+ extrusion by the sarcolemmal NCX and plasma membrane Ca2+-ATPase (PMCA)   originates in the sinoatrial node and propagates through the heart. In the ventricle the initial depolarization opens voltage-gated sodium channels leading to further depolarization which, in turn, opens the L-type Cage (NCX) . This Cae (PMCA) . This no storms) .2+ in generating and maintaining cardiac arrhythmias from basic arrhythmia mechanisms to recent progresses in pharmacological challenges and possible future therapies.Here we review the role of Ca2+ oscillations, developing into propagating Ca2+ waves arise when the molecular and cellular dynamics merge at the tissue and organ level. The lower and higher scales tend to have a bidirectional information flow. A good example is when EADs arising during an AP due to abnormal ion currents and Ca2+ dynamics, can bring an extra amount of Ca2+ into the cell due to L-type Ca2+ channel reopening and potentiate Ca2+ waves. These multiscale dynamics can lead to life threatening complex arrhythmias.Arrhythmia mechanisms have multiscale dynamics in the heart. The lower end is the molecular scale, originating from the stochastic behavior of ion channels, resulting from thermodynamic fluctuations . Next is2+]i. This “coupled-clock” pacemaker system is produced by membrane proteins, driving the AP and the intracellular Ca2+ cycling molecules  node. If SA node impulse generation is impaired, atrioventricular node (AV node) and Purkinje fibers can show automatic activity. These secondary pacemakers are also called latent or subsidiary pacemakers . SA nodeolecules .f), working mainly during early diastolic depolarization. The consequent depolarization opens Ca2+ channels  and the pacemaker (slow type) action potential occurs. As in the case of the working myocardium, K+ currents repolarize the membrane. In the last two decades it has become clear that spontaneous Ca2+ release in a cardiac cell is not always pathological. In the “calcium clock” mechanism, spontaneous SR Ca2+ release events, the Ca2+ sparks activate INCX and cause late diastolic membrane depolarization. Coupled clock pacemaker system comprises functional interactions between the membrane and calcium clock  is an important arrhythmogenic mechanism and consists of abnormal automaticity and triggered activity.In the normal human heart, the SA node generates the propagating APs and determine the heart rate. In the case of parasystole, when the primary pacemaker is bordered by ischemic, infarcted regions the impulse cannot leave the SA node. Under these conditions, parasystolic pacemakers can take over pacemaker activity and fire APs at a lower rate compared to that of the SA node . The AV Aside from the abnormal automaticity, the most common causes of focal activity are the early and delayed afterdepolarizations . EADs occur before the terminal repolarization (phase 2 and phase 3 repolarization) of the AP, while DADs occur after the repolarization when membrane potential reaches the resting levels  and/or increased inward currents   and are considered as a hybrid between EAD and DAD (NCX (and ICl(Ca)), these Ca2+-mediated currents are weakly inward. However, if APD is abbreviated, they become strongly inward, allowing an INCX-driven depolarizing current, when the shorter repolarization allows a stronger (and not spontaneous) Ca2+ release from the SR  , that istivation  and as ativation  , Ca2+ release during an EAD is not governed by sarcolemmal Ca2+ influx, so that it is spontaneous, which resembles as a heterogenous pattern, just like in the case of DADs , activation of Na+ current  (veratridine), and activation of ICa,L (BAY K8644) at slow pacing rates. Additional application of the Ca2+ chelator BAPTA-AM decreased [Ca2+]i as expected, but either reduced EAD frequency in the presence of dofetilide and veratridine or further increased EAD frequency in the presence of BAY K8644 . Since BAPTA-AM decreased EAD frequency in the presence of veratridine, but failed to shorten APD, these results contradicts the exclusive role of APD in EAD generation and indicate that an increase in [Ca2+]i is a significant factor not only for generating DADs, but for evoking EADs as well (2O2 significantly increased APD and relative short term beat-to-beat variability (SV)  induced EADs  the reactivation of ICa,L during the plateau phase of AP and (2) SR Ca2+ overload  anemone toxin II (ATX-II) facilitates INa,L that caused elevated systolic Ca2+ transient and SR load, (b) the spontaneous Ca2+ wave precedes the first EAD, and (c) Ca2+ buffering with BAPTA in the patch pipette abolished EADs   and incrncidence . Furtherced EADs . The twooverload . In our hed EADs .2+]i and the amplitude of Ca2+ transient, along with the APD and beat-to-beat variability of APD, AP morphology and plateau potential, net membrane current, and the actual availability of L-type Ca2+ channels. Regardless of the type of EAD mechanisms, if the depolarizing effect of the EAD on the membrane potential is sufficient to activate INa, the result will be an abnormal impulse generation, triggered activity , consequently these regions are more prone to AP prolongation  and chloride current (ICl(Ca)) may also be involved in DAD generation (ti), which is responsible for the membrane depolarization  . The clinical significance of DAD generation lies in triggered activity that contributes to arrhythmogenesis with catecholaminergic polymorphic ventricular tachycardia (CPVT), atrial fibrillation (AF), and HF. In CPVT and HF, intracellular Ca2+ load combines with RyR dysfunction (“leaky” RyR). Under circumstances when the SR becomes loaded  and/or RyR becomes leaky, spontaneous Ca2+ release is favored.DADs are the other common causes of focal activity and were originally described as oscillatory afterpotentials . They ocode INCX . Other nneration . These tigitalis , catechoigitalis , hypokaligitalis , hypertrigitalis , and rapnt (IK1) . Subthrent (IK1) . However2+ release, there are two main patterns. First, focal Ca2+ release, when Ca2+ signal acts locally , while it was increased by INa and Ito, and in general, increased if repolarization reserved was compromised. RSV was also increased at faster rates and at increased [Ca2+]i. Transient changes of [Ca2+]i due to Ca2+ released from the SR were the dominant contributor to this process  in AP configuration can cause disturbances in Casettings , as recesettings . Higher  process . High RS2+ cycling. At the cellular level, alternans is manifested as beat-to-beat alternations in contraction amplitude , APD , and Ca2+ transient amplitude  at constant heart rate. However, because of the bidirectional information flow between membrane voltage and Ca2+ cycling, electrical alternans is always influenced by Ca2+ alternans, and vice versa . The net result is a decrease of SR Ca2+ content. The next beat therefore arises from a depleted SR resulting in a smaller Ca2+ transient and decreased INCX, so that the cell will gain Ca2+ resulting in a larger SR content and Ca2+ transient for the third beat  and the other without an obstacle (phase-2 reentry and reflection). The key difference is in refractoriness. Circus movement reentry travels around an anatomic or functional obstacle and all cells are recovered from inactivation, while cells involved in reflection or phase-2 reentry show large differences in recovery from refractoriness with no obstacle in the way of the reentrant wave. In addition, classic nomenclature distinguishes between microreentry and macroreentry, where the reentrant circuit does not or does appear on the surface ECG, respectively.2+]i, reduced pH, or lower ATP levels  . Later wInitiation of reentry requires a trigger and a substrate. The trigger can be a premature contraction, while tissue substrate is the dispersion of refractoriness. On top of that, fundamental settings are needed for reentry excitation with anatomical obstacle: (1) the impulse initiating the circus movement must propagate in one direction  and (2) the proportion of absolute and relative refractoriness in the tissue, that is, the reentrant circuit must be long enough to let all areas—within the circuit, distal from the stimulus—recover from refractory (excitable gap), so the circuit can return to its origin and continue as a new cycle . These cIn the cases, when there is no anatomical barrier present, functional reentry can still form, maintained only by the electrical properties (dispersion of refractoriness) of the tissue. The best known examples are the leading circle, spiral wave, and figure-of-8 reentry  current, and overall by the competitive behavior between INa and Ito , so that the slow pulse propagates to the regions of short APs until it triggers a fast pulse  on the slow Ca2+ pulse  and switches between them (+- and Ca2+-mediated (fast) or a Ca2+-mediated (slow) propagating wavefront. This bi-stable behavior might serve as an explanation for the two different possible outcomes of torsade de pointes. According to Chang et al., in cases where the Ca2+-mediated slow spiral wave is terminated, leads to termination of the torsade de pointes, while if the tissue is sufficiently heterogenous, Na+ and Ca2+-mediated fast spiral waves degenerate torsade de pointes to VF  is a severe arrhythmogenic disorder, manifesting as a bidirectional or polymorphic VT, mainly in young patients with structurally healthy hearts after exercise or acute emotional stress . As hearThe main criteria for CPVT diagnosis are as follows: structurally normal heart , normal QT interval, and adrenergic induced bidirectional or polymorphic VT . CPVT is2+ release from the SR leading to a DAD. The fundamental feature of this process is the Ca2+ release unit (Ca2+ sparks), where the spontaneous Ca2+ release occurs. If sufficient number of release units are activated, a Ca2+ wave is born, which depends on the SR Ca2+ content and the SR Ca2+ threshold  and protein kinase A (PKA)  and activates RyR. Simultaneous activation of ICa,L, SERCA (increases SR Ca2+ content), and RyR therefore increases the possibility of spontaneous Ca2+ release  . In addi release . Experimhythmias .Several CPVT subtypes have been described to date, albeit the two most common types are the CPVT-1 and CPVT-2 (2+ binding protein (HRC), junctin and triadin  and a defective mutation in the RyR 3D conformation  CSQ2 is a monomer, which is converted to a dimer (0.6–3 mmol/L) or polymer (> 3 mmol/L) at higher Ca2+ concentrations   and is first seen at an early age with high likelihood of infant sudden cardiac death . When CPe below) . Mutatioe of RyR . TRDN-en+ influx .2+ sensitivity was shown  and is the only β-blocker to have RyR inhibitory action, albeit it is a less potent β-blocker after all (2+-channel blocker (verapamil) combination therapy was shown to be beneficial  was shown to prevent arrhythmias in mouse models by reducing RyR opening, SERCA activity and ICa,L  implantation in patients with diagnosis of CPVT who experience VT, syncope, or cardiac arrest despite the optimal medical treatment . HoweverKs, IKr, or INa. As mentioned in a previous section, EADs can form if the repolarization reserve is compromised, outward currents are reduced and/or inward currents are increased. In the case of LQTS, inhomogeneity of refractoriness combined with EADs establishes the arrhythmia substrate for VT, torsade de pointes.Congenital long QT syndrome (LQTS) is an inherited cardiac ion channelopathy. LQTS is characterized by a prolonged QT interval on the surface ECG, reflecting the ventricular APD prolongation, which gives rise to risk for syncope, seizures, VT or torsade de pointes and finally VF and sudden cardiac death . Prolongv7.1) that encodes IKs (v11.1), encoding IKr (+ channel (Nav1.5) encoding INa  results in an abnormal response upon β-stimulation, as mutation reduces interaction between AKAP9 and KvLQT1 cannel α subunit  leading to dysfunctional response to cAMP and a prolonged APD (QT)  .+ channel Navβ4 β-subunit) together resemble the LQTS-3 phenotype as QT prolongation is achieved by increased Na+ current (+ current (Nav1.5)  channel function. IKAch is formed by Kir3.1 and Kir3.4. Mutation in Kir3.4 function disrupts membrane targeting and stability, i.e. reduced membrane expression has been suggested as the cause of LQTS-13 (LQTS-9 and LQTS-10 (gain-of-function mutation in SCN4B-encoded Na current . Mutatio(Nav1.5) . LQTS-7 interval . In LQTS LQTS-13 .+ and Na+ channels , there are several Ca2+-signaling proteins that are linked to the occurrence of long QT intervals, typically causing LQTS-8, LQTS-14, LQTS-15, LQTS-16, and LQTS-17 (Although most of the LQTS mutant genes are related to KC subunit of L-type Ca2+ channel (Cav1.2) and is generally associated with Timothy syndrome. Timothy syndrome is a rare (less than 30 patients reported worldwide), but severe multisystem disorder, involving QT prolongation, syndactyly, congenital heart defects, cardiomyopathies, bradycardia (caused by AV block rather than sinus bradycardia), and autism (v1.2 leads to (1) a significant reduction in voltage-dependent inactivation of ICa,L, (2) enhanced ICa,L, (3) decreased current density with enhanced window current, and (4) a steeper APD restitution curve . Mutations in CALM1-encoding CaM1, CALM2-encoding CaM2, and CALM3-encoding CaM3 are responsible for producing LQTS-14, LQTS-15, and LQTS-16, respectively. Patients diagnosed with these conditions are usually young and have a high rate of cardiac arrest with severe QT prolongation  may contribute to APD prolongation and that LQTS associated CaM mutations can lead to electrical alternans, a pathological feature of LQTS , increased spontaneous Ca2+ release via RyR, and promotion of SR Ca2+ loading by NCX. It is not clear yet, whether the arrhythmogenic feature is mediated by DAD or EAD, but in a TRDN-null mice model, nifedipine aborted SR Ca2+ overload and spontaneous Ca2+ release   . LQTS-reKs) .+ channel blockade with limited effects on K+ channels)  . It shouhannels) . It was hannels) . As an ahannels) , lidocaihannels) , flecainhannels) , phenytohannels) , or ranohannels)  can be uhannels) . In LQTScorandil  or RPR26corandil  were shocorandil . Left cacorandil .to over INa and ICa,L) is suggested in the epicardium of the right ventricular outflow tract, causing an enhanced transmural dispersion of repolarization, i.e. ST elevation (Brugada syndrome (BrS) is characterized by ST elevation in V1–V3 ECG leads and is associated with elevated risk of polymorphic VT, VF, and sudden cardiac death . Two hyplevation . The arrlevation  The depolevation .+ channel (Nav1.5) and is accountable for about one third of all BrS , the CACNB2-encoded β2-subunit , and the CACNA2D1-encoded α2δ1-subunit  of the L-type Ca2+ channel  have been described with a concomitant reduction of ICa,L  can either be gain-of-function or loss-of-function (2+ is elevated). Therefore, TRPM4 mutation may change the AP dome and be arrhythmogenic. TRMP4 may also slow down conduction by altering the availability of Na+ channels  mutations have been described generating BrS-1–BrS-23 . The mos all BrS . Genes, of cases  , some of them were effective in preventing recurrent episodes of VF or electrical storms, but did not reduce the overall risk of VF . GuideliCa,L , the CACNB2-encoded β2-subunit , and the CACNA2D1-encoded α2δ1-subunit  of the L-type Ca2+ channel, similar to the BrS-3, BrS-4, and BrS-11 phenotype. These mutations decrease ICa,L , cause heterogenous shortening of APD and QT interval, therefore increases dispersion of repolarization (+ channel (Nav1.5) causes an overlapping phenotype of SQTS and BrS.Short QT syndrome (SQTS) is a rare inherited syndrome characterized by QT intervals essentially shorter than 360 ms and by an increased incidence of VT/VF mainly in youngsters . There arization . Transmu2+ binding and/or binding of target proteins, thus generating a rather insensitive CaM and that the gene anomaly is more pronounced in the Purkinje system  is characterized by J-point and ST segment elevation in two or more contiguous leads on ECG . The eare system . Recentle system . RyR2-H2J-point elevation associated malignant arrhythmias have recently been proposed with a new classification, as J-wave syndrome .In addition to the congenital form, LQTS can also be acquired. The prevalence of acquired LQTS is greater than that of congenital forms . It is gv11.1 channel, responsible for IKr while effects on enhanced INa,L has also been reported (The above mentioned effects are often seen for the hERG-encoded (human ether-à-go-go-related gene or KCNH2) Kreported . The rolreported . It was via phase-2 prolongation of AP  can favor the formation of EADs.Several other causes of acquired LQTS have been described, including electrolyte disorders , such ason of AP , also lo2+ handling defects such as triggered activity , conduction block (reentry), and Ca2+-driven cardiac alternans and altered Ca2+ buffering , increased SERCA function, increased diastolic SR Ca2+ leak and spontaneous SR Ca2+ release, increase in Ca2+ sparks and waves, enhanced CaMKII function (with subsequent RyR hyperphosphorylation), or reduced ICa,L . These ectopic activities can serve as a trigger for reentry which is considered to be the main arrhythmogenic mechanism in AF. Also, ICa,L reduction in AF causes APD shortening and promotes reentrant activity  after rapid pacing. This transcriptional downregulation of Ca2+ channel has been proposed to be mediated by activation of calcineurin by Ca2+/CaM, which in turn, regulates nuclear translocation of NFAT  and this can be classified as paroxysmal (spontaneously self-terminates into sinus rhythm in less than 7 days), persistent (lasts for more than 7 days), long-lasting persistent (AF lasts for more than a year) or permanent AF (without active rhythm control) . AF is muffering . DAD-meded ICa,L . Involveed ICa,L . As in mactivity . Reducti of NFAT .2+ signaling silencing, as an antiarrhythmic adaptive mechanism in AF  rather than “lone AF” itself in those cases when unstable Ca2+ signaling occurs in AF. Ca2+ signaling silencing process includes the failure of centripetal intracellular Ca2+ signal propagation , remodeling of the RyR complex (reduced protein expression and CaMKII-mediated phosphorylation), and lower Na+ concentration   or destabilize it (arrhythmogenic Ca2+ instability). Therefore, future therapeutic approaches should identify the substrate  and tailor therapies for individual AF patients  . The decoponin I . The autpatients .2+ in the pathophsiology of AF see the review of For an excess review about the role of Ca2+ in cardiac E-C-coupling focusing on those defects which lead to cardiac arrhythmias in inherited and acquired syndromes. In the last few decades there have been great advances in the understanding of these arrhythmias, however, there is still a need for more work investigating the physiology and pathophysiology of Ca2+ related events. Designing drugs to treat a specific disease type has never been simple; it is enough to think of the early disappointing attempts to block the Na+ or K+ channels . Multiple characteristics of novel therapeutic approaches have to be determined and to be considered as a complex, systems problem.In summary, we have reviewed the roles of CaAlong with the generally used β-blockers, newly developed selective drugs without proarrhythmic side effects are necessary. While implantable cardiac defibrillators provide longer life expectancy, they cannot prevent the onset of cardiac events. An additional helpful tool would be reliable and effective risk stratification and clinical guidance for all of the syndromes discussed. It should not be overlooked that in the future other genetic mutations may be discovered requiring novel biological therapies. Because of the diversity of inherited and acquired mutations individually tailored therapeutic approaches  will be required.2+ in the cardiac arrhythmias data from basic science should meet the clinical practice; translational aspects must be key in all fields of science.To gain a better understanding of the role of CaKK conceived the review and drafted the manuscript. KK, RV, BH, TB, PN, and DE revised the manuscript critically for important intellectual content. DE contributed to the critical review of the literature, editing of the manuscript text and review of the figures. All authors approved the final version of the manuscript submitted.This work was funded by the National Research Development and Innovation Office (NKFIH-K115397). Further support was obtained from GINOP-2.3.2.-15-2016-00040 and EFOP-3.6.2-16-2017-00006 projects, which are co-financed by the European Union and the European Regional Development Fund. The research was financed by the Thematic Excellence Programme of the Ministry for Innovation and Technology in Hungary (ED_18-1-2019-0028), within the framework of the Space Sciences thematic program of the University of Debrecen. This work was supported by the British Heart Foundation Chair Award (grant number: CH/200004/12801).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.


Using qPCR we detected transcripts of all Trpc genes in CFs; Trpc1, Trpc3 and Trpc4 being the most abundant ones. We show that the AngII-induced Ca2+ entry but also Ca2+ release from intracellular stores are critically dependent on the density of CFs in culture and are inversely correlated with the expression of the myofibroblast marker α-smooth muscle actin. Our Ca2+ measurements depict that the AngII- and thrombin-induced Ca2+ transients, and the AngII-induced Ca2+ entry and Ca2+ release are not affected in CFs isolated from mice lacking all seven TRPC proteins (TRPC-hepta KO) compared to control cells. However, pre-incubation with GSK7975A (10 µM), which sufficiently inhibits CRAC channels in other cells, abolished AngII-induced Ca2+ entry. Consequently, we conclude the dispensability of the TRPC channels for the acute neurohumoral Ca2+ signaling evoked by AngII in isolated CFs and suggest the contribution of members of the Orai channel family as molecular constituents responsible for this pathophysiologically important Ca2+ entry pathway.TRPC proteins form cation conducting channels regulated by different stimuli and are regulators of the cellular calcium homeostasis. TRPC are expressed in cardiac cells including cardiac fibroblasts (CFs) and have been implicated in the development of pathological cardiac remodeling including fibrosis. Using Ca Remodeling processes comprising the activation of fibroblasts and the consequent changing in the extracellular matrix (ECM) composition are key processes during the development of different pathologies in several organs. Abnormal deposition of ECM proteins including collagens, or fibrosis occurs for example during non-alcoholic fatty liver disease (NAFLD) ,2 or patAmong the different cell types found in the heart, cardiac fibroblasts (CFs) which represent the big majority are important mediators regulating the secretion of growth factors with direct impact on cardiomyocyte function, cardiac hypertrophy development and ECM formation during both normal cardiac growth and pathological conditions ,6. The t2+ entry pathways are important in CFs and are considered to contribute to the development of fibrosis. In CFs, Ca2+ entry pathways include receptor-operated Ca2+ entry (ROCE) channels and store-operated Ca2+ entry (SOCE) channels imidazole, Cat.Nr. T320722, Sigma-Aldrich). In experiments where the TRPC- or CRAC-antagonists were used CFs were pre-incubated for 10 min in TS solution containing the blockers which were also included in the solutions used during the Ca2+ imaging experiments.The following compounds were used: angiotensin II , thrombin , TGF-p < 0.05. p-values are depicted as * p < 0.05; ** p < 0.01, *** p < 0.001. Statistical analysis was performed using the two-tailed unpaired Student’s t-test except in Data were analyzed and processed with Excel 2007  and OriginPro 2015G ver. 9.2.257 (OriginLab). Values are shown as mean ± standard error of the mean (SEM) except for 2+ response in isolated primary murine CFs we first determined the conditions of cell culture and analysis using CFs obtained from adult wild type (WT) mice. For that purpose cells isolated by a Langendorff-based perfusion system and subjected to a pre-plating strategy were cultured in different cell densities and subsequently analyzed with respect to their Ca2+ homeostasis  and TRPC5 (IC50 3.1 µM) compared to TRPC3 (IC50 30.4 µM) and TRPC6 (IC50 57.1 µM). Pre-treatment (10 min) of CFs with 10 µM T320722 had no effect on the AngII-induced Ca2+ release but it was able to significantly reduce the AngII-induced Ca2+ entry in WT cells  mice [2+ transients evoked either by AngII or by thrombin were not different between WT and TRPC-hepta-KO CFs after 5 or 6 days in culture  mice . The acu culture . Moreoveol cells . TogetheGSK7975A  that wasGSK7975A ,79. Pre-mulation D, indica2+ entry following stimulation with neurohumoral agonists relevant during development of cardiac remodeling in primary cardiac fibroblast isolated from TRPC-KO mice has not been studied so far. In this study we therefore aimed to assess the causal contribution of TRPC proteins for Ca2+ entry following acute AngII application by comparative analysis of primary isolated CFs from several Trpc-deficient mouse models and WT controls. We showed that CFs isolated from global TRPC-hepta KO (Trpc1/2/3/4/5/6/7–/–) mice exhibit no impairment in AngII-induced Ca2+ entry nor Ca2+ release.There are several reports analyzing the role of TRPC proteins in cardiac fibroblasts (CFs) and in fibrosis development using TRPC antagonist, siRNA or shRNA; however, the causal contribution of TRPC isoforms for Ca2+ entry was only observed at the increased cell density >300 cells/mm2 and that the Ca2+ release from intracellular stores was largely increased under high density condition. When the maximal peaks of the Ca2+ signals of the Ca2+ release and the Ca2+ entry are compared, the maximum peak of the Ca2+ entry is much lower than the one from the Ca2+ release. However, the Ca2+ release signals decay much faster in comparison to the Ca2+ entry; the Ca2+ entry is maintained elevated at least over minutes and it could account for higher Ca2+ concentrations over time triggering specific transcriptional programs and/or extracellular matrix production. Obviously, one or more steps in the signaling cascade triggered by AngII in CFs are enhanced under these high-density conditions. In addition, the magnitude of the Ca2+ signals induced by AngII is inversely correlated with the expression of the myofibroblast marker α-SMA. These correlation between Ca2+ signaling and cell density is not surprising due to published observations e.g., from endothelial cells where the importance of cell to cell contact for Ca2+ signaling was shown [2+ signaling. For example, changes in the starvation conditions, which were also optimized in our approach in CFs, have profound effects on the secretome of human prostatic stromal myofibroblasts and other cell types [2+ signaling, this is another important variable determining the responses in primary cells; different culture substrates have significant effects on expression profiles as revealed by a transcriptome analysis from human dermal fibroblasts cells [To define the CF cell model used in this study we could prove a high homogeneity of our cultured cell population by positive immunostaining for the established CF-specific markers P4HB and DDR2 that was comparable with findings from other studies using similar approaches ,80. We aas shown . Defininll types . Despitets cells .2+ release and Ca2+ entry, we first examined the expression of the TRPC channels and found transcripts from all seven Trpc genes with Trpc1, Trpc3 and Trpc4 being most abundant. Since it has been reported that TRPC3 and TRPC6 are involved in fibroblasts function we first analyzed CFs from TRPC3/C6-DKO mice, to also circumvent possible compensation of TRPC6 in TRPC3-KOs as described [2+ entry or Ca2+ release compared to WT CFs. In addition, the TRPC3/C6/C7 blocker SAR7334 [2+ responses. Based on our results obtained with this TRPC3/C6/C7 channel inhibitor it can be assumed that also TRPC7 has no essential role in the AngII-induced Ca2+ signals, because the acute blockage of all three channels (in WT cells) or the genetic inhibition of TRPC3/C6 plus pharmacological inhibition of TRPC7 (TRPC3/C6-DKO cells) did not affect the Ca2+ responses. In contrast to our results, it is published that targeting TRPC3 channels by pharmacological inhibition using Pyr3 in cardiac fibroblast from adult rats is able to blunt AngII-induced Ca2+ entry and α-SMA expression [2+ imaging experiments were reported. Remarkably, both Pyr compounds also block Orai1-mediated store-operated Ca2+ currents, with Pyr10 being more selective for TRPC3  [2+ entry, as well as the AngII-induced increase in expression of α-SMA, CTGF and Fibronectin [2+ entry. TRPC6 is regarded as an important regulator of fibroblasts function especially in terms of myofibroblast differentiation [2+concentration in human ventricular fibroblasts [2+ signaling was not impaired, but ROCE induced by AngII following siRNA-mediated knock down of TRPC6 was not analyzed in the study from Ikeda and co-workers [2+ entry, but using a 100-fold higher AngII concentration in human cells. In vivo evidence for the role of TRPC6 during fibrosis development comes from observations using TRPC6-KO mice. TRPC6-deficient dermal fibroblasts isolated from TRPC6-KO mice are refractory to TGF-β and AngII-induced transdifferentiation (58); similar to that study we also observed that TGF-β treatment leads to a differentiation of the CFs to myofibroblasts with increased expression of α-SMA [2+ entry in CFs produced by inhibiting the Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) was enhanced by TGF-β or AngII treatment and it was attributed to increased TRPC6 expression. In our experiments the Ca2+ entry after TGF-β pre-treatment was reduced in WT CFs and it was not different in the absence or pharmacological inhibition of TRPC6. Moreover, in primary murine lung fibroblasts AngII treatment does not affect TRPC6 levels whereas TGF-β changes TRPC6 expression levels [2+ entry mechanism between the entry evoked by AngII or by SERCA blockage. Inhibition of TRPC6 by BI-749327 was shown to reduce cardiac fibrosis produced by pressure overload as well as fibrosis developed in a model of renal injury [2+ signaling. Other groups also attempted to determine the activity of TRPC1 in right atrial human fibroblasts; however, the treatment with siRNAs against TRPC1 did not alter Ca2+ influx that was evoked in these fibroblasts by application of a bath solution containing 20 mM Ca2+ [With our established protocol to culture and measure the AngII-induced Caescribed . We obse SAR7334  elicitedpression . Moreovepression , the carpression . In linepression . Discrep3.08 µM) . Furtherronectin . These rntiation ,58. Apprntiation , reductintiation  and reduroblasts . One canroblasts ; however-workers . In contn levels . Those dl injury ; however mM Ca2+ . 2+ signaling in the absence of both TRPC3/C6, and even in the absence of all seven TRPC proteins in CFs does not completely preclude that these TRPC proteins might be involved in the regulation of fibrosis-related gene expression in CFs. It is possible that conformational changes and signaling mediated thereby independent from ionic signaling conveyed by a channel protein can also play a role in stimulating transcription [The fact that we observed no differences in acute AngII-evoked Cacription . Along tcription . 2+ rise provoked by capsaicin is enhanced; additionally, in those mice the Isoproterenol-induced cardiac fibrosis and cardiac hypertrophy in vivo are reduced [2+ concentration as well as the in vitro proliferation of CFs. In contrast, treatment with siRNA directed against TRPV3 reduces the AngII-induced collagen expression [2+ rise, but this response is entirely absent in CFs from TRPV4-KO mice indicating that TRPV4 is functionally active in these CFs [2+ signaling. However, the role of TRPV4 for Ca2+ entry in CFs triggered by neurohumorally acting agonist has not been reported yet.Other candidates outside the TRPC group that could mediate the AngII-induced ROCE are found in other TRP subfamilies like TRPV, TRPM or TRPA, therefore below we discuss their implications in CFs functioning and fibrosis. For several TRP proteins different mechanisms for their involvement in fibroblast function and fibrosis development have been proposed. In CFs from TRPV1 over-expressing mice the sustained Ca reduced . In vitr reduced . In ratspression . Anotherpression , was alspression . The TRPpression . Stimulahese CFs  and coul2O2 induces a higher Ca2+ elevation in hypoxia-exposed adult rat CFs compared to normoxia-exposed cells, which was also attributed to TRPM2 [Astragalus), and that its inhibition by siRNA prevents fibrotic features in NIH-3T3 mouse fibroblasts [2O2 and AngII also are able to provoke a sustained increase in the intracellular Ca2+ and elevated expression of fibrogenic growth factors  that were either blunted or reduced in fibroblasts in which TRPM7 was silenced via shRNA [Trpm7 mRNA with shRNA treatment reduces TGF-β1-induced differentiation and proliferation [2 treatment [2+ concentration that is largely reduced by treatment with HC030031, a selective TRPA1 antagonist or by TRPA1 knockdown using siRNA [, cardiac fibrosis is reduced in TRPA1-KO mice after myocardial infarction but is increased in WT mice treated with the TRPA1 agonist cinnamaldehyde [A hypoxia-evoked cation current was described in cultured primary fibroblasts from adult rat hearts and siRNA against TRPM2 prevented the development of such current . H2O2 into TRPM2 . In addito TRPM2 ,99,100. roblasts . Additioia shRNA . In humaferation . Using nreatment . These rng siRNA . In CFs aldehyde . However2+ signaling might occur. For example, TRPC1/C4-DKO mice show reduced development of cardiac fibrosis after pressure-overload as well as reduced expression of collagen genes after chronic neurohumoral stimulation with AngII most likely due to a reduced background Ca2+ entry in cardiomyocytes [2+ dependent processes in CFs, such as expression of fibrogenic growth factors and development of tissue fibrosis, are mediated by channels composed of TRPC proteins. To circumvent such problems and to address such questions the use of new conditional Trpc alleles  that allow for a cell type-specific deletion of selective TRPC proteins would be a promising approach. Nonetheless, such approaches can be challenging in CFs due to the diversity of CFs found in the healthy heart and during disease progression [2+ homeostasis in isolated cells such as CFs. Here we observed that the TRPC blocker (T320722) reduces the AngII-induced Ca2+ entry; however, our interpretation in light of the observation from TRPC-deficient cardiac fibroblasts is that such effect is more likely due to unspecific effects of that compound rather than the inhibition of a TRPC4/C5-mediated Ca2+ entry. Recently, an inhibitor of TRPC4- and TRPC5-homomeric channels as well TRPC1/TRPC4- and TRPC1/TRPC5-heteromeric channels has been developed [50 in the picomolar range) and specific within the TRP channel family. However, its effectiveness in primary cells and in vivo has not yet been demonstrated. Apart from C31, other TRPC channel blockers such as ML204 and recent new derivates of C31 such as A54 [A main general limitation in studies about the role of TRPC channels for complex in vivo phenotypes and associated cellular mechanisms is the use of global KO mice. Global deletion of TRPC proteins during early development might evoke mechanisms and pathways that compensate for the loss of TRPC-containing channels. Furthermore, differences in processes depending on cell-cell interactions including Camyocytes . Neverthgression . Alternaeveloped ,107. Thih as A54  or AM237h as A54  were rep2+ signaling in isolated adult murine CFs. Furthermore, they prove the importance of cell density for intracellular Ca2+ homeostasis and transdifferentiation. CFs cultured at low density show markedly increased expression of the myofibroblast-marker α-SMA. Our work suggests that other Ca2+ conducting channels such as TRPV4, TRPM2, TRPM7 or members of the ORAI family of Ca2+ channels may mediate Ca2+ entry following activation of AngII-induced signaling cascades in CFs. Finally, by using the CRAC inhibitor GSK7975A we could show an important contribution of CRAC channels to the AngII-evoked Ca2+ signaling in cardiac fibroblasts. Taken together, our results reveal the dispensability of TRPC proteins for the acute AngII-induced Ca


This designer platform synergizes the TMDC monolayer and plasmonic nanosieves to empower tunable geometric phases and large field enhancement, paving the way toward multifunctional and ultracompact nonlinear optical devices.The growing demand for tailored nonlinearity calls for a structure with unusual phase discontinuity that allows the realization of nonlinear optical chirality, holographic imaging, and nonlinear wavefront control. Transition-metal dichalcogenide (TMDC) monolayers offer giant optical nonlinearity within a few-angstrom thickness, but limitations in optical absorption and domain size impose restriction on wavefront control of nonlinear emissions using classical light sources. In contrast, noble metal-based plasmonic nanosieves support giant field enhancements and precise nonlinear phase control, with hundred-nanometer pixel-level resolution; however, they suffer from intrinsically weak nonlinear susceptibility. Here, we report a multifunctional nonlinear interface by integrating TMDC monolayers with plasmonic nanosieves, yielding drastically different nonlinear functionalities that cannot be accessed by either constituent. Such a hybrid nonlinear interface allows second-harmonic (SH) orbital angular momentum (OAM) generation, beam steering, versatile polarization control, and holograms, with an effective SH nonlinearity  In the past decade, nonlinear signals  from nanomaterials and artificially structured materials, namely, metamaterials and metasurfaces, have attracted extensive attention for their wavelength tunability, coherence, and ultrafast responses –6. TheseAB2, A = Mo, W, Ta, and Nb and B = S, Se, and Te). Their broken centrosymmetry enables a second-order susceptibility χ(2) around 1~100 nm/V [μm in size; it is quite challenging to spatially control the amplitude, polarization, phase, and even angular momentum of the emitted SH from monolayer TMDC. A paradigm-shift mechanism is needed to boost, manipulate, and control the SH photons of atomically thin materials.On the other hand, emerging 2D materials, such as transition-metal dichalcogenide (TMDC) monolayers, are well known for their strong light-matter interactions and valley-polarized optoelectronic properties –44. Of p100 nm/V –48, whic100 nm/V . However2 monolayer with a plasmonic Au nanosieve; the bonded assembly behaves as a hybrid nonlinear optical interface for multifunctional SHG manipulation. SHG is in principle forbidden in Au nanosieves since they are made of centrosymmetric rectangular nanoholes, but the integrated hybrid optical interface offers highly efficient SHG emission, whose properties may be precisely controlled at a hundred-nanometer pixel-level resolution. SHG, originating from the WS2 monolayer, is enhanced by the strong local fields within each Au nanoholes, and simultaneously, the geometric phase determined by individual Au nanoholes can be locally engineered and imparted to the WS2 monolayer on top of the Au nanosieves. Our experimental results indicate that an effective SHG susceptibility of ~25 nm/V is achieved, 3 orders of magnitude larger than the one of conventional plasmonic metasurfaces [Nature actually provides a hint towards such a mechanism. Epoxy is a type of strong glue made from two resins, with neither individually able to make the glue sticky. Putting those two resins together, a strong and powerful adhesive is obtained, because a chemical bond is produced. Inspired by the working principle of epoxy, we experimentally report a recipe for full control of SH photons via an integrated nonlinear optical interface composed of plasmonic nanosieves strongly coupled to a TMDC monolayer. As a proof-of-concept validation, TOC illustrates the schematic of bonding a WSsurfaces . Based o2 metasurface, which can be easily prepared by a material transferring method (see Methods for details). An atomic force microscope (AFM) image of the WS2 flake shown in 2 monolayer. An optical image of the WS2 monolayer on the Au nanosieve is shown in 2 monolayer with an area of 100 μm2. 2 monolayer can be clearly observed. 2 monolayer under the excitation at 810 nm, showing the peak wavelength at ~620 nm. 2 monolayer excited at 532 nm. Two typical signatures located at 352.3 and 417.3 cm−1 can be observed, which are attributed to the E2g1 and Ag1 phonon modes from the WS2 monolayer, respectively. In general, the characterization results prove the high quality of the WS2 monolayer, and their intrinsic properties are not changed in the hybrid Au-WS2 interface.2 interface (red curve), a bare WS2 monolayer (blue curve) and pure Au nanoholes (black curve). It can be clearly seen that both curves have sharp peaks at 405 nm, which is the SHG wavelength. Note that no SHG can be observed in the bare Au metasurface under the same excitation (black curve). As shown in 2 interface is increased linearly as the square of incident power. Thus, it can be concluded that the emitted signal is ascribed to the SHG of the WS2 monolayer. More importantly, the measured SHG intensity from the Au-WS2 interface is 5 times stronger than the one from the pure WS2 monolayer under the same excitation condition, which is ascribed to the strong field confinement in Au nanoholes. The area ratio of the nanohole and the whole unit cell is around 10%. Without considering the local-field enhancement effect, the SHG intensity of the Au-WS2 interface should be 10%2 = 0.01 in comparison with that of a pure WS2 monolayer. Therefore, the SHG intensity in the area of nanoholes of the Au-WS2 interface is estimated to be enhanced by two orders of magnitude (5/0.01 = 500 times). Previous work indicates that the WS2 monolayer has a large second-order susceptibility of ~5 nm/V [2 interface is estimated to be ~25 nm/V which is three orders of magnitude larger than the one of typical plasmonic metasurfaces (~pm/V). ~5 nm/V , and the2 interface is illustrated in θ, is defined as the angle between the long axis of the rectangular nanohole and the x-axis. The nanohole can be considered as an ideal polaroid with an extinction ratio  tx ≫ ty, and letting tx = 1, the Jones' matrix can be calculated byθ is the rotation angle of a nanohole andσω) and right-handed circularly polarized  beams, with geometric phase discontinuity ±2θ, and a LP beam without geometric phase, which are written as ei2θ) = −χxyy(2) = −χyxy(2) = −χyyx(2) = χ(2), and we haveIt indicates that the transmitted beam can be decomposed into three components: left-handed circularly polarized . The ±1st-order and ±2nd-order deflected beams are CP beams (LCP and RCP) with a phase delay of ∓2θ and ∓4θ, respectively. Due to the conservation of angular momentum, these deflected SHG beams have a polarization state orthogonal to the excitation fundamental beams. Therefore, the ±1st-order and ±2nd-order deflected beams can be written as eiθ)(±2 σω)(±4 σω). Note that the intensity of the ±1st-order deflected beams is much stronger than the one of the ±2nd-order beams, so we mainly focus on the manipulation of ±1st-order SHG beams in the following. We stress that the results as well as its underlying mechanisms are fundamentally different from conventional nonlinear metasurfaces made of symmetric nanostructures  along the x-axis [Next, we elaborate on how this strategy can be implemented to realize multifunctional nonlinear optical devices. To demonstrate SHG manipulation, we designed a linear plasmonic nanosieve with a gradient phase as shown in the SEM image in e x-axis , 50. A hz-axis. It can be seen clearly that the emitted SH signals are split into three beams. The inset images captured at different positions along the z-axis present a clear illustration of the SHG spots, indicating a low divergence angle of the SHG beams. Specifically, the 0th-order SHG beam propagates along the z-axis without any deflection due to the fundamental laser not carrying a geometric phase, while the rest travel along two sides of the z-axis with the same deflection angle (±1st order). The deflection angle α is measured to be 10°, in good agreement with the theoretical design (10°). Furthermore, the polarization states of the SH beams are analyzed. The Stokes parameters S3(S3 = (IRCP − ILCP)/(IRCP + ILCP)) of -1-, 0-, and 1-order SH beams are determined to be -0.90, -0.32, and 0.95, respectively. This indicates that the ±1st-order deflected beams have opposite CP with almost pure polarization, while the 0th-order SH beam is approximately LP. In Figures α = 5°, 10°, and 15°, while the RCP component of the 0th-order beam propagates without deflection and the LCP component is blocked by a quarter-wave plate coupled with a Glan-laser polarizer. Interestingly, a weak 2nd-order SH beam can also be observed with a deflection angle of ~10° . By reversing the order of the supercells arranged on the Au nanosieve, the SAM of the deflected SHG beams can be reversed, while keeping the same deflection angle. As a result, this integrated interface can generate a LP beam by a superposition of two SHG beams with opposite SAMs, whose polarization can be arbitrarily controlled by adjusting the phase difference of the two SHG beams. y-axis polarized). Specifically, by reversing the direction of the phase gradient of the supercells arranged on the odd lines of the Au metasurface, half are converted to deflect an opposite SAM while keeping the same deflection angle (α = 10°), thus generating a LP beam. Note that both deflected LP beams have the same polarization. The phase difference of σω+,2 and σω−,2 can be controlled by adjusting the optical path difference, dsin(α), where d is the offset of the odd and even lines of the metasurfaces with an inversed order of the supercells. Figures β, at phase differences, dsin(α) = 0λ, 0.25λ, and 0.5λ, respectively. β = 0° implies that the SHG beam is linearly polarized along the y-axis. All measured SHG beams are approximately purely linearly polarized. The polarization directions for each beam are indicated by the long axis of the patterns at 10°, 55°, and 100°, respectively. This is in good agreement with the design rule β = ((dsin(α))/λ)π, where λ is the SHG wavelength. We find the same deviation angle of 10° for each SHG beam, which is attributed to the orientation of the WS2 monolayer [Following the beam steering results, two deflected SHG beams propagate along different sides of the onolayer . This isonolayer . Such veonolayer .st-order RCP component of the SH emission from each pixel under a LP pumping (y-axis polarized), which can be described asx is the coordinates of the nanoholes, λ is the wavelength of SHG, α is the deflection angle, n is the topological charge, and γ is the azimuth angle in a circular coordinate system. Here, the deflection angle is α = 10° and the geometric topological charge is n = 1. In y-axis polarized), and five SHG beams are clearly seen as predicted by our theory. Specifically, the 0th-order beam with a circular spot is the LP beam without deflection. The ±1st-order SH beams are propagated along both sides of the z-axis with the same deflection angle, indicating a doughnut-shaped spot due to the properties of OAM beam propagation. Both beams have opposite CP with the same value of deflection angle, α = 10°, which is consistent with our design. Importantly, the topological charge is determined to be ±1 by the interference pattern between the 0th-order and ±1st-order beams . The solution of polymethyl methacrylate  was drop-coated on the WS2-sapphire substrate. It was placed at room temperature for 1.5 h and then baked at 120°C for 0.5 h. The WS2-sapphire substrate was immersed in NaOH aqueous solution (3 mol/L) at room temperature for more than 4 h to etch the sapphire surface, and PMMA-WS2 films can be naturally lifted off from the sapphire substrate. The PMMA-WS2 films were fished out with a glass slide and immersed in deionized water for 3 times in order to remove the residual NaOH solutionTheWSA 60 nm thick gold film with a 5 nm thick Cr adhesion layer was deposited on a quartz substrate by using an e-beam evaporator. Then, the Au nanohole array with different arrangements was milled by the focused ion beam milling method 2 films were fished out by the fabricated metasurfaces, and then they were aligned quickly with the Au metasurface under the microscope before the water was dry. Finally, they were baked at 120°C for 1 h to improve the combination of the WS2 monolayers and the Au nanosieve and immersed in acetone for 3 times to remove the PMMA filmWith the help of a motorized stage-controlled needle, the PMMA-WSμm to 200 μm along the z-axis with a step-size of 0.5 μm.A mode-locked Ti-sapphire femtosecond laser centered at 810 nm  was used for the fundamental beam source. The polarization was adjusted by a quarter-wave plate . A home-built optical system was used to measure an emitted SH signal under an excitation of a plane-wave laser. The fundamental beam was focused by an 8 cm lens or an objective  to generate different sizes of focal spots. The emitted signal was collected by an objective lens , filtered spectrally, and imported to a CMOS camera  or to a spectrometer  through a fiber. The emitted SH signal was extracted by a Glan-laser polarizer with a quarter-wave plate at the SH wavelength . To measure the spatial intensity of the SH signals, we captured the SHG images at the different planes from 0


TRIC-A mainly expresses in excitable cells. TRIC-A−/− mice survive normally but show abnormal SR Ca2+ handling in both skeletal and cardiac muscle cells. Importantly, TRIC-A mutations have been identified in human patients with stress-induced arrhythmia. In the past decade, important discoveries have been made to understand the structure and function of TRIC channels, especially its role in regulating intracellular Ca2+ homeostasis. In this review article, we focus on the potential roles of TRIC-A in regulating cardiac function, particularly its effects on intracellular Ca2+ signaling of cardiomyocytes and discuss the current knowledge gaps.Trimeric intracellular cation (TRIC) channels have been identified as monovalent cation channels that are located in the ER/SR membrane. Two isoforms discovered in mammals are TRIC-A (TMEM38a) and TRIC-B (TMEM38b). TRIC-B ubiquitously expresses in all tissues, and TRIC-B These STIM1 clusters would recruit and activate the Orai1 channels on the plasma membrane and trigger Ca2+ entry into the cytosol [2-induced Ca2+ oscillations in HEK293 cells required the SOCE machinery. Upon ER Ca2+ store depletion, TRIC-A channels co-clustered with the STIM1/Orai1 complex within ER–PM junctions. The association of STIM1 with TRIC-A reduced the co-localization of STIM1 and Orai in the cells, thus suppressing SOCE. Furthermore, they demonstrated that knocking down of TRIC-A in HL-1 would promote SOCE, mimicking the effects of STIM1 overexpression.ER/SR Caechanism . They fo complex . STIM1 i cytosol , 94. UsiSeveral studies have established the physiological and pathological relevance of STIM1 in the heart , 72. Sil2+ signaling plays an important role in physiological and pathological conditions [2+ signaling [2+ uptake by mitochondria is an important messenger for matching energy supply to demand during various physiological workloads. This is achieved through Ca2+-induced activation of Krebs cycle dehydrogenases and pyruvate dehydrogenase [2+ has also been indicated to enhance the activities of complexes I, II, IV, and V along the respiratory chain [2+ handling in cardiomyocytes, triggered by repetitive isoproterenol treatment, chronic pressure overload, or ischemia/reperfusion, would lead to pathological mitochondrial Ca2+ overload, which is not a simple compensatory mechanism to increase energy output [2+ overload enhanced the activity of Na+/Ca2+/Li+ exchanger (NCLX), leading to Ca2+ extrusion at the cost of increased matrix Na+. Matrix Na+ interacts with phospholipids, such as phosphatidylcholine, of the inner mitochondria membrane, leads to reduced membrane fluidityand, the diffusion of ubiquinol (coenzyme Q) from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or complex II to complex III of the respiratory chain, and elevation of ROS production of complex III at Qo site [2+-ATPase, NCX, and other Ca2+ signaling–related proteins [The crosstalk between intracellular organelles has drawn great attention in cell biology and physiology studies in recent years. The crosstalk between ER/SR and mitochondria-mediated Canditions , 75. Li ignaling . Mitochoignaling . In cardrogenase , 36, 58.ry chain , 32. Howy output , 96, 108y output , 63. Mor Qo site . ROS thuproteins , 33, 99.proteins , 39, 92.proteins .−/− mice. However, heart from TRIC-A-deficient mice showed altered SR Ca2+ regulation such as (SR Ca2+ overload) under stress conditions. For example, chronic treatment with β-adrenergic receptor agonist isoproterenol (ISO) leads to extensive fibrosis development in the TRIC-A−/− heart. Such effect is likely caused by increased death of cardiomyocytes and fibrotic remodeling, which may be linked to the overload of SR Ca2+ associated with the ablation of TRIC-A. SR Ca2+ overload due to TRIC-A depletion likely exacerbates mitochondria Ca2+ overload, promoting mitochondria ROS production and facilitating apoptosis and/or necrosis of the TRIC-A−/− cardiomyocytes. However, the underlying mechanism is still largely unknown. Therefore, it is worthwhile to further explore the role of TRIC-A in Ca2+ mediated SR-mitochondrial crosstalk.There are no significant abnormalities in the heart under basal conditions in TRIC-ATRIC-A is not only located in the ER/SR membrane but also heavily expressed in the membrane of the nuclear envelope of muscle cells . Althoug2+ regulation. Since TRIC-A is known to regulate ER/SR Ca2+ signaling, its role in the nuclear Ca2+ regulation is plausible. It has been shown that Ca2+ signaling in the nucleoplasm regulates events that are distinct from the ones mediated by cytosolic Ca2+ [2+ store called nucleoplasmic reticulum that expresses RyR and IP3R [3-induced Ca2+ release from nucleoplasmic reticulum facilitates protein kinase C (PKC) translocation to the nuclear envelope [2+ signaling is also involved in cardiomyocyte hypertrophy. Diminishing Ca2+ level in the nuclei leads to swelling of the nuclei in neonatal cardiomyocytes, accompanied by increased calcineurin expression and increased nuclear enrichment of NFAT [2+ regulation and gene translational regulation. Further studies are required for understanding the exact role that TRIC-A plays in the nuclear envelope of cardiomyocytes. The potential of TRIC-A regulating nuclear Ca2+ signaling would be an interesting direction for future investigation.Another possible role of TRIC-A on the nuclear envelope could be related to nuclear Calic Ca2+ . For exaand IP3R , 54. IP3envelope . Abnorma of NFAT . It is w of NFAT , 62. Rec of NFAT , althougTable 2+ release, the identification of direct interactions between TRIC-A and RyR2 support a pivotal role of TRIC channels in regulating ER/SR Ca2+ homeostasis. Due to the growing interest in the physio-pathological roles of TRIC channels, novel regulatory mechanisms mediated by TRIC-A have started to emerge, including direct control of RyR2 activity, SOCE, SOICR functions, and its potential role in modulating mitochondria Ca2+ signaling. In addition to SR/ER localization, TRIC-A is also identified on the nuclear envelope and may participate in excitation-transcriptional regulation of genes involved in myogenesis and adaptive responses of the muscle and heart under physiologic and pathophysiologic settings.We depict a diagram to demonstrate the proposed functions of TRIC-A in cardiomyocytes Fig. . Althoug


The Ca2+ signalling is an evolutionarily conserved process from prokaryotes to eukaryotes. Ca2+ at high concentration is deleterious to the cell; therefore, cell maintains a low resting level of intracellular free Ca2+ concentration ([Ca2+]c). The resting [Ca2+]c is tightly regulated, and a transient increase of the [Ca2+]c initiates a signalling cascade in the cell. Ca2+ signalling plays an essential role in various processes, including growth, development, reproduction, tolerance to stress conditions, and virulence in fungi. In this review, we describe the evolutionary aspects of Ca2+ signalling and cell functions of major Ca2+ signalling proteins in different fungi.Calcium (Ca Ca2+ has formed when oxygen and neon fused with successive particles  at resting state is maintained at ∼100 nM; therefore, cells maintain a more than 10,000-fold gradient across the plasma membrane , mitochondria, and vacuoles (Cornelius and Nakashima 2+ concentration ([Ca2+]ER) is maintained at several hundred µM. Besides, cells also need to maintain intracellular Ca2+ homoeostasis to avoid severe Ca2+ fluctuations and their effects  and the ryanodine receptors (RyR) induces Ca2+ release from the internal stores (Mikoshiba and Hattori 2+-ATPase (PMCA), the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), the Na+/Ca2+ exchanger, and the mitochondrial uniporter are responsible for sequestering the excess Ca2+ from the cytosol by transporting Ca2+ either to external medium or into different cellular compartments , phospholipase B (PLB), phospholipase C (PLC), and phospholipase D (PLD)  into two important second messengers, inositol 1,4,5-triphosphates (IP3) that induces Ca2+ release from internal stores and diacylglycerol (DAG) that activates protein kinase C (PKC), and triggers a range of cellular activities -dependent enzymes , which shows sequence homology to mammalian PI-PLC-δ isoforms  or higher temperatures (43°C), and reduced growth in medium containing galactose, but not glucose, as the sole carbon source  have been identified , sarco (endo) plasmic reticulum Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are ATP-driven pumps involved in the active transport and homoeostasis of Ca2+ , which is involved in germ tube formation causing morphogenesis of C. albicans; however, calmodulin inhibitor trifluoperazine (TFP) inhibits the phosphorylation and germ tube formation  lacking the coding region for the C-terminal calmodulin-binding site and the autoinhibitory domain  irradiation, and defect in the sexual development causing female sterility  that is essential for cell survival , ergosterol-depletion agents voriconazole and itraconazole, and the ergosterol intercalating agent amphotericin B; however, did not affect the virulence , which belongs to the family of EF-hand containing protein, binds to CaBurgoyne . The NCS2+ and CaM for its activity  subunits encoding genes CNA1 and CNA2 showed an increased sensitivity to growth arrest induced by the mating pheromone α-factor , and the regulatory (cnb1) subunit, cause sensitivity to high concentrations of Mn2+ (MnCl2), because the mutants were not able to block the entry of Mn2+ into the cell   causing damage of the fungal cell walls  contains a serine-proline-rich-region (SPRR) unique to the filamentous fungi, but absent in the human calcineurin α-catalytic subunit  in A. fumigatus  plays a role in mycelial growth, conidiation, formation of specialised infection structure called appressorium, and pathogenicity  inhibits the immunophilins FKBP12 – FK506 binding to Cnb1, which revealed the mechanism of drug action in C. neoformans (Fox et al. C. albicans (Cruz et al. C. albicans, disruption of cna-1 results in the loss of viability in the presence of antifungal agents fluconazole, amorolfine, itraconazole, terbinafine, and voriconazole (Sanglard et al. C. albicans (Singh et al. C. albicans (Jia et al. In addition, calcineurin plays a critical role in fungal pathogenicity. us Chang . In M. oS. cerevisiae, the calcineurin target Crz1p, also known as Tcn1p, drives the expressions of PMC1, PMR1, PMR2A, and FKS2 genes, which play a vital role in the tolerance to high Ca2+, Mn2+, Na+, and cell wall damage, respectively (Matheos et al. S. pombe, the null mutant of prz1, the crz1 homologue, did not show any defects in morphology and sexual development; however, the Δprz1 mutant was hypersensitive to Ca2+ (Hirayama et al. M. grisea, the CRZ1 homologue MgCRZ1 partially complemented the S. cerevisiae Δcrz1 mutant and suppressed the Li+ sensitivity (Zhang et al. 2+ stress tolerance, and full virulence (Zhang et al. C. neoformans, crz1 drives the expression of the chitin synthase gene chs6, and crz1 deletion mutant showed a defect in cell wall synthesis (Lev et al. C. albicans, the CRZ1 deletion mutant was more sensitive to Ca2+, Li+, and Mn2+ cations, hypersensitive to anionic detergents SDS and antifungal azoles, including fluconazole and miconazole (Santos and de Larrinoa Aspergillus parasiticus, crzA is required for vegetative growth, asexual development, and aflatoxin production under the Ca2+ stress condition (Chang A. fumigatus, knock out of crzA results in a defect in germination and polarised hyphal growth, and reduced sporulation (Cramer et al. A. fumigatus ΔcrzA mutants also displayed sensitivity to heat shock conditions (Soriani et al. A. flavus, which produces aflatoxin, the deletion of crzA renders the strain more vulnerable to cell wall stress and osmotic pressure (Lim et al. A. flavus ΔcrzA mutants, conidiophore production was reduced as the head of the conidiophore become short, along with a decrease in the number of conidia (Lim et al. There are different target proteins of calcineurin found across different organisms (Li et al. on Chang . In A. fM. grisea, when infected to onion surface or rice leaves, Δmocrz1 showed a significant reduction in the appressorial penetration rate (Choi et al. M. grisea, lipid droplets are transported from conidia to nascent appressorium, which fully melanised within 12–24 h (Zhang et al. M. grisea Δcrz1 mutant, the lipid droplets were undegraded even after 48 h (Zhang et al. M. grisea (Zhang et al. B. bassiana, Δcrz1 mutant showed defects in growth and conidiation, suppressed growth in the presence of osmotic salts, sensitivity to stress-inducing chemicals and carbendazim and osmotic salts, decreased thermotolerance, reduced resistance to UV-B irradiation, changed cell wall composition, and longer virulence period (Li et al. Crz 1 also plays a critical role in fungal virulence. In 2+ signalling regulates multiple cell functions ranging from growth, development, fertility, stress tolerance, and virulence in fungi. Phospholipases are enzymes that act on membrane phospholipids and classified into four types. In response to specific signals, PLC produces IP3 and DAG, which mediate Ca2+ release and activation of PKC, respectively. There are also six major types of Ca2+ transporters that are required for Ca2+ homoeostasis and signalling. The increase in [Ca2+]c activates various Ca2+ sensors such as CaM and NCS-1 that interact with specific downstream proteins as a response to a signal. Calcineurin, which consists of catalytic A and regulatory B subunits, is one of the major downstream Ca2+ signalling proteins required for multiple cell functions, including growth, stress tolerance, and virulence in fungi. Calcineurin is also identified as a target for antifungal drugs. Calcineurin activates the Crz 1 transcription factor to activate expressions of target genes required for growth, developments, tolerance to stress conditions, and pathogenicity. Further research on the Ca2+ signalling machinery will unravel its complex molecular network under different cellular conditions.Ca


IKZF1 deletion has been demonstrated recurrent, but whether IKZF1 mutation also exists in AML remained largely unknown. Herein, we analyzed the IKZF1 mutation in AML. In our cohort, the frequency of IKZF1 mutation was 2.6% (5/193), and 5 frameshift/nonsense mutations as well as 2 missense mutations were identified in total. Molecularly, IKZF1 mutation was absent in fusion gene-positive AML, but it was demonstrated as the significant concomitant genetic alteration with SF3B1 or bi-alleleCEBPA mutation in AML. Clinically, two IKZF1, PTPN11 and SF3B1-mutated AML patients exhibited one aggressive clinical course and showed primary resistant to chemotherapy. Furthermore, we confirmed the recurrent IKZF1 mutation in AML with cBioPortal tool from OHSU, TCGA and TARGET studies. Interestingly, OHSU study also showed that SF3B1 mutation was the significant concomitant genetic alteration with IKZF1 mutation, indicating their strong synergy in leukemogenesis. In conclusion, IKZF1 mutation recurrently affected AML.IKZF1 belongs to the IKAROS family of transcription factors, and its deletion/mutation frequently affects acute lymphoblastic leukemia. In acute myeloid leukemia,  IKZF1 deletions and mutations have been reported to affect B-cell precursor ALL and contribute to its poor prognosis [IKZF1 alterations are less studied in AML. Recurrent IKZF1 deletions have been identified in AML [IKZF1 mutations affect AML in general remains unknown. Herein, we analyzed IKZF1 mutation in AML.IKZF1 belongs to the IKAROS family of transcription factors. It contains four zinc fingers at the N-terminal that directly bind to DNA at the core motif A/GGAAA and additional two zinc fingers at the C-terminal required for forming homo- and hetero-dimerization between different IKZF proteins [rognosis . IKZF1 ad in AML , 4, but de novo AML, 10 with refractory/relapsed AML, 6 with MDS/AML, 5 with MLL (5 de novo cases), and 3 with MS/AML . The panel of TES included 236 genes recurrently mutated in hematological malignancies, and TES was displayed by NovaSeq platform (Illumina). The average raw sequencing depth on target per sample was ≥ 1000, and VAF ≥ 1% was considered significant. For TES, 184 samples were collected from BM and 9 samples from PB. In addition, fusion gene screening for common rearrangements in AML was employed.A total of 193 adult AML patients, who subjected to TES, were retrospectively analyzed in our center (01/05/2018–29/02/2020), while APL was excluded. Among these patients, 100 were male and 93 were female, and the median age was 56 (range 18–82). A total of 169 patients were diagnosed with IKZF1 mutation affected 2.6% (5/193) or 1.8% (3/169) of all AML patients or de novo AML patients from our cohort, respectively  , but it was absent in COSMIC and our study . The frIKZF1 alteration is well studied in ALL. Churchman et al. reported that IKZF1 alteration affected 25% of childhood and 44% of young adult pre-B-cell ALL, especially BCR-ABL1-positive ALL with frequency of over 80%. In ALL, the most common type of alterations in IKZF1 is deletions, whereas IKZF1 mutations accounted only 2.6% of childhood and 3.4% of young adult ALL. The latter were observed in 11.9% of BCR-ABL1-negative and 2.2% of BCR-ABL1-positive ALL cases [IKZF1 often occurred at the N-terminal or the region between DNA binding and dimerization domains, while missense mutations affected both domains. Consistently, IKZF1 mutations followed the same pattern in AML. Similarly to IKZF1N159S in AML, IKZF1N159Y is a hotspot mutation in ALL that affects its DNA binding domain. IKZF1N159Y-ALL exhibited one unique transcriptional profile characterized by downregulation of B-cell receptor and JAK-STAT signaling and upregulation of SALL1 [IKZF1N159S-AML could be defined as one independent subtype remains to be investigated.Compared to AML, IKZF1 deletion, IKZF1 mutation is also recurrent in AML.In conclusion, besides of Additional file 1: Table S1. The variant allele frequency of IKZF1 mutation in AML from cBioPortal database.


From a physiological point of view, the open state of CALHM1 depends not only on voltage but also on the extracellular concentration of calcium ([Ca2+]) ions. At low [Ca2+]e or depolarization, the channel is opened, allowing Ca2+ influx; however, high extracellular [Ca2+]e or hyperpolarization promote its resting state. The unique Ca2+ permeation of CALHM1 relates to the molecular events that take place in brain ischemia, such as depolarization and extracellular changes in [Ca2+]e, particularly during the reperfusion phase after the ischemic insult. In this study, we attempted to understand its role in an in vitro model of ischemia, namely oxygen and glucose deprivation, followed by reoxygenation (OGD/Reox). To this end, hippocampal slices from wild-type Calhm1+/+, +/−Calhm1, and −/−Calhm1 mice were subjected to OGD/Reox. Our results point out to a neuroprotective effect when CALHM1 is partially or totally absent. Pharmacological manipulation of CALHM1 with CGP37157 reduced cell death in Calhm1+/+ slices but not in that of −/−Calhm1 mice after exposure to the OGD/Reox protocol. This ionic protection was also verified by measuring reactive oxygen species production upon OGD/Reox in Calhm1+/+ and −/−Calhm1 mice, resulting in a downregulation of ROS production in −/−Calhm1 hippocampal slices and increased expression of HIF-1α. Taken together, we can conclude that genetic or pharmacological inhibition of CALHM1 results in a neuroprotective effect against ischemia, due to an attenuation of the neuronal calcium overload and downregulation of oxygen reactive species production.Calcium homeostasis modulator 1 (CALHM1) is a calcium channel involved in the regulation of cytosolic Ca Ischemic stroke is the most frequent cause of cerebrovascular disease . It is t+/K+/ATPase and Ca2+/ATPase, promoting membrane depolarization due to Na+ influx. This depolarization provokes the opening of voltage-dependent calcium channels (VDCCs) and makes the Na+/Ca2+ exchanger work in its reverse way; these two processes lead to intracellular Ca2+ overload o). In the presence of physiological [Ca2+]o (~1.5 mM), CALHM1 is closed at resting membrane potentials but can be activated by strong depolarizations. On the contrary, hyperpolarization can trigger its inactivation, which prevents Ca2+ entry into the cell [2+ add-back strongly elevates [Ca2+]i in CALHM1-transfected HT-22 and N2A cells [2+]o oscillations can also regulate the CALHM1 state.CALHM1 is an octamer whose monomers form the functional ion channel . CALHM1 the cell ,17. Furt2A cells . Finally2+]o. Reducing [Ca2+]o from 1.5 to 0.2 mM reduces the input resistance by ~50% and increases excitability in neurons from wild-type mice but fails to enhance the excitability of cortical neurons from −/−Calhm1 mice i after the removal and following the add-back of Ca2+ (conditions by which CALHM1 is activated) in Hela cells overexpressing CALHM1 c entry but, in addition, contributes to lowering ROS production, opening a new neuroprotective pathway for CALHM1.It is well described that under hypoxic conditions, such as ischemia, there is intracellular Cal damage . Once de2+ dependent protein such as HIF-1α. Under hypoxic conditions, HIF-1α accumulates and dimerizes with HIF-1β, and translocates into the nucleus where it binds to the hypoxic-response elements (HREs), inducing the expression of genes that have been linked with the adaptation to hypoxia, such as antioxidant genes, as well as genes involved in angiogenesis and glycolytic energy metabolism. This metabolic adaptation decreases the formation of ROS and mitochondrial toxicity c [2+] [2+] c ,16,17, m+]c [2+]  and ER [2+] [2+] . Now we rization , but itschannels . Ca2+ enoduction . In fact2+ elevation, triggering cell survival under low oxygen concentrations [−/−Calhm1 mice, subjected to OGD/Reox, compared to Calhm1+/+. An increase in HIF-1α levels might be beneficial because it could further induce antioxidant protein expression [−/−Calhm1 samples is conflicting. However, intracellular Ca2+ increase is not the only molecular mechanism that induces HIF-1α expression and stabilization. HIF-1α stabilization in the cytosol promotes metabolic adaptation to hypoxia, proliferation, survival, and resistance to apoptosis, although the lack of CALHM1 leads to less Ca2+ entry into the cell, it is no surprising that the expression levels of HIF-1α protein are increased in knock-out mice. Also, HIF-1α regulates the expression of some Ca2+ permeant channels, such as the transient receptor potential (TRP) C1 (TRPC1), stromal interaction molecule-1 (STIM1), or the Golgi Ca2+ pump secretory pathway Ca2+-ATPase 2 (SPCA2). Based on the latter observations, we can hypothesize that the deletion of CALHM1 could trigger an elevation in HIF-1α levels as a compensatory mechanism in order to restore Ca2+ signaling through other Ca2+ channels [Another key marker activated after exposure to OGD/Reox is HIF-1α. This protein is inducible upon hypoxia and cytosolic Catrations . We founpression . The finchannels 2+ and Co2+, their high toxicity and non-specificity make them inadequate for use in the clinic. Something similar occurs with 2-APB and Ruthenium Red [Although different CALHM1 antagonists have been described in the literature such as Ninium Red , they blnium Red , indicat2+ entry through CALHM1 contributes to cell death upon ischemic conditions. The neuroprotective molecular pathway results from lowering ROS production and activating of HIF-1α. Taken together, these results open a novel neuroprotective pathway for ischemic stroke, which includes the inhibition of CALMH1. Genetic or pharmacological inhibition of CALHM1 in hippocampal slices from mice, was neuroprotective in the ODG/Reox protocol, suggesting that Ca


FLT3-ITD has been recently used as a molecular prognostic marker for risk classification in acute myeloid leukemia (AML) therapy. In this study we aimed to investigate the association of FLT3-ITD gene mutation with bone marrow blast cell count, CD34 expression as malignant cell burden, cyclin D1 and Bcl-xL expressions as indexes of cell proliferation and anti-apoptosis and human equilibrative nucleoside transporter 1 (hENT1) expression as cytarabine transporter during AML treatment.FLT3-ITD mutations, bone marrow blast cell count, CD34, cyclin D1, Bcl-xL and hENT1 expression in bone marrow aspirates from 22 de novo AML patients in a cross sectional study.We investigated FLT3-ITD mutations were observed in 5 out of 22 de novo AML patients (22.7%). Patient with FLT3-ITD mutations had higher blast cell counts . In patients with FLT3-ITD mutations, CD34 and cyclin D1 expressions were higher  than the patients without mutations. hENT1 expression in AML with FLT3-ITD mutation was lower  than in mutation-free AML. There was no significant difference in Bcl-xL expression between patients with and without mutations (P=0.61).FLT3-ITD gene mutations in AML patients with bone marrow blast cell count, CD34, cyclin D1 and hENT1 expressions, however no association was obtained with Bcl-xL expression. These findings support the role of such mutation in pathogenesis of AMLand its contribution in rearrangement of standard therapy with cytarabine in management of AML.A significant association was found between  CEBPA biallelic gene mutation and NPM1 gene mutation without FLT3-ITD mutation. Findings in intermediate risk cases include normal cytogenetics, +8 and t and the findings in poor risk cases include complex cytogenetic abnormalities , -5/5q-, -7, 7q- and 11q23 rearrangement except t, inv(3) or t, t, t and normal cytogenetics with FLT3-ITD gene mutation (Acute myeloid leukemia (AML) is a hematologic malignancy with clonal abnormalities in hematopoietic stem cells with heterogeneous clinical features and basic genetic aberrations. The incidence of AML is 4.2/100.000 persons/year globally and its mortality rate is 2.8/100.000 persons/year  expression in children with acute lymphoblastic leukemia , Bcl-xL (an anti-apoptotic agent) and hENT1 (a cytarabine transporter) expressions that lead to poor prognosis and determine treatment outcomes in AML patients with this mutation.In this study, we investigated the association of This study was a cross sectional study and was ethically approved by the ethical committee, faculty of medicine, Airlangga University. Appropriate informed consents were obtained before samples procurement. Bone marrow aspirates were collected from 22 newly diagnosed AML patients from August 2019 – January 2020 from a private hospital around Surabaya, Indonesia. All bone marrow samples investigated in the study were obtained at the time of first diagnosis. The laboratory examination was performed in department of biochemistry – molecular biology and clinical pathology, faculty of medicine, Airlangga University, Dr. Soetomo general hospital Surabaya.® using monoclonal antibody anti-CD45 conjugated to peridinin chlorophyll protein (PerCP), anti-CD34 conjugated to phycoerythrin (PE) from Becton Dickinson®, anti-hENT1 conjugated to fluorescein isothiocyanate (FITC), anti-Bcl-xL conjugated to PE and anti-cyclin D1 conjugated to FITC provided from Santa Cruz Biotechnology Inc®. Diagnosis of AML was established based on bone marrow aspirate cytology analysis. Diagnosis of AML was established observing more than 20% blast cell count as cut off point. Subtypes of AML were classified based on French American British (FAB) criteria. Bone marrow blast cells were counted among 500 of nucleated cells. The expressions of CD34, hENT1, Bcl-xL and cyclin D1 were examined with BD FACSCaliburFLT3-ITD gene mutation was done with bone marrow specimen. DNA was extracted and purified using QIAmp DNA blood mini kit Qiagen®. In brief, 1 µL of DNA was mixed with 10 µL of PCR kit Go Taq 2x PCR master mix solution (Promega®), 1 µL of forward and 1 µL of reverse primer in a total volume of 20 µL. The forward FLT3?????100 V electrophoresis was done with a 100 bp DNA ladder (Promega®). The presence of FLT3-ITD mutation was determined by visualization of 329 bp wild type gene fragment and a fragment larger than 329 bp. The PCR investigation for ®) in the first tube and 2, 5 µL of PerCP-labeled anti-CD45, 2, 5 µL of anti-Bcl-xL and 2, 5 µL of anti-cyclin D1  in the second tube. Blast gating strategy was used to evaluate CD34, hENT1, Bcl-xL and cyclin D1 expressions in the blast cells. Median Fluorescent Intensity (MFI) was applied to examine CD34, hENT1, Bcl-xL and cyclin D1 expressions  mixture was added to 2, 5 µL of PerCP-labeled anti-CD45, 2, 5 µL of PE-labeled anti-CD34 and FITC-labeled anti-hENT1 . FLT3-ITD gene mutation, blast cell count, CD34, Bcl-xL, cyclin D1 and hENT1 expressions. Total AML patients included 13 males and 9 females. Both adult and pediatric AML cases (ages 4–84) were included in this study. According to FAB classification for AML, 6 cases were diagnosed with AML M1 (27.3%), 5 cases were diagnosed with AML M2 (22.7%), 5 cases were diagnosed with AML M3 (22.7%), 1 case was diagnosed with AML M4 (4.5%) and 5 cases were diagnosed with AML M5 (22.7%). We analyzed bone marrow specimens obtained from 22 newly diagnosed AML patients for the presence of FLT3-ITD were found in 5 (22.7%) AML patients based on the detectable amplicon with 329 bp and larger than 329 bp in 3% agarose gel electrophoresis. All of those patients were classified as FLT3-ITD mutants. The cases result of gel electrophoresis is presented in Mutations in FLT3-ITD was frequently found in AML-M2 patients (3 out of 5 patients). The other subtypes of AML patients with FLT3-ITD mutations were AML-M3 (1 out of 5 patients) and AML-M5 (1 out of 5 patients). This mutation is found in 1 pediatric 5-year-old patient. The flowcytometry analysis of AML cases with and without FLT3-ITD gene mutation are presented in The mutation of FLT3-ITD gene mutation was significantly higher than mutation-free AML patients . The expressions of CD34 and cyclin D1 in AML cases with FLT3-ITD mutation were higher than in AML cases without this mutation . hENT1 expression in AML with FLT3-ITD mutation was lower  than mutation-free AML. The Bcl-xL expression didn’t have a significant difference between AML patients with and without FLT3 mutation (P=0.61). The analytical data are summarized in The blast cell count in AML patients with FLT3. The first mutation is internal tandem duplication (ITD) in juxtamembrane (JM) domain and a point mutation in tyrosine kinase domain. FLT3-ITD gene mutation has been known as a strong prognostic factor in AML patients and it is related to disease progression, high relapse rate and low overall survival for 10 months . Our result is slightly higher than the published studies in Asian populations in which the frequency of FLT3-ITD gene mutation varies between 10 to 21% . The other AML subtypes with FLT3-ITD gene mutation include AML-M3 (1 patient) and AML-M5 (1 patient). This result is different from the result of a large study in Germany. Thiede C. et al. reported that AML-M5 is the most common FAB subtype in AML with FLT3-ITD gene mutation  and the regulation of cycline D1, Bcl-xL, c-MYC, PIM serine-threonine kinase and p21WAF1/CIP1 (inhibitor of cyclin dependent kinase) transcription (Mutant FLT3 receptor inhibits the function of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), a co-repressor that interacts with promyelocytic leukemia zinc finger (PLZF) and eight twenty one protein (ETO) that are related to the cell proliferation blockade . Mutatedcription . FLT3-ITD mutation induces constitutive receptor activation, ligand independent dimerization and autophosphorylation which supports uncontrolled leukemic cells proliferation and apoptosis. The leukemic cells burden is the resultant of uncontrolled proliferation and apoptotic-antiapoptotic activity (FLT3-ITD mutation had higher bone marrow blast cell count and CD34 expression level than mutation-free patients. Blast cell count and CD34 expression had significant association with the FLT3-ITD gene mutation in AML. Some studies have also reported that the presence of FLT3-ITD gene mutation is significantly associated with the higher blast cell count and CD34 expression (FLT3 and negative expression of CD34 predict a longer survival (activity ,21. In tactivity ,23. AML pression ,17,18,24survival . FLT3-ITD mutation than in patients without mutation. Cyclin D1 expression had a significant association with this mutation. This result supports the excessive leukemic cells proliferation in the pathogenesis of AML with FLT3-ITD mutation (FLT3-ITD gene mutation impairs the auto-inhibitory function in JM domain, causing ligand independent activation of the receptor and the receptor related pathway. The expression of Bcl-xL as the marker of anti-apoptotic activity was not significantly different in AML cases with and without FLT3-ITD gene mutations. The FLT3-ITD mutation was not associated with the expression of Bcl-xL. Based on this study, the excessive proliferation activity is the prominent pathogenesis of the AML with FLT3-ITD gene mutation.In this study, cyclin D1 expression was significantly higher in AML with mutation ,22. FLT3FLT3-ITD mutation have poor prognosis with high relapse rate, low overall survival (FLT3-ITD gene mutation had significant association with expression of hENT1, the important cytarabine transporter. AML patients with FLT3-ITD gene mutation had significantly lower expression level of hENT1 than mutation-free patients. Previous study proposed that FLT3-ITD mutation in AML cell lines had association with hENT1, an important cytarabine transporter in AML therapy (FLT3-ITD specifically induced cytarabine resistance in leukemic cells through repression of hENT1 expression. Its mechanism might be due to upregulation of HIF-1α (FLT3-ITD gene mutation and the resistance to cytarabine, one of the essential chemotherapy drugs in the current strategy of AML treatment in vivo. hENT1 is responsible for transporting cytarabine to the leukemic cells with up to 80% influx. Intracellular concentrations of cytarabine depend on the uptake process by hENT1 in AML induction treatment with standard doses of cytarabine (AML patients with survival ,26. Our  therapy . This fif HIF-1α ,27. Cytaf HIF-1α . Our restarabine -35. FLT3-ITD gene mutation in AML patients was associated with blast cell count, CD34 and cyclin D1 expression, therefore it supports the role of FLT3-ITD gene mutation in excessive proliferation activity responsible for the pathogenesis of AML. This mutation was not associated with Bcl-xL expression.FLT3-ITD gene mutation in AML is associated with hENT1 expression. The expression of hENT1 in AML patients with FLT3-ITD mutation is significantly lower than in mutation-free AML patients. This phenomenon supports the pathogenesis of cytarabine resistance in AML with mutated FLT3-ITD during AML induction therapy. It is important that either addition of FLT3 inhibitor in standard induction therapy and resetting cytarabine doses or development of novel therapeutic approaches should be considered in AML patients with FLT3-ITD gene mutation.